Methods and compositions for stem cell differentiation

Abstract

Provided herein are compositions, methods, kits, pharmaceutical, formulations comprising stem cells expressing one or more transcription factors for differentiating pluripotent stem cells (PSC) into hematopoietic stem cell / progenitor or hematopoietic-like stem cell lineages.

Description

METHODS AND COMPOSITIONS FOR STEM CELL DIFFERENTIATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 617,402, filed on January 03, 2024, which is incorporated by reference herein in its entirety for all purposes.SUMMARY OF THE INVENTION

[0002] Induced pluripotent stem cells (iPSCs) can be programmed into various phenotypes via the introduction of one or more transcription factors or one or more molecules that modulate transcription or transcription factors. For example, iPSCs can be programmed into immune cells, which may be used in, for example, immunotherapeutic applications.

[0003] Disclosed herein are provided engineered stem cells compositions, methods, kits, formulations for inducing differentiation of a pluripotent stem cell to differentiate into immune cell lineages.

[0004] This disclosure provides a cell or methods of generating a hematopoietic stem cell, a progenitor cell, or a hematopoietic-like stem cell. An aspect of the present disclosure is an engineered cell comprising a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise LM02 and SPI1. In some embodiments, an engineered cell comprises one or more transcription factors further comprising TALI . In some embodiments, an engineered cell comprises one or more transcription factors further comprising GATA2. In some embodiments, an engineered cell comprises one or more transcription factors further comprising HOXA10.

[0005] An aspect of the present disclosure is an engineered cell comprising a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1. In some embodiments, an engineered cell comprises one or more transcription factors comprise CEBPA. In some embodiments, an engineered cell comprises one or more transcription factors comprise GATA2. In some embodiments, an engineered cell comprises one or more transcription factors comprise SPI1. In some embodiments, anengineered cell comprises one or more transcription factors comprising at least one of LM02 or TALI . In some embodiments, an engineered cell comprising one or more transcription factors is a human stem cell. In some embodiments, an engineered cell comprising one or more transcription factors a human induced pluripotent stem cell. In some embodiments, an engineered cell comprising one or more transcription factors a human progenitor cell. In some embodiments, an engineered cell comprising one or more transcription factors comprises a human fetal stem cell derived from a human stem cell.

[0006] In some embodiments, an engineered cell comprises an erythroid lineage cell. In some embodiments, an engineered cell comprises myeloid lineage cell. In some embodiments, an engineered cell comprises a granulocyte-macrophage lineage cell. In some embodiments, an engineered cell comprising a granulocyte lineage cell. In some embodiments, an engineered cell comprising a erythrocyte lineage cell. In some embodiments, an engineered cell comprising a monocyte lineage cell. In some embodiments, an engineered cell comprising a megakaryocyte lineage cell. In some embodiments, an engineered cell comprising one or more transcription factors induces differentiation of the PSC into the engineered cell. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune- cell-like markers 2 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers 1 day or less. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers comprising CD34. In some embodiments, an engineered cell expresses one or more immune cell or immune -cell-like markers comprising CD43. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers comprising CD45. In some embodiments, an engineered cell expresses one or more immune cell or immune -cell-like markers comprising THY1. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers comprising ITGA6. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers comprising CD33. In some embodiments, an engineered cell expresses one or more immune cell or immune -cell-like markers comprising PEC AMI . In some embodiments, an engineered cell expresses CD34 and CD43. In some embodiments, an engineered cell expresses CD34 and CD45. In some embodiments, an engineered cell expresses CD43 and CD45. In some embodiments, an engineered cell expresses CD34, CD43. In some embodiments, an engineered cell expresses CD45. In some embodiments, an engineered cell expresses is derived from a patient. In some embodiments, an engineered cell is not derived from a patient.

[0007] An aspect of the present disclosure is an engineered cell comprising a pharmaceutical composition comprising an engineered cell. In some embodiments, a pharmaceutical composition comprising an engineered cell further comprises an excipient.

[0008] An aspect of the present disclosure is a method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, comprising administering the engineered cell to a subject in need thereof, thereby treating a disease associated with dysfunctional bone marrow condition. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises multiple myeloma. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises Hodgkin lymphoma. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises nonHodgkin lymphoma. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises acute myeloid leukemia. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises acute lymphocytic leukemia. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises any indication or disease disclosed herein. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises sickle cell anemia. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises Multiple Sclerosis. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises systemic lupus erythematosus. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises another autoimmune disorder. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises any other autoimmune disorder.

[0009] An aspect of the present disclosure is a method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acid molecules encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise LM02 and SPH ; inducing expression of the expression cassette, thereby generating an engineered cell. In some embodiments, the one or more transcription factors further comprise TALI . In some embodiments, the one or more transcription factors further comprise GATA2. In some embodiments, the one or more transcription factors further comprise HOXA10.

[0010] An aspect of the present disclosure is a method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acid molecules encoding one or more transcription factors, oneor more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1 ; inducing expression of the expression cassette, thereby generating an engineered cell. In some embodiments, the one or more transcription factors comprise CEBPA. In some embodiments, the one or more transcription factors comprise GATA2. In some embodiments, the one or more transcription factors comprise SPI1. In some embodiments, the one or more transcription factors comprise at least one of LM02 or TALI . In some embodiments a method comprising an engineered cell comprising a human stem cell. In some embodiments a method comprising an engineered cell comprising a human induced pluripotent stem cell. In some embodiments a method comprising an engineered cell comprising a human progenitor cell. In some embodiments a method comprising an engineered cell comprising a human fetal stem cell. In some embodiments a method comprising an engineered cell comprising an embryonic stem cell derived from a human stem cell. In some embodiments, an engineered cell is an erythroid lineage cell. In some embodiments, an engineered cell is myeloid lineage cell. In some embodiments, an engineered cell is a granulocyte-macrophage lineage cell. In some embodiments, an engineered cell is a granulocyte lineage cell. In some embodiments, an engineered cell is erythrocyte lineage cell. In some embodiments, an engineered cell is monocyte lineage cell. In some embodiments, an engineered cell is a megakaryocyte lineage cell. In some embodiments, an engineered cell is induced to express an expression cassette. In some embodiments, inducing an expression cassette induces expression of one or more transcription factors. In some embodiments, inducing expression of one or more transcription factors induces differentiation of a PSC into an engineered cell. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in 2 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in or 1 day or less. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD34. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD43. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD45. In some embodiments, one or more immune cell or immune-cell-like markers comprise THY1. In some embodiments, one or more immune cell or immune-cell-like markers comprise ITGA6. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD3 or PECAM1. In some embodiments, an engineered cell expresses CD34 and CD43. In some embodiments, an engineered cell expresses CD34 and CD45. In some embodiments, an engineered cell expresses CD43 and CD45. In some embodiments, an engineered cell expressesCD34, CD43, and CD45. In some embodiments, an engineered cell does not express TRA-1-60. In some embodiments, an engineered cell comprises a pluripotent stem cell (PSC). In some embodiments, a PSC is derived from a patient. In some embodiments, a PSC is not derived from a patient. In some embodiments, an engineered cell is an adherent cell. In some embodiments, an engineered cell is a suspension cell. In some embodiments, a method further comprises generating a population of engineered cells. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the population of engineered cells express CD34. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the population of engineered cells express CD43. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of engineered cells express CD45. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD43. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45 -positive cells of the population of engineered cells express CD34 and CD43. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45. In some embodiments, a method further comprises generating a population of engineered cells comprising at most 10%, 9%, 80%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% of the population of engineered cells express TRA-1-60. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02.In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1 . In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI . In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express CEBPA. In some embodiments, a method further comprises generating a population of engineered cells comprising at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HOXA10. In some embodiments, a method further comprises generating a population of engineered cells comprising erythroid lineage cells. In some embodiments, a method further comprises generating a population of engineered cells comprising myeloid lineage cells. In some embodiments, a method further comprises generating a population of engineered cells comprising granulocyte-macrophage lineage cells, or any combination thereof. In some embodiments, myeloid lineage cells comprise granulocyte lineage cells. In some embodiments, myeloid lineage cells comprise erythrocyte lineage cells. In some embodiments, myeloid lineage cells comprise monocyte lineage cells. In some embodiments, myeloid lineage cells comprise megakaryocyte lineage cells. In some embodiments, myeloid lineage cells comprise or any combination thereof. In some embodiments, a method comprises generating a population of engineered cells comprising at least 5% erythroid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% erythroid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 50% erythroid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 5% myeloid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 30% myeloid lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 40%granulocyte-macrophage lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising atleast40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte-macrophage lineage cells. In some embodiments, a method comprises generating a population of engineered cells comprising at least 90% granulocytemacrophage lineage cells. In some embodiment, erythroid lineage cells express CD71, CD235a, or both. In some embodiment, myeloid lineage cells express CD13, CD14, CD15, or any combination thereof. In some embodiment, myeloid lineage cells express CD14 and CD15.

[0011] An aspect of the present disclosure is a method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, the method comprising: administering an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise LM02 and SPI1. In some embodiments, the one or more transcription factors further comprise TALI . In some embodiments, the one or more transcription factors further comprise GATA2. In some embodiments, the one or more transcription factors further comprise HOXA10.

[0012] An aspect of the present disclosure is a method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, the method comprising: administering an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1 . In some embodiments, the one or more transcription factors comprise CEBPA. In some embodiments, the one or more transcription factors comprise GATA2. In some embodiments, one or more transcription factors comprise SPI1 . In some embodiments, the one or more transcription factors comprise at least one of LM02 or TALI . In some embodiments, a method comprises an engineered cell comprising a human stem cell. In some embodiments, a method comprises an engineered cell comprising a human induced pluripotent stem cell. In some embodiments, a method comprises an engineered cell comprising a human progenitor cell. In some embodiments, a method comprises an engineered cell comprising a human fetal stem cell. In some embodiments, a method comprises an engineered cell comprising an embryonic stem cell from a human stem cell. In some embodiments, a method comprises an engineered cell comprising an erythroid lineage cell. In some embodiments, a method comprises an engineered cell comprising a myeloid lineage cell ora granulocyte-macrophage lineage cell. In some embodiments, a method comprises an engineered cell comprising a granulocyte lineage cell. In some embodiments, a method comprises an engineered cell comprising an erythrocyte lineage cell. In some embodiments, a method comprises an engineered cell comprising a monocyte lineage cell or a megakaryocyte lineage cell. In some embodiments is a method comprising inducing expression of an expression cassette. In some embodiments, inducing expression of an expression cassette induces expression of one or more transcription factors. In some embodiments, inducing expression of one or more transcription factors induces differentiation of an engineered cell. In some embodiments, an engineered cell comprises a pluripotent stem cell (PSC). In some embodiments, a PSC comprises an engineered cell. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune -cell-like markers in 2 days or less. In some embodiments, an engineered cell expresses one or more immune cell or immune-cell-like markers in 1 day or less. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD34. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD43. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD45. In some embodiments, one or more immune cell or immune-cell-like markers comprise THY 1 . In some embodiments, one or more immune cell or immune-cell-like markers comprise ITGA6. In some embodiments, one or more immune cell or immune-cell-like markers comprise CD33 or PEC AMI . In some embodiments, an engineered cell expresses CD34 and CD43. In some embodiments, an engineered cell expresses CD34 and CD45. In some embodiments, an engineered cell expresses CD43 and CD45. In some embodiments, an engineered cell expresses CD34, CD43, and CD45. In some embodiments, an engineered cell comprises a PSC. In some embodiments, a PSC is derived from a patient. In some embodiments, a PSC is not derived from a patient. In some embodiments, a method of a disease associated with dysfunctional bone marrow comprises multiple myeloma. In some embodiments, a disease associated with dysfunctional bone marrow comprises Hodgkin lymphoma. In some embodiments, a disease associated with dysfunctional bone marrow comprises non-Hodgkin lymphoma. In some embodiments, a disease comprises acute myeloid leukemia or acute lymphocytic leukemia. In some embodiments, a disease comprises sickle cell anemia. In some embodiments, a disease comprises Multiple Sclerosis. In some embodiments, a disease comprises systemic lupus erythematosus. In some embodiments, a disease comprises another autoimmune disorder. Disclosed herein some embodiments is an engineered cell that is not derived from the subject in need thereof. In some embodiments, an engineered cell is derived from the subject in need thereof. In some embodiments, a methodfurther comprises administering a population of engineered cells. In some embodiments, a population of engineered cells express at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the population of engineered cells express CD34. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the population of engineered cells express CD43. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of engineered cells express CD45. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD43. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45-positive cells of the population of engineered cells express CD34 and CD43. In some embodiments, a population of engineered cells express at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45. In some embodiments, a population of engineered cells express at most 10%, 9%, 80%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% of the population of engineered cells express TRA-1-60. In some embodiments, a population of engineered cells express at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02. In some embodiments, a population of engineered cells express atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1. In some embodiments, a population of engineered cells express atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI . In some embodiments, a population of engineered cells express atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2. In some embodiments, a population of engineered cells express atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2. In some embodiments, a population of engineered cells express atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%of the population of engineered cells express CEBPA. In some embodiments, a population of engineered cells express at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HO XA10. In some embodiments, a population of engineered cells comprise erythroid lineage cells. In some embodiments, a population of engineered cells comprise myeloid lineage cells. In some embodiments, a population of engineered cells comprise granulocyte-macrophage lineage cells or any combination thereof. In some embodiments, a population of engineered cells comprising myeloid lineage cells comprise granulocyte lineage cells. In some embodiments, a population of engineered cells comprising myeloid lineage cells comprise erythrocyte lineage cells. In some embodiments, a population of engineered cells comprising myeloid lineage cells comprise monocyte lineage cells. In some embodiments, a population of engineered cells comprising myeloid lineage cells comprise megakaryocyte lineage cells or any combination thereof. In some embodiments, a population of engineered cells comprise at least 5% erythroid lineage cells. In some embodiments, a population of engineered cells comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% erythroid lineage cells. In some embodiments, a population of engineered cells comprise at least 50% erythroid lineage cells. In some embodiments, a population of engineered cells comprise at least 5% myeloid lineage cells. In some embodiments, a population of engineered cells comprise at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells. In some embodiments, a population of engineered cells comprise at least 30% myeloid lineage cells. In some embodiments, a population of engineered cells comprise at least 40% granulocytemacrophage lineage cells. In some embodiments, a population of engineered cells comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte-macrophage lineage cells. In some embodiments, a population of engineered cells comprise at least 90% granulocyte-macrophage lineage cells. In some embodiments, a population of engineered cell comprise erythroid lineage cells expressing CD71, CD235a, or both. In some embodiments, a population of engineered cell comprise myeloid lineage cells expressing CD13, CD 14, CD 15, or any combination thereof. In some embodiments, a population of engineered cell comprise myeloid lineage cells expressing CD 14 and CD15.

[0013] Disclosed herein are provided engineered stem cells compositions, methods, kits, formulations for inducing differentiation of a pluripotent stem cell to differentiate into immune cell lineages. Provided herein is a pluripotent stem cell (PSC) comprising a recombinant polynucleotide comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of said one or more open reading frames encoding said one or more transcription factors, wherein said recombinantpolynucleotide comprising one or more open reading frames encoding one or more transcription factors, said one or more transcription factors, or said activator of transcription of said one or more open reading frame encoding said one or more transcription factors comprise one or more transcription factors, and wherein said recombinant polynucleotide comprising one or more open reading frames encoding one or more transcription factors, said one or more transcription factors, or said activator of transcription of said one or more open reading frame encoding said one or more transcription factors induces differentiation of said PSC into an immune cell or immune-like cell, and expresses one or more immune cell or immune-cell like markers in 28 days or less; and wherein said one or more immune cell or immune-cell like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, and PEC AMI . In some embodiments, the immune cell or immune-cell like expresses both CD34 and CD43. In some embodiments, the immune cell or immune-like cell expresses both CD34 and CD45. In some embodiments, the immune cell or immune-cell like expresses both CD43 and CD45. In some embodiments, the immune cell or immune-cell like expresses CD34, CD43, and CD45. In some embodiments, the immune cell or immune-like cell is a hematopoietic progenitor cell. In some embodiments, the immune cell or immune-cell like is a lymphoid progenitor cell. In some embodiments, the immune cell or immune-cell like is a B cell. In some embodiments, the immune cell or immune-cell like is a T cell. In some embodiments, the immune cell or immune-cell like is a natural killer cell. In some embodiments, the immune cell or immune-cell like is a myeloid progenitor cell. In some embodiments, the immune cell or immune-cell like is a megakaryocyte-erythrocyte progenitor cell. In some embodiments, the immune cell or immune-cell like is an erythroid progenitor cell. In some embodiments, the immune cell or immune-cell like is an erythrocyte. In some embodiments, the immune cell or immune-cell like is an endothelial cell of lymphatic vessel. In some embodiments, the immune cell or immune-cell like is a cardiac endothelial cell. In some embodiments, the immune cell or immune-cell like is a platelet. In some embodiments, the immune cell or immune-cell like is a mast cell. In some embodiments, the immune cell is or immune-cell like a neutrophil. In some embodiments, the immune cell or immune-cell like is a basophil. In some embodiments, the immune cell or immune-cell like is an eosinophil. In some embodiments, the immune cell or immune-cell like is a monocyte. In some embodiments, the immune cell or immune-cell like is a macrophage. In some embodiments, the immune cell or immune-cell like is a dendritic cell. In some embodiments, the immune cell or immune-cell like is a regulatory T cell. In some embodiments, the immune cell or immune-cell like is an alphabeta T cell. In some embodiments, the immune cell or immune-cell like is a gamma-delta T cell. In some embodiments, the one or more transcription factors comprises one or more of the transcription factors listed in Table 1. In some embodiments, the one or more transcriptionf actors comprises one or more transcription factors from at least one family member of any one of the transcription factors listed in Table 1. In some embodiments, the one or more transcription factors, consists of one or more transcription family members of one or more transcription factors listed in Table 1. In some embodiments, the one or more transcription factors, comprises one or more transcription factors listed in Table 1, one or more transcription factor family members of one or more transcription factors listed in Table 1, or both. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, HOPX, LM02, FLU, NOTCH family member, and TALI . In some embodiments, the one or more transcription factors consists of SPI1, HOPX, LM02, FLI1, NOTCH family member, and TALI . In some embodiments, the one or more transcription factors or transcription factor family members comprises ERG, GATA2, TFDP1, HLF, RFX2, HOXAIO, RUNX1, FOXP1 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members consists of ERG, GATA2, TFDP1, HLF, RFX2, HOXAIO, RUNX1, FOXP1 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members comprises ERG, GATA2, HOXAIO and RFX2. In some embodiments, the one or more transcription factors or transcription factor family members consists of ERG, GATA2, HOXAIO and RFX2. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1 and NOTCH family member. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1 and NOTCH family member. In some embodiments, the one or more transcription factors or transcription factor family members comprises HOPX, LM02, TALI, FLI1, TFDP1, HOXAIO, GATA2, HLF and RUNX1. In some embodiments, the one or more transcription factors or transcription factor family members consists of HOPX, LM02, TALI, FLI1, TFDP1, HOXAIO, GATA2, HLF and RUNX1. In some embodiments, the one or more transcription factors or transcription factor family members comprises NOTCH family member, SPH , RUNX2, HOPX and TFDP1. In some embodiments, the one or more transcription factors or transcription factor family members consists of NOTCH family member, SPI1, RUNX2, HOPX and TFDP1. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, LM02, TALI, NOTCH family member, GATA2, HOPX, RUNX1, TFDP1, HLF, RFX2, FLU, NFE2, MYB and ERG. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPH, LM02, TALI, NOTCH family member, GATA2, HOPX, RUNX1, TFDP1, HLF, RFX2, FLU, NFE2, MYB and ERG. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPH, LM02, TALI, RUNX1, TFDP1, FLI1, HLF, HOPX, NOTCH family member, ERG, GATA2, RUNX2, MYB and NFE2. In someembodiments, the one or more transcription factors or transcription factor family members consists of SPI1, LM02, TALI, RUNX1, TFDP1, FLU, HLF, HOPX, NOTCH family member ,ERG, GATA2, RUNX2, MYB and NFE2. In some embodiments, the one or more transcription factors or transcription factor family members comprises TALI, LM02, NOTCH family member, HOPX, FLU, TFDP1, GATA2, HLF, HOXAIO, H0XA5 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members consists of TALI, LM02, NOTCH family member, HOPX, FLU, TFDP1, GATA2, HLF, HOXAIO, H0XA5 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPH, NOTCH family member, LM02, RUNX1, MYB, TALI and HOPX. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1 , NOTCH family member, LM02, RUNX1, MYB, TALI and HOPX. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, LM02, TALI, NOTCH family member, RUNX1, HOPX and H0XA5. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, LM02, TALI, NOTCH family member, RUNX1, HOPX and H0XA5. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, TALI, LM02, TFDP1, HOPX, FLU and RUNX2. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, TALI, LM02, TFDP1, HOPX, FLI1 and RUNX2. In some embodiments, the one or more transcription factors comprises one or more NOTCH family member. In some embodiments, the one or more transcription factors consists of NOTCH family member. In some embodiments, the one or more transcription factors comprises N0TCH1. In some embodiments, the one or more transcription factors comprises N0TCH1 fragments thereof. In some embodiments, the one or more transcription factors comprises N0TCH1 ICD. In some embodiments, the one or more transcription factors consists of N0TCH1 ICD. In some embodiments, the one or more transcription factors comprises N0TCH1. In some embodiments, the one or more transcription factors consists of N0TCH1. In some embodiments, the one or more transcription factors consists of N0TCH1. In some embodiments, the one or more transcription factors consists of N0TCH1 fragments thereof. In some embodiments, the NOTCH family member comprises NOTCH1,NOTCH1 ICD, or both. In some embodiments, the one or more transcription factors comprises N0TCH1, N0TCH1 ICD, NOTCH family member, or any combination thereof. In some embodiments, the one or more transcription factors are one or more transcription factors selected from the group consisting of N0TCH1, N0TCH1 ICD, NOTCH family member, and any combination thereof. In some embodiments, the one or moretranscription factors comprises NOTCH family ICD. In some embodiments, the one or more transcription factors comprises N0TCH1 or N0TCH1 fragments thereof.

[0014] Disclosed herein provided is a method of generating a population of immune cells or immune-cells like, the method comprising providing one or more pluripotent stem cells (PSCs); expressing in the one or more PSCs: a recombinant polynucleotide comprising one or more open reading frames encoding one or more transcription factors; one or more transcription factors, or an activator of transcription of said one or more open reading frames encoding said one or more transcription factors; and generating said population of immune cells or immune-cell like from the one or more PSCs, wherein said recombinant polynucleotide comprising one or more open reading frames encoding one or more transcription factors, said one or more transcription factors, or said activator of transcription of said one or more open reading frames encoding said one or more transcription factors comprise one or more transcription factors listed in Table 1; and wherein said PSC differentiates into an immune cell or immune-like cell and expresses one or more immune cell or immune-cell like markers in 28 days or less, wherein said one or more immune cell or immune-cell like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, and PECAM1 . In some embodiments, the recombinant polynucleotide comprising the one or more open reading frame encoding the one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding the one or more transcription factors induces differentiation of the one or more PSCs into the population of immune cells or immune-cell like in 28 days or less, 11 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. In some embodiments, the population of immune cells or immune-cell like are adherent cells. In some embodiments, the population of immune cells or immune-cell like are suspension cells. In some embodiments, at least 2%, 3%, 4%, 5%, 10%, or 15% TRA- 1-60-negative cells of the population of immune cells or immune-cell like express CD34. In some embodiments, atleast2%, 3%, 4%, 5%, 10%, 11%, 12%, 13%, 14%, or 15% or about 20% of TRA-1 -60-negative cells of the population of immune cells or immune-cell like express CD43. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or about 15% of TRA-1 -60-negative cells of the population of immune cells or immune-cell like express CD45. In some embodiments, at least 1% 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6% or about 8% ofTRA-1-60- negative cells of the population of immune cells or immune -cell like express both CD34 and CD43. In some embodiments, at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6% or about 8% of TRA-1 -60-negative cells of the population of immune cells or immune-cell like express both CD34 and CD45. In some embodiments, atleast O.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6% or about 8% of TRA-1 -60-negative cells of the population of immune cells or immune-cell like express both CD43 and CD45. In some embodiments, atleastO.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6% or about 8% of TRA-1 -60-negative cells of the population of immune cells or immune -cell like express both CD34 and CD43. In some embodiments, at least 0.25%, 0.5%, 0.75%, 1%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.75%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or about 6% of TRA-1 -60-negative cells of the population of immune cells or immune -cell like express CD34, CD43 and CD45. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, HOPX, LM02, FLU, NOTCH family member, TALI, ERG, GATA2, TFDP1, HLF, RFX2, HOXA10, RUNX1, FOXPlor TFEC. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, HOPX, LM02, FLU, NOTCH family member, TALI, ERG, GATA2, TFDP1, HOXA10, HLF, RFX2, or RUNX1. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express NOTCH family member, FLI1, HOPX, TALI, SPH, LM02, GATA2, ERG, HOXA10, orRFX2. In some embodiments, atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express FLU, NOTCH family member, TALI, SPH, HOPX, LM02, GATA2, HOXA5, ERG, TFDP1 or RUNX1. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, NOTCH family member, HOPX, LM02, TALI, FLU, TFDP1, HOXA10, GATA2, HLF, or RUNXl . In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express NOTCH family member, SPI1, RUNX1, or HOPX. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express NOTCH family member, SPI1, HOPX or TFDP1. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, LM02, TALI, NOTCH family member, GATA2, HOPX, RUNX1, TFDP1, HLF, RFX2, FLU, NFE2, MYB, or ERG. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, LM02, TALI, RUNX1, TFDP1, FLU, HLF, HOPX, NOTCH family member, ERG, GATA2, RUNX2, MYB, or NFE2. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, TALI, LM02, NOTCH family member, HOPX, FLU, TFDP1, GATA2, HLF, HOXA10, HOXA5, TFEC. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPH, NOTCH familymember, LM02, RUNX1, MYB, TALI, or HOPX. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, LM02, TALI, NOTCH family member, RUNX1, HOPX or HOXA5. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPH, TALI, LM02, or TFDP1. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, TALI, LM02, HOPX, TFDP1, or FLIl. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1 orLMO2. In some embodiments, atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express SPI1, LM02, TALI, or TFDP1. In some embodiments, at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said population of immune cells express LM02, TALI, or RUNX2. In some embodiments, the immune cell or immune-cell like expresses both CD34 and CD43. In some embodiments, the immune cell or immune-like cell expresses both CD34 and CD45. In some embodiments, the immune cell or immune-cell like expresses both CD43 and CD45. In some embodiments, the immune cell or immune-cell like expresses CD34, CD43, and CD45. In some embodiments, the immune cell or immune-like cell is a hematopoietic progenitor cell. In some embodiments, the immune cell or immune-cell like is a lymphoid progenitor cell. In some embodiments, the immune cell or immune-cell like is a B cell. In some embodiments, the immune cell or immune-cell like is a T cell. In some embodiments, the immune cell or immunecell like is a natural killer cell. In some embodiments, the immune cell or immune-cell like is a myeloid progenitor cell. In some embodiments, the immune cell or immune-cell like is a megakaryocyte-erythrocyte progenitor cell. In some embodiments, the immune cell or immunecell like is an erythroid progenitor cell. In some embodiments, the immune cell or immune -cell like is an erythrocyte. In some embodiments, the immune cell or immune-cell like is an endothelial cell of lymphatic vessel. In some embodiments, the immune cell or immune-cell like is a cardiac endothelial cell. In some embodiments, the immune cell or immune -cell like is a platelet. In some embodiments, the immune cell or immune-cell like is an erythrocyte. In some embodiments, the immune cell or immune-cell like is a mast cell. In some embodiments, the immune cell is or immune-cell like a neutrophil. In some embodiments, the immune cell or immune-cell like is a basophil. In some embodiments, the immune cell or immune-cell like is an eosinophil. In some embodiments, the immune cell or immune-cell like is a monocyte. In some embodiments, the immune cell or immune-cell like is a macrophage. In some embodiments, the immune cell or immune-cell like is a dendritic cell. In some embodiments, the immune cell or immune-cell like is a regulatory T cell. In some embodiments, the immune cell or immune-celllike is an alpha-beta T cell. In some embodiments, the immune cell or immune-cell like is a gamma-delta T cell. In some embodiments, the one or more transcription factors comprises one or more of the transcription factors listed in Table 1. In some embodiments, the one or more transcription factors comprises one or more transcription factors from at least one family member of any one of the transcription factors listed in Table 1. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, HOPX, LM02, FLU, NOTCH family member, and TALI . In some embodiments, the one or more transcription factors or transcription factor family members consists of: SPI1, HOPX, LM02, FLI1, NOTCH family member, and TALI . In some embodiments, the one or more transcription factors or transcription factor family members comprises ERG, GATA2, TFDP1, HLF, RFX2, HOXAIO, RUNX1, FOXP1 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members consists of ERG, GATA2, TFDP1, HLF, RFX2, HOXAIO, RUNX1, FOXP1 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members comprises ERG, GATA2, HOXAIO and RFX2. In some embodiments, the one or more transcription factors or transcription factor family members consists of ERG, GATA2, HOXAIO and RFX2. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1 and NOTCH family member. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1 and NOTCH family member. In some embodiments, the one or more transcription factors or transcription factor family members comprises HOPX, LM02, TALI, FLU, TFDP1, HOXAIO, GATA2, HLF and RUNX1. In some embodiments, the one or more transcription factors or transcription factor family members consists of HOPX, LM02, TALI, FLU, TFDP1, HOXAIO, GATA2, HLF and RUNX1. In some embodiments, the one or more transcription factors or transcription factor family members comprises NOTCH family member, SPI1, RUNX2, HOPX and TFDP1. In some embodiments, the one or more transcription factors consists of NOTCH family member, SPI1, RUNX2, HOPX and TFDP1. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPH, LM02, TALI, NOTCH family member, GATA2, HOPX, RUNX1, TFDP1, HLF, RFX2, FLU, NFE2, MYB and ERG. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, LM02, TALI, NOTCH family member, GATA2, HOPX, RUNX1, TFDP1, HLF, RFX2, FLU, NFE2, MYB and ERG. In some embodiments, the one or more transcription factors comprises SPI1, LM02, TALI, RUNX1, TFDP1, FLU, HLF, HOPX, NOTCH family member, ERG, GATA2, RUNX2, MYB and NFE2. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, LM02, TALI, RUNX1, TFDP1, FLU, HLF, HOPX, NOTCH familymember, ERG, GATA2, RUNX2, MYB and NFE2. In some embodiments, the one or more transcription factors or transcription factor family members comprises TALI, LM02, NOTCH family member, HOPX, FLU, TFDP1, GATA2, HLF, HOXAIO, H0XA5 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members consists of TALI, LM02, NOTCH family member, HOPX, FLU, TFDP1, GATA2, HLF, HOXAIO, H0XA5 and TFEC. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPH, NOTCH family member, LM02, RUNX1, MYB, TALI and HOPX. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1 , NOTCH family member, LM02, RUNX1, MYB, TALI and HOPX. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, LM02, TALI, NOTCH family member, RUNX1, HOPX and H0XA5. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, LM02, TALI, NOTCH family member, RUNX1, HOPX and H0XA5. In some embodiments, the one or more transcription factors or transcription factor family members comprises SPI1, TALI, LM02, TFDP1, HOPX, FLI1 and RUNX2. In some embodiments, the one or more transcription factors or transcription factor family members consists of SPI1, TALI, LM02, TFDP1, HOPX, FLI1 and RUNX2. In some embodiments, the one or more transcription factors comprises one or more NOTCH family member. In some embodiments, the one or more transcription factors consists of NOTCH family member. In some embodiments, the one or more transcription factors comprises N0TCH1 ICD. In some embodiments, the one or more transcription factors consists of N0TCH1 ICD. In some embodiments, the one or more transcription factors comprises N0TCH1. In some embodiments, the one or more transcription factors consists of N0TCH1. In some embodiments, the NOTCH family member comprises NOTCH1,NOTCH1 ICD, or both. In some embodiments, the one or more transcription factors comprises N0TCH1, N0TCH1 ICD, NOTCH family member, or any combination thereof. In some embodiments, the one or more transcription factors are one or more transcription factors selected from the group consisting of N0TCH1, N0TCH1 ICD, NOTCH family member, and any combination thereof. In some embodiments, the one or more transcription factors comprises NOTCH family ICD. In some embodiments, the one or more transcription factors consists of NOTCH family ICD. In some embodiments, the one or more transcription factors, comprises one or more transcription family member of one or more transcription factors listed in Table 1. In some embodiments, the one or more transcription factors, consists of one or more transcription family members of one or more transcription factors listed in Table 1. In some embodiments, the one or more transcription factors, comprisesone or more transcription factors listed in Table 1, one or more transcription factor family members of one or more transcription factors listed in Table 1, or both.INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0017] FIG. 1A-1B illustrates that transcription factors (TFs) differentiate pluripotent stem cells (PSCs) into CD34-expressing cells. Differentiation of PSCs into CD34-expressing cells was observed following TFs induction (induced cells, FIG. 1A). Among TRA- 1-60-negative cells, a higher percentage of the induced cells displayed robust expression of CD34 (FIG. IB) relative to unstained or uninduced cells.

[0018] FIG. 2A-2B illustrates that transcription factors (TFs) differentiate pluripotent stem cells (PSCs) into CD43 -expressing cells. Differentiation of PSCs into CD43 -expressing cells was observed following TFs induction (induced cells, FIG. 2A). Among TRA- 1-60-negative cells, a higher percentage of the induced cells displayed robust expression of CD43 (FIG. 2B) relative to unstained or uninduced cells.

[0019] FIG. 3A-3B illustrates that transcription factors (TFs) differentiate pluripotent stem cells into CD45 -expressing cells. Differentiation of PSCs into CD45 -expressing cells was observed following TFs induction (induced cells, FIG. 3A). Among TRA- 1-60-negative cells, a higher percentage of the induced cells displayed robust expression of CD45 (FIG. 3B) relative to unstained or uninduced cells.

[0020] FIG. 4A-4C illustrates that transcription factors (TFs) differentiate pluripotent stem cells into double-positive cells for key marker of hematopoietic stem / progenitor cells andhematopoietic lineage cells. Among TRA- 1-60-negative cells, a higher percentage of the TFs induced cells displayed robust expression of both CD34 and CD43 (FIG. 4A), both CD34 and CD45 (FIG. 4B), or both CD43 and CD45 (FIG. 4C) relative to unstained or uninduced cells.

[0021] FIG. 5 illustrates that transcription factors (TFs) differentiate pluripotent stem cells into triple-positive cells for key marker of hematopoietic stem / progenitor cells and hematopoietic lineage cells. The percentage of TRA-1 -60-negative cells that expressed CD34, CD43 and CD45 was higher in TFs induced cells relative to unstained or uninduced cells.

[0022] FIG. 6 illustrates an exemplary assessment of PSC differentiation using RNA sequencing of cells enriched by MACS. Uniform manifold approximation and projection (UMAP projection) displayed distinct populations of cells enriched for CD34, for CD45, for TRA-1 -60 negative, in relation to reference CD34+ cord blood cells and unenriched populations.

[0023] FIG. 7 A-7G illustrates an exemplary uniform manifold approximation and projections (UMAP projections) displaying distinct populations of cells that expressed hematopoietic / immune cell lineage markers CD34 (FIG. 7A), CD43 (SPN) (FIG. 7B), CD45 (FIG. 7C), THY1 (FIG. 7D), ITAG6 (FIG. 7E), CD33 (FIG. 7F) and PECAM1 (FIG. 7G).

[0024] FIG. 8A-8D illustrates an exemplary UMAP projections displaying cells exiting pluripotency. Exit of cells from pluripotency was assessed based on loss of stem cell markers NANOG (FIG. 8A), OCT4 (FIG. 8B), SOX2 (FIG. 8C), or TRA-1 -60 (PODXL) (FIG. 8D).

[0025] FIG. 9A-9B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in PSCs. UMAP projects confirmed that cells expressing CD34 (FIG. 9A) were enriched for specific TFs (FIG. 9B).

[0026] FIG. 10A-10C illustrates an exemplary experiment showing that cells purified based on CD34 expression by MACS were enriched for specific transcription factors (TFs). Expression of some specific TFs remained consist regardless of the sample tested, as seen in sample 1 (FIG. 10 A), sample 2 (FIG. 10B), and sample 3 (FIG. 10C).

[0027] FIG. 11A-11B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in pluripotent stem cells (PSCs). UMAP projections confirmed that cells expressing CD45 (FIG. 11 A) were enriched for specific TFs (FIG. 11B).

[0028] FIG. 12A-12B illustrates an exemplary experiment showing that cells purified based on CD45 expression by MACS were enriched for specific TFs. Expression of some specific TFs remained consistent regardless of the sample tested, as seen in in sample 1 (FIG. 12A) and sample 2 (FIG. 12B).

[0029] FIG. 13A-13B illustrates an exemplary uniform manifold approximation and projections (UMAP projections) and transcription factors (TFs) expressed in PSCs. UMAP projections confirmed that cells expressing CD43 (FIG. 13A) were enriched for specific TFs (FIG. 13B).

[0030] FIG. 14A-14B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in PSCs. UMAP projections confirmed that cells expressing both CD34 and CD43 (FIG. 14A) were enriched for specific TFs (FIG. 14B).

[0031] FIG. 15A-15B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in PSCs. UMAP projections confirmed that cells expressing both CD34 and CD45 (FIG. 15A) were enriched for specific TFs (FIG. 15B).

[0032] FIG. 16A-16B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in PSCs. UMAP projections confirmed that cells expressing both CD43 and CD45 (FIG. 16A) were enriched for specific TFs (FIG. 16B).

[0033] FIG. 17A-17B illustrates an exemplary UMAP projections and transcription factors (TFs) expressed in PSCs. UMAP projections confirmed that cells expressing CD34, CD43 and CD45 (FIG. 17A) were enriched for specific TFs (FIG. 17B).

[0034] FIG. 18A-18B illustrates an exemplary machine learning classifier. The classifier as seen in UMAP projections (FIG. 18A) confirmed identities of hematopoietic stem cells (FIG.18B)

[0035] FIG. 19A-19J illustrates an exemplary machine learning classifier that confirmed identities of hematopoietic stem cells. Cells classified as hematopoietic stem cells were enriched for specific TFs based on various classification probability thresholds including: Top classification (FIG. 19A), probability > 0.25 (FIG. 19B), probability > 0.50 (FIG. 19C), probability > 0.75 (FIG. 19D), or probability > 0.90 (FIG. 19E). Additionally, the specific transcription factors (TFs) that were enriched among cells classified as hematopoietic stem cells were investigated and these TFs are shown for Top classification (FIG. 19F), probability > 0.25 (FIG. 19G), probability > 0.50 (FIG. 19H), probability > 0.75 (FIG. 191), or probability > 0.90 (FIG. 19J). P-values, in parenthesis, were seen between observed versus expected; tg is transgene or exogenous TFs separated by as illustrated in the figures.

[0036] FIG. 20A-20D illustrates an exemplary machine learning classifier used to confirm identity of additional hematopoietic / immune cell lineage and progenitor states expressing specific transcriptional factors (TFs). The classifier confirmed additional hematopoietic / immune cell lineage and progenitor states including: common myeloid progenitor cell (FIG. 20A), endothelial cell of lymphatic vessel (FIG. 20B), erythroid progenitor cell (FIG. 20C) and cardiacendothelial cell (FIG. 20D) in which specific TFs are induced and / or expressed. The identity of the specific TFs will be examined and / or confirmed.

[0037] FIG. 21A-FIG. 21F illustrates flow cytometry data generated from engineered cell line HZN2-7. Pluripotent stem cells engineered to express transcription factor (TF) combinations were created and the HZN2-7 cell line was assayed to evaluate differentiation into hematopoietic stem cell (HSC). Cells were induced and analyzed using flow cytometry. Cells were gated based on CD34+ (of live cells; FIG. 21A), CD45+ (of live cells; FIG. 21B), CD43+ (of live cells; FIG. 21C), CD34+CD43+ (of live cells; FIG. 21D), CD34+CD45+ (of live cells; FIG. 21E), and CD34+CD43+ (of CD45+ cells; FIG. 21F). Expression of markers was quantified as a percentage for induced and uninduced cells, error bars denote the standard deviation.

[0038] FIG.22A-FIG.22L illustrates representative flow plots from engineered cell line HZN2- 7. Pluripotent stem cells engineered to express transcription factor (TF) combinations were created and the HZN2-7 cell line was assayed to evaluate differentiation into hematopoietic stem cells (HSCs or HSC). Cells were induced and analyzed using flow cytometry. The flow plots show CD34 marker expression in (i) uninduced (FIG. 22 A) vs. induced (FIG. 22B) HZN2 or HZN2-7 cell line; (ii) uninduced (FIG. 22C) vs. induced (FIG.22D) HZN3 cell line; (iii) uninduced (FIG. 22E) vs. induced (FIG. 22F) HZN4 cell line; (iv) uninduced (FIG. 22G) vs. induced (FIG. 22H) HZN5 cell line; (v) uninduced (FIG. 221) vs. induced (FIG. 22J) HZN6 cell line; (vi) uninduced (FIG. 22K) vs. induced (FIG. 22L) HZN7 cell line. All cells were gated on live.

[0039] FIG. 23A-FIG. 23D illustrates flow cytometry data generated from engineered cell lines HZN2B, HZN6B, HZN8-11 . Pluripotent stem cells engineered to express transcription factor(TF) combinations were created and the HZN2B, HZN6B, HZN8-11 cell lines were assay edto evaluate differentiation of cells into hematopoietic stem cells (HSCs or HSC). Cells were induced and analyzed using flow cytometry. Cells were gated based on CD34+ (of live cells; FIG. 23A), CD45+ (of live cells; FIG. 23B), CD43+ (oflive cells; FIG. 23C), CD34+CD43+CD45+ (oflive cells; FIG. 23D) and percentages of cells expressing the markers is indicated for uninduced and induced cells. Shown are three technical replicates represented as dots.

[0040] FIG. 24A-FIG. 24R shows representative flow plots from cell lines HZN2B, HZN6B, HZN8-11. Pluripotent stem cells engineered to express transcription factor (TF) combinations were created and the HZN2B, HZN6B, HZN8-11 cell lines were assayed to evaluate differentiation into hematopoietic stem cells (HSCs or HSC). Cells were induced and analyzed using flow cytometry. Shown is the expression of HSC markers among the HZN2B cells (CD34, FIG. 24A; CD43, FIG. 24B; CD45, FIG. 24C); HZN6B cells (CD34, FIG. 24D; CD43, FIG. 24E; CD45, FIG. 24F); HZN8 cells (CD34, FIG. 24G; CD43, FIG. 24H; CD45, FIG. 241);HZN9 cells (CD34, FIG. 24J; CD43, FIG. 24K; CD45, FIG. 24L); HZN10 cells (CD34, FIG. 24M; CD43, FIG. 24N; CD45, FIG. 240); HZN11 cells (CD34, FIG. 24P; CD43, FIG. 24Q; CD45, FIG. 24R). Only induced samples are shown.

[0041] FIG. 25A-25B illustrates an exemplary study showing colony formation of cell lines expressing combinations of transcription factors. Cell lines created using specific combinations of transcription factors (TFs) were cultured and cultivated in a colony forming unit (CFU) assays. CFU assay is one of the assays to determine hematopoietic stem cell (HSC) function in vitro. Cell lines HZN2 and HZN6 were tested. Colony formation was absent when 10,000 uninduced cells were plated (without a TF induction step) (FIG. 25A). Induced cells from HZN2 were cultured for 10 days in STEMCELL Technologies Methocult™ and plated to generate CFUs. Induced cell lines were able to form all colony types; multipotent (CFU-GEMM), erythroid (CFU- / BFU-E) and myeloid lineage (CFU-GM) (FIG. 25B, HZN2 after CD34+ purification shown). Note: BFU- E includes erythroid lineage cells, CFU-GM includes granulocyte-macrophage lineage cells, CFU-GEMM includes myeloid lineage cells e.g., granulocytes, erythrocyte, monocyte, megakaryocyte lineages.

[0042] FIG. 26A-26B illustrates an exemplary study showing colony formation of cell lines expressing combinations of transcription factors. Cell lines were created using specific combinations oftran scription factors (TFs) and 10,000 (10k) to 100,000 (100k) induced cells from lines HZN2 and HZN6 were cultured for 10 days in STEMCELL Technologies Methocult™, imaged and counted. Quantitative results of colony forming unit (CFU) assays shows the relative ratio of CFU-GEMM, CFU-GM and BFU-E colonies in FIG. 26A, while the total colony yield per 1000 (Ik) input cells forHZN2 is represented in FIG. 26B. Note: BFU-E includes erythroid lineage cells, CFU-GM includes granulocyte-macrophage lineage cells, CFU-GEMM includes myeloid lineage cells e.g., granulocytes, erythrocyte, monocyte, megakaryocyte lineages.

[0043] FIG. 27A-27D illustrates an exemplary study showing differentiation potential of cell lines HZN2 expressing combinations of transcription factors. Shown is the performance of engineered cell line HZN2 following culture in STEMCELL Technologies HemaTox™ mediafor lineage-specific differentiation. After 7 days of culture in HemaTox™ Erythroid medium, the percentage of erythroid cells positive for CD71marker and / or CD235a marker was determined (FIG. 27A). The HemaTox™ Myeloid kit was used to assess overall percentage number of cells positive for CD 13 and the percent producing or forming CD14 and CD15 myeloid lineage cells (FIG. 27B) Shown are representative flow cytometry plots and gating percentages after 7 days of culture in HemaTox Erythroid and HemaTox Myeloid media / kit. GlyA (FIG. 27C) and CD71 (FIG. 27D) are markers of erythroblasts while CD 13 and CD 15 markers of myeloblasts.

[0044] FIG. 28A-28E illustrates flow cytometry data generated from engineered cell lines HZN2B and HZN8. Top performing cell lines engineered to express transcription factor (TF) combinations was created. Cells were induced and analyzed using flow cytometry. Cells were gated based on CD34+(of live cells; FIG. 28A), CD45+(of live cells; FIG.28B), CD43+ (of live cells; FIG. 28C), CD34+CD43+CD45+ (of live cells; FIG. 28D), and TRA- 1-60+ (of live cells; FIG. 28E) and gate percentages were represented as shown.

[0045] FIG. 29A-29E illustrate flow cytometry data of top performing cells generated from cell lines HZN2B and HZN8. Top performing cell lines engineered to express transcription factor (TF) combinations was created. Cells were induced and analyzed using flow cytometry. Cells were gated based on CD34+(of live cells; FIG. 29A), CD45+(of live cells; FIG.29B), CD43+ (of live cells; FIG. 29C), CD34+CD43+CD45+ (of live cells; FIG. 29D), and TRA- 1-60+ (of live cells; FIG. 29E) and gate percentages are shown.

[0046] FIG. 30A-30F illustrate flow cytometry data of exemplary top performers 6 A6, 4C8, 4G4, and 4G3. Top performing clones 6A6, 4C8, 4G4, and 4G3 engineered to express transcription factor (TF) combinations were created, induced, and analyzed using flow cytometry. Representative flow plots show expression of (i) CD34 in the 6 A6 and 4C8 cell lines in uninduced vs. induced cells (FIG. 30A), expression of CD34 in the 4G4 and 4G3 cell lines in uninduced vs. induced cells (FIG.30B); show expression of (ii) CD43 in the 6 A6 and 4C8 cell lines in uninduced vs. induced cells (FIG. 30C), expression of CD43 in the 4G4 and 4G3 cell lines in uninduced vs. induced cells (FIG. 30D); and (iii) show expression of CD45 in the 6A6 and 4C8 cell lines in uninduced vs. induced cells (FIG. 30E), expression of CD45 in the 4G4 and 4G3 cell lines in uninduced vs. induced cells (FIG. 30F). All plots were gated on live cells.

[0047] FIG. 31A-31C illustrates an exemplary study showing colony forming ability of the top performing cell lines, HNZ2B and HZN8. Cell lines comprising combinations of transcription factors were induced for 4 days, cultured for 10 days in Methocult™ and colonies were counted then normalized to colony forming yield per 1 million cells by multiplying the colony forming ability of CD34+ enriched cells with the amount of CD34+ cells yielded per million total cells, and CD34-enriched batches were used as input for colony forming unit (CFU) assay . Top performing clones from HZN8 (4C8, 4G3, 4G4) generated about or approximately 14,000 colonies per 1 million induced cells (FIG. 31A). Top performing clones from HZN8 generated about or approximately 700 colonies per 10,000 input cells (7%, FIG.31B) and showed about or approximately 13,000 colonies per 1 million induced cells (FIG. 31C).DETAILED DESCRIPTION

[0048] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are providedby way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. Additionally, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

[0049] Disclosed herein are various compositions, methods, pharmaceutical, formulations, kits, and methods that facilitate efficient differentiation of a cell out of pluripotency and into a different cell comprising of the blood cell lineage. In some embodiments, provided herein is a cell comprising a pluripotent stem cell. In some embodiments, a pluripotent stem cell can comprise an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, a pluripotent stem cell can comprise a genetically modified cell or a genetically engineered cell. In some embodiments, a pluripotent stem cell can comprise an engineered cell disclosed herein. In some embodiments, an engineered cell can express one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an open reading frame encoding one or more transcription factors. In some embodiments, the open reading frame described herein encodes an activator of transcription of the open reading frame encoding one or more transcription factors. In some embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXAIO, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2 and at least one of CEBPA, GATA2, SPI1 , or any combination thereof.

[0050] Disclosed herein in some embodiment is provided a method of treating a disease associated with a blood-associated condition, disease, or disorder described herein. In some embodiments, a blood-associated condition, disease, or disorder can comprise a dysfunctional bone marrow described herein. In some embodiments, provided is a method for treating a subject with a dysfunctional bone marrow condition. In some embodiments, a subject can comprise a mammal e.g., an animal described herein. In some embodiments, an animal comprises a primate, or a non-primate animal described herein. In some embodiments, a primate animal comprises a human e.g., a human subject. In some embodiments a method of treating comprises administering an engineered cell engineered to express one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid moleculescomprise an open reading frame described herein encoding one or more transcription factors. In some embodiments, the open reading frame encodes activator of transcription of the open reading frame encoding the one or more transcription factors described herein, embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXAIO, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2 and at least one of CEB PA, GATA2, SPI1, or any combination thereof. In some embodiments, a method for treating a subject comprises administering an engineered cell described herein to a subject in need thereof. In some embodiments, the administering of an engineered cell described herein can comprise injecting an engineered cell described herein into a subject in need thereof. In some embodiment, injecting the engineered cell comprises intra-venous infusion into a subject. In some embodiments, the engineered cell injected can comprise frozen cells or fresh cells. In some embodiment, injecting the engineered cell described herein can conducted following chemotherapy. In some embodiments, the chemotherapy can comprise total body irradiation or may not comprise total body irradiation. In some embodiments, administering injections of an engineered cell described herein can lead to treating a disease associated with dysfunctional bone marrow condition.

[0051] Disclosed herein in some embodiments is provided a method of generating an engineered cell. In some embodiments, the method comprises contacting a cell e.g., a pluripotent stem cell (PSC) described herein with an expression cassette comprising one or more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules described herein comprises an open reading frame described herein such as an open reading frame encoding one or more transcription factors described herein. In some embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXAIO, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2 and at least one of CEBPA, GATA2, SPI1, or any combination thereof.

[0052] Disclosed herein in some embodiments is provided a cell comprising a pluripotent stem cell comprising an expression cassette described herein can comprise one or more transcription factors. In some embodiments, the expression of the one or more transcription factors can generate an induced pluripotent stem cells described herein, or an engineered cell described herein. In some embodiments, an iPSC or engineered cell can comprise a cell with a different phenotype. In some embodiments, the different phenotype can comprise a cell that has differentiated into a different cell lineage e.g., a blood-cell lineage or hemocytoblast described herein. In some embodiments, a blood-cell lineage can comprise a cell of the hematopoietic progenitor, hematopoietic stem cell, or a hematopoietic -like stem cell lineage described herein.In some embodiments, an engineered cell can comprise a cell lineage comprising a hematopoietic stem cell (HSP) or a hematopoietic-like stem cell described herein. In some embodiments, a hematopoietic stem cell can comprise a cell of the HSC lineage described herein. In some embodiments, a hematopoietic stem cell can comprise a myeloid progenitor cell or a lymphoid progenitor cell. In some embodiments, a common myeloid progenitor cell or myeloid progenitor can comprise an erythrocyte (e.g., a reticulocyte, an erythrocyte). In some embodiments a myeloid progenitor can comprise a mast cell. In some embodiments, a myeloid progenitor can comprise a megakaryocyte. In some embodiments, a megakaryocyte can comprise a thrombocyte (e.g., a platelet). In some embodiments, a myeloid progenitor can comprise a myeloblast. In some embodiments, a myeloblast can comprise a basophil. In some embodiments, a myeloblast can comprise a neutrophil. In some embodiments, a myeloblast can comprise an eosinophil. In some embodiments, a myeloblast can comprise a monocyte. In some embodiments, a monocyte can comprise a macrophage. In some embodiments, a monocyte can comprise a dendritic cell. In some embodiments, a hematopoietic cell can comprise a lymphoid cell lineage. In some embodiments, a lymphoid cell lineage can comprise a lymphoid progenitor or a common lymphoid progenitor, a large granular lymphocyte, (e.g., a natural killer cell,), a small lymphocyte (e.g., a B lymphocyte cell, a plasma cell, or a T lymphocyte cell), or any combination thereof.

[0053] In some embodiments, a hematopoietic stem cell (HSC), progenitor, or hematopoietic - like stem cell that is exiting out of pluripotency can exhibit an ability to differentiate into colony forming units (CFUs) or colonies of multipotent lineage. In some embodiments, a cell with capability of differentiating into CFU or colonies of multipotent lineage can be capable of producing final cell types on CFUs. In some embodiments, final cell types on CFU can comprise erythroid lineage cells (BFU-E), granulocyte-macrophage lineage cells (CFU-GM) and myeloid lineage cells (i.e. granulocyte, erythrocyte, monocyte, megakaryocyte lineages (CFU-GEMM).

[0054] In some embodiments, a genetically modified cell comprises programming factors that can allow differentiation of the cell. The programming factors can comprise one or more exogenous factors. For example, the programming factors can comprise one or more transcription factors, one or more transcription factor family members, or combinations thereof. In some embodiments, the one or more exogenous programming factors can comprise at least one activator of one or more transcription factors or one or more transcription factor family members. In some embodiments, the programming factors comprise exogenous, native, wildtype, heterologous or not heterologous. In some embodiments, the programming factors can comprise any desired factors to improve differentiation, transcription, or any other desired programming factors as known by a skilled artisan. The programming factors can be desired fordifferentiation of a stem cell. In some embodiments, the programming factors are expressed in an expression cassette. In some embodiments, programming factors, recombinant polynucleotides, as disclosed, can be presented into a cell via genetic modification or genetic engineering to comprise a genetically modified cell. In some cases, the genetically modified cell can comprise exogenous programming factors which can induce differentiation of the cell. In some embodiments, the programming factors are provided as soluble factors to a stem cell . The programming factors can comprise one or more transcription factors, transcription factor family members, or both.

[0055] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0056] An engineered cell: Disclosed herein in some embodiments is provided a cell comprising a pluripotent stem cell comprising an expression cassette comprising one or more transcription factors. In some embodiments, expression of the one or more transcription factors can generate an induced pluripotent stem cells. In some embodiments, an induced pluripotent stem cell can comprise a modified cell or an engineered cell described herein. In some embodiments, an engineered cell can be inducedto express the one or more transcription factors described herein. In some embodiments, expressing the one or more transcription factors can induce differentiation of the pluripotent stem cell into a cell with a different phenotype. In some embodiments, the different phenotype can comprise a cell with a different lineage described herein e.g., a blood-cell lineage.

[0057] In some embodiments, a blood-cell lineage described herein can comprise a cell of the hematopoietic progenitor, hematopoietic stem cell, or a hematopoietic -like stem cell lineage. In some embodiments, a hematopoietic stem cell can comprise any one of the cell lineages described herein. In some embodiments, a hematopoietic stem cell or progenitor can comprise hemacytoblast. In some embodiments, a hemocytoblast can comprise a megakaryoblast. In some embodiments, a megakaryoblast can comprise a megakaryocyte e.g., a thrombocyte or platelet. In some embodiments, a hemocytoblast a monoblast. In some embodiments, a monoblast can comprise a monocyte. In some embodiments, a monocyte can comprise a macrophage or a dendritic cell. In some embodiments, a hemocytoblast can comprise a myeloblast. In some embodiments, a myeloblast can comprise a progranulocyte comprising a basophil, an eosinophil, and a neutrophil. In some embodiments a hemacytoblast can comprise a proerythroblast comprising polychromatic erythroblast e.g., an erythrocyte. In some embodiments, a hemocytoblast can comprise a lymphoblast comprising lymphocytes. In some embodiments, alymphocyte can comprise a T cell, a B cell, or a natural killer cell. In some embodiments, disclosed herein is an HSC cell comprising any one of HSC described herein.

[0058] Provided in some embodiments herein is a pluripotent stem cell described herein. In some embodiments, a pluripotent stem cell (PSC) can comprise an embryonic stem cell. In some embodiments, aPSC can comprise an induced pluripotent stem cell (iPSC). In some embodiments, an iPSC can comprise an engineered cell or an HSC described herein. In some embodiments, an engineered cell provided herein can comprise an expression cassette comprising one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, the one or more transcription factors comprise LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further comprise TALI . In some embodiments, the one or more transcription factors described herein further comprise GATA2. In some embodiments, the one or more transcription factors described herein further comprise HOXAIO. In some embodiments, an engineered cell provided herein can comprise an expression cassette comprising one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an open reading frame encoding one or more transcription factors, one ormore transcriptionfactors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, the one or more transcription factors comprise LM02, SPI1, TALI, GATA2, HOXAIO, or any combination thereof. In some embodiments, an engineered cell provided herein can comprise an expression cassette comprising one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an openreadingframe encoding one ormore transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, the one or more transcription factors comprise NFE2 and at least one of CEB PA, GATA2, SPH, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2. In some embodiments, the one or more transcription factors described herein comprise CEBPA. In some embodiments, the one ormore transcription factors described herein comprise GATA2. In some embodiments, the one ormore transcription factors described herein comprise SPI1. In some embodiments, the one or more transcription factors described herein further comprise LM02, TALI, or a combination thereof. In some embodiments, the one or more transcription factors consists of LM02 and SPH . In someembodiments, the one or more transcription factors described herein further consists of TALI. In some embodiments, the one or more transcription factors described herein further consists of GATA2. In some embodiments, the one or more transcription factors described herein consists of HOXAIO. In some embodiments, an engineered cell provided herein can comprise an expression cassette comprising one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an openreadingframe encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, the one or more transcription factors consists of LM02, SPI1, TALI, GATA2 and HOXAIO, or any combination thereof. In some embodiments, an engineered cell provided herein can comprise an expression cassette comprising one or more nucleic acid molecules described herein. In some embodiments, the one or more nucleic acid molecules can comprise an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, the one or more transcription factors consists of NFE2 and at least one of CEBPA, GATA2 and SPI1 , or any combination thereof. In some embodiments, the one or more transcription factors described herein consists of NFE2. In some embodiments, the one or more transcription factors described herein consists of CEBPA. In some embodiments, the one or more transcription factors described herein consists of GATA2. In some embodiments, the one or more transcription factors described herein consists of SPI1 . In some embodiments, the one or more transcription factors described herein further consists of LM02 and TALI, or a combination thereof.

[0059] Provided in some embodiments herein is an engineered cell described herein. In some embodiments, an engineered cell can comprise a human stem cell. In some embodiments, an engineered cell can comprise a human induced pluripotent stem cell. In some embodiments, an engineered cell can comprise a human progenitor cell. In some embodiments, an engineered cell can comprise a human fetal stem cell. In some embodiments, an engineered cell can comprise an embryonic stem cell. In some embodiments, an engineered cell can comprise a hematopoietic stem cell or progenitor derived from a human stem cell.

[0060] Provided in some embodiments herein is an engineered cell described herein comprising one or more transcription factors. In some embodiments, the one or more transcription factors can undergo induction or be induced to differentiate into a new cell lineage described herein. In some embodiments, the new cell lineage can comprise a different cell phenotype e.g, a blood cell lineage. In some embodiments, the blood cell lineage can comprise one or morehematopoietic stem cell or progenitors or hematopoietic -like stem cell. In some embodiments, an engineered cell described herein can differentiate into an HSC (or a hematopoietic-like stem cell or hematopoietic progenitor, both included). In some embodiments, an engineered cell described herein can comprise a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic- like stem cell. In some embodiments, a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic-like stem cell can comprise an immune cell or immune -cell-like. In some embodiments, an engineered cell described herein can comprise an erythroid lineage cell, myeloid lineage cell, or granulocyte-macrophage lineage cell. In some embodiments, an engineered cell described herein can comprise is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

[0061] In some embodiments, an engineered cell comprising an HSC can differentiate and express one or more HSC markers. In some embodiments, the one or more HSC markers described herein can be expressed in 4 days or less, 3 days or less, 2 days or less, or 1 day or less following induction of a cell in culture e.g., induction of an engineered cell described herein. In some embodiments, expression of one or more markers defining a blood cell lineage, e.g. , CD34, CD43, CD45, CD71 or CD235a, CD13, CD14 and CD15 markers can be indicative an engineered cell has differentiated to a blood cell lineage e.g., an HSC cell lineage e.g., an immune cell or a white blood cell described herein, In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD34, CD43, CD45, THY1, ITGA6, CD33, PEC AMI, THY1, ITGA6, CD33, or PECAM1 described herein. In some embodiments, expression of the CD34 surface marker is indicative an engineered cell differentiated to a blood cell e.g., an HSC cell lineage such as an immune cell or HSC cell lineage described herein In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD43. In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD45. In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD34 and CD43. In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD34 and CD45. In some embodiments, the one or more HSC markers of an engineered cell describe herein can comprise CD43 and CD45 (of CD45 positive cells). In some embodiments, one or more HSC markers of an engineered cell describe herein can be triple positive for CD34, CD43, and CD45 markers. In some embodiments, one or more HSC markers of an engineered cell describe herein can comprise THY 1 , ITGA6, CD33, or PECAM1.

[0062] Provided herein in some embodiments herein is an engineered cell described herein that can be derived from a patient. In some embodiments, an engineered cell described herein may not be derived from a patient.

[0063] Methods of generating or producing a cell: Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein. In some embodiments a method for generating an engineered cell can comprise contacting a pluripotent stem cell (PSC) disclosed herein. In some embodiments, aPSC can comprise an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, aPSC can comprise an expression cassette described herein comprising one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules described herein can encode one or more transcription factors or an activator of transcription of an open reading frame encoding the one or more transcription factors. In some embodiments, one or more transcription factors can comprise LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further comprise TALI . In some embodiments, the one or more transcription factors described herein further comprise GATA2. In some embodiments, the one or more transcription factors described herein further comprise HOXA10. In some embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXA10, or any combination thereof.

[0064] Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein. In some embodiments a method for generating an engineered cell can comprise contacting a pluripotent stem cell (PSC) disclosed herein. In some embodiments, a PSC can comprise an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, a PSC can comprise an expression cassette described herein comprising one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules described herein can encode one or more transcription factors or an activator of transcription of an open reading frame encoding the one or more transcription factors described herein. In some embodiments, one or more transcription factors can comprise NFE2 and at least one of CEBPA, GATA2, SPI1, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2. In some embodiments, the one or more transcription factors described herein comprise CEBPA. In some embodiments, the one or more transcription factors described herein comprise GATA2. In some embodiments, the one or more transcription factors described herein comprise SPI1. In some embodiments, the one or more transcription factors described herein further comprise LM02, TALI, or a combination thereof.

[0065] Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein. In some embodiments a method for generating an engineeredcell can comprise contacting a pluripotent stem cell (PSC) disclosed herein. In some embodiments, a PSC can comprise an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, a PSC can comprise an expression cassette described herein comprising one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules described herein can encode one or more transcription factors or an activator of transcription of an open reading frame encoding the one or more transcription factors. In some embodiments, one or more transcription factors can consist of LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further consist of TALI . In some embodiments, the one or more transcription factors described herein further consist of GATA2. In some embodiments, the one or more transcription factors described herein further consist of HOXAIO. In some embodiments, the one or more transcription factors described herein consist of LM02, SPI1, TALI, GATA2 and HOXAIO, or any combination thereof.

[0066] Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein. In some embodiments a method for generating an engineered cell can comprise contacting a pluripotent stem cell (PSC) disclosed herein. In some embodiments, a PSC can comprise an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, a PSC can comprise an expression cassette described herein comprising one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules described herein can encode one or more transcription factors or an activator of transcription of an open reading frame encoding the one or more transcription factors described herein. In some embodiments, one or more transcription factors consist of NFE2 and at least one of CEBPA, GATA2 and SPI1, or any combination thereof. In some embodiments, the one or more transcription factors described herein consist of NFE2. In some embodiments, the one or more transcription factors described herein consists of CEBPA. In some embodiments, the one or more transcription factors described herein consist of GATA2. In some embodiments, the one or more transcription factors described herein consist of SPI1. In some embodiments, the one or more transcription factors described herein further consist of LM02 and TALI, or a combination thereof.

[0067] Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein. In some embodiments, an engineered can comprise a stem cell described herein. In some embodiments, a stem cell can comprise a mammalian stem cell. In some embodiments, a mammalian stem cell can comprise an animal stem cell. In some embodiments, an animal stem cell can comprise a pluripotent stem cell described herein. In some embodiments, a pluripotent stem cell (PSC) can comprise an embryonic stem cell or aninduced pluripotent stem cell. In some embodiments, an induced pluripotent stem cell (iPSC) can comprise a human progenitor cell, a human fetal stem cell, or a cell derived from any adult cell. In some embodiments, an engineered cell described herein can comprise a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic -like stem cell. In some embodiments, a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic -like stem cell can comprise an immune cell or immune-cell-like. In some embodiments, an engineered cell described herein can comprise an erythroid lineage cell, myeloid lineage cell, or granulocytemacrophage lineage cell. In some embodiments, an engineered cell described herein can comprise is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

[0068] In some embodiments, an engineered is generated by inducing the expression of an expression cassette described herein. In some embodiments, an expression cassette can comprise one or more transcription factors described herein. In some embodiments inducing expression of the expression cassette induces expression of one or more transcription factors induces or triggers differentiation of a cell described herein, e.g., a PSC. Disclosed herein in some embodiments is provided a method of generating an engineered cell described herein can trigger or induce differentiation of a cell e.g., a PSC to become an engineered cell with a different cellular phenotype or cell lineage, e.g., an engineered PSC differentiating into a blood cell lineage described herein. In some embodiments, a blood cell lineage can comprise a cell of the hematopoietic progenitor, hematopoietic stem cell, or a hematopoietic-like stem cell lineage. In some embodiments, a hematopoietic stem cell can comprise any one of the cell lineages described herein. In some embodiments, a hematopoietic stem cell or progenitor can comprise hemacytoblast. In some embodiments, a hemocytoblast can comprise a megakary oblast. In some embodiments, a megakary oblast can comprise a megakaryocyte e.g., a thrombocyte or platelet. In some embodiments, a hemocytoblast a monoblast. In some embodiments, a monoblast can comprise a monocyte. In some embodiments, a monocyte can comprise a macrophage or a dendritic cell. In some embodiments, a hemocytoblast can comprise a myeloblast. In some embodiments, a myeloblast can comprise a progranulocyte comprising a basophil, an eosinophil, and a neutrophil. In some embodiments a hemacytoblast can comprise a proerythroblast comprising polychromatic erythroblast e.g., an erythrocyte. In some embodiments, a hemocytoblast can comprise a lymphoblast comprising lymphocytes. In some embodiments, a lymphocyte can comprise a T cell, a B cell, or a natural killer cell. In some embodiments, disclosed herein is an HSC cell comprising any one of HSC described herein. In a non-limiting example, an engineered cell described herein can be generated or created then analyzed to assess the differentiation capability of the engineered cell. In some embodiments, an engineered cellcan be analyzed to assess expression of one or more markers of differentiation to the desired cell lineage. In some embodiments, an engineered cell can be analyzed (standard methods of analyzing a differentiated cell are available to an artisan of skill) to assess if an engineered cell expresses one or more markers indicative of sternness or pluripotency of a cell. In some embodiments, a cell e.g., engineered cell described herein may not be induced to express the one or more transcription factors described herein. In some embodiments, an engineered cell that is not induced may expresses one or more markers. In some embodiments, an uninduced engineered cell can express one or more marker comprising one or more stem cell or pluripotency markers. In some embodiments, one or more markers sternness or pluripotency markers can comprise one or more cell lineage-specifying marker. In some embodiments, one or more cell lineage-specifying marker markers can comprise pluripotency or sternness markers e.g., a TRA-1-60 marker. In some embodiments, an engineered cell can be analyzed to assess if an engineered cell expresses one or more markers indicative of a cell exiting out of pluripotency e.g., a cell on a differentiation trajectory, a committed differentiation cell lineage, a progenitor of a cell lineage, or any indication that the cell is moving out of sternness. Provided in some embodiments herein is a method of generating, inducing, and analyzing differentiation of a PSC or engineered cell described herein. In some embodiments, an engineered cell described herein can express one or more markers of a hematopoietic stem cell markers, a hematopoietic progenitor, or a hematopoietic-like stem cell.

[0069] In some embodiments, an engineered cell can express one or immune cell or immune- cell-like markers described herein. In some embodiments, an engineered cell can express one or immune cell or immune-cell-like markers in 4 days or less, 3 days or less, 2 days or less, or 1 day or less. In some embodiments, an engineered cell can express one or more immune cell or immune-cell-like markers comprising CD34, CD43, CD45, THY1, ITGA6, CD33, orPECAMl . In some embodiments, an engineered cell expresses CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45. In some embodiments, an engineered cell can express comprising one or more markers can comprise a differentiating cell-markers e.g., markers indicating a cell is exiting pluripotency or is committed to differentiating to a different cell lineage. In some embodiments, an engineered cell does not express TRA-1-60. In some embodiments, a PSC can be derived from a patient. In some embodiments, a PSC may not be derived from a patient. In some embodiments, a PSC or engineered cell is an adherent cell. In some embodiments, an engineered cell is a suspension cell. In some embodiments, an engineered cell further comprising generating a population of engineered cells. The present disclosure provides a method of generating or producing a population of cells, e.g., PSC orengineered cells as described herein. In some embodiments, a population of cells described herein can be created, induced, and analyzed to assess expression of one or more cell lineagespecifying markers. In some embodiments, a population of cells is induced to differentiate by expressing one or more transcription factors described herein. In some embodiments, a population of cells can be induced to express one or more lineage specifying markers. In some embodiments, a cell line can be selected or isolated that expression one or more transcription factors, also described herein as transcription factor combinations or recipes. In some embodiments, a cell line expressing a specific cell line can be isolated and further analyzed to assess if the cell has differentiated to a different cell lineage e.g., by assess any number of parameters or metrices e.g., expression levels of one or more markers, ability for colony forming unit (CFU) e.g., a CFU assay which can determine hematopoietic stem cell function in vitro as described herein. In non-limiting examples, a cell described herein e.g., a PSC, an engineered cell, an iPSC, a population of cells, can be induced and allowed to differentiate to a new cellular phenotype such as a new cell lineage (developmental trajectory as well) and then investigated for colony forming unit (CFU) assay. In a non-limiting example, STEMCELL Technologies Methocult™ or STEMCELL Technologies HemaTox™ kits or assay can be applied to assess that a cell described herein has differentiated into an erythroid lineage, myeloid lineage, or both .

[0070] In some embodiments, one or more transcription factors (TFs) engineered in the cell as described herein can determine degree or rate of differentiation of a cell. In non-limiting examples, a cell described herein can comprise one or more TFs. In some embodiments, one or more TFs in an engineered cell can be assessed to determine integration into the cell as described herein. In some embodiments, a combination or recipe of TFs can be used to generate a cell described herein.

[0071] Disclosed herein in some embodiments, the recipe comprise one or more TFs described herein. In some embodiments, a cell line comprise a “top performing” or “top performer” cell line described herein. In some embodiments, a “top performer” can comprise one or more cell. In some embodiments, a cell line comprising top performers have better performance or capability from expressing a certain TF recipes or combination. In some embodiments, a cell line comprising top performers have better functionality. In some embodiments, better functionality comprises better colony formation, higher expression of bone marrow or hematopoietic stem cell associated genes or transcripts. In some embodiments, better functionality comprises better colony formation, higher expression of bone marrow or hematopoietic stem cell associated gene products, e.g., marker expression levels (qualities) or quantities. In some embodiments, a cell line comprises TFs inserted in one or more particularsites of integration of the construct within the cell. In some embodiments, “top performers” cell lines are used to determine differentiation of a cell described herein. Non-limiting examples of “top performers” or “top performing” cell lines are described herein. In non -limiting examples, “top performers” can comprise one or more TFs e.g., the exemplary TF recipes of cell lines described in Table 2 to Table 5. In some embodiments, exemplary cell lines and the respective exemplary TFs are shown in Table 2 to Table 5. Non-limiting examples cell lines are listed in Table 2 to Table 5.

[0072] Provided in some embodiments herein is a cell that can be induced and then differentiate to express one or more markers. At least one cell in a population of cells may express CD34. In some embodiments, a population of cells described herein can express two or more markers. In some embodiments, a population of cells described herein can express three or more markers. In some embodiments, a population of cells can comprise CD34-expressing cells described herein. In some embodiments, atleast25% of a population of engineered cells express CD34. In some embodiments, at least 30% of a population of engineered cells express CD34. In some embodiments, at least 95% of a population of engineered cells express CD34. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of a population of engineered cells express CD34. In some embodiments, at least 1% of a population of engineered cells express CD34 about 5% of a population to about 60% of a population of engineered cells express CD34. In some embodiments, at least 3 % of a population of engineered cells express CD34 about 5% of a population to about 10% of a population, about 5% of a population to about 15% of a population, about 5% of a population to about 20% of a population, about 5% of a population to about25% of a population, about 5% of a population to about 30% of a population, about 5% of a population to about 35% of a population, about 5% of a population to about 40% of a population, about 5% of a population to about 45% of a population, about 5% of a population to about 50% of a population, about 5% of a population to about 55% of a population, about 5% of a population to about 60% of a population, about 10% of a population to about 15% of a population, about 10% of a population to about 20% of a population, about 10% of a population to about 25% of a population, about 10% of a population to about 30% of a population, about 10% of a population to about 35% of a population, about 10% of a population to about 40% of a population, about 10% of a population to about 45% of a population, about 10% of a population to about 50% of a population, about 10% of a population to about 55% of a population, about 10% of a population to about 60% of a population, about 15% of a population to about 20% of a population, about 15% of a population to about 25% of a population, about 15% of a populationto about 30% of a population, about 15% of a population to about 35% of a population, about 15% of a population to about 40% of a population, about 15% of a population to about 45% of a population, about 15% of a population to about 50% of a population, about 15% of a population to about 55% of a population, about 15% of a population to about 60% of a population, about 20% of a population to about 25% of a population, about 20% of a population to about 30% of a population, about 20% of a population to about 35% of a population, about 20% of a population to about 40% of a population, about 20% of a population to about 45 % of a population, about 20% of a population to about 50% of a population, about 20% of a population to about 55% of a population, about 20% of a population to about 60% of a population, about 25% of a population to about 30% of a population, about 25% of a population to about 35% of a population, about 25% of a population to about 40% of a population, about 25% of a population to about 45% of a population, about 25% of a population to about 50% of a population, about 25% of a population to about 55% of a population, about 25% of a population to about 60% of a population, about 30% of a population to about 35% of a population, about 30% of a population to about 40% of a population, about 30% of a population to about 45% of a population, about 30% of a population to about 50% of a population, about 30% of a population to about 55% of a population, about 30% of a population to about 60% of a population, about 35% of a population to about 40% of a population, about 35% of a population to about 45% of a population, about 35% of a population to about 50% of a population, about 35% of a population to about 55% of a population, about 35% of a population to about 60% of a population, about 40% of a population to about 45% of a population, about 40% of a population to about 50% of a population, about 40% of a population to about 55% of a population, about 40% of a population to about 60% of a population, about 45% of a population to about 50% of a population, about 45% of a population to about 55% of a population, about 45% of a population to about 60% of a population, about 50% of a population to about 55% of a population, about 50% of a population to about 60% of a population, or about 55% of a population to about 60% of a population of engineered cells express CD34. In some embodiments, at least 1 % of a population of engineered cells express CD34 about 5% of a population, about 10% of a population, about 15% of a population, about 20% of a population, about 25% of a population, about 30% of a population, about 35% of a population, about 40% of a population, about45% of a population, about 50% of a population, about 55% of a population, or about 60% of a population of engineered cells express CD34. In some embodiments, at least 3 % of a population of engineered cells express CD34 at least about 5% of a population, about 10% of a population, about 15% of a population, about 20% of a population, about 25% of a population, about 30% of a population, about 35% of a population, about 40% of a population, about 45% of a population, about 50% of a population, or about 55% of a population ofengineered cells express CD34. In some embodiments, at least 6 % of a population of engineered cells express CD34 about 10% of a population, about 15% of a population, about 20% of a population, about 25% of a population, about 30% of a population, about 35% of a population, about 40% of a population, about 45% of a population, about 50% of a population, about 55% of a population, or about 60% of a population of engineered cells express CD34 marker. In some embodiments, at least 62 % of a population of engineered cells express CD34 about 65% of a population to about 99% of a population of engineered cells express CD34 marker. In some embodiments, at least 63 % of a population of engineered cells express CD34 about 65% of a population to about 70% of a population, about 65% of a population to about 75% of a population, about 65% of a population to about 80% of a population, about 65% of a population to about 85% of a population, about 65% of a population to about 90% of a population, about 65% of a population to about 95% of a population, about 65% of a population to about 96% of a population, about 65% of a population to about 97% of a population, about 65% of a population to about 98% of a population, about 65% of a population to about 99% of a population, about 70% of a population to about 75% of a population, about 70% of a population to about 80% of a population, about 70% of a population to about 85% of a population, about 70% of a population to about 90% of a population, about 70% of a population to about 95% of a population, about 70% of a population to about 96% of a population, about 70% of a population to about 97% of a population, about 70% of a population to about 98% of a population, about 70% of a population to about 99% of a population, about 75% of a population to about 80% of a population, about 75% of a population to about 85% of a population, about 75% of a population to about 90% of a population, about 75% of a population to about 95% of a population, about 75% of a population to about 96% of a population, about 75% of a population to about 97% of a population, about 75% of a population to about 98% of a population, about 75% of a population to about 99% of a population, about 80% of a population to about 85% of a population, about 80% of a population to about 90% of a population, about 80% of a population to about 95% of a population, about 80% of a population to about 96% of a population, about 80% of a population to about 97% of a population, about 80% of a population to about 98% of a population, about 80% of a population to about 99% of a population, about 85% of a population to about 90% of a population, about 85% of a population to about 95% of a population, about 85% of a p opulation to about 96% of a population, about 85% of a population to about 97% of a population, about 85% of a population to about 98% of a population, about 85% of a population to about 99% of a population, about 90% of a population to about 95% of a population, about 90% of a population to about 96% of a population, about 90% of a population to about 97% of a population, about 90% of a population to about 98% of a population, about 90% of a population to about 99% of apopulation, about 95% of a population to about 96% of a population, about 95% of a population to about 97% of a population, about 95% of a population to about 98% of a population, about 95% of a population to about 99% of a population, about 96% of a population to about 97% of a population, about 96% of a population to about 98% of a population, about 96% of a population to about 99% of a population, about 97% of a population to about 98% of a population, about 97% of a population to about 99% of a population, or about 98% of a population to about 99% of a population of engineered cells express CD34. In some embodiments, at least 61% of a population of engineered cells express CD34 about 65% of a population, about 70% of a population, about 75% of a population, about 80% of a population, about 85% of a population, about 90% of a population, about 95% of a population, about 96% of a population, about 97% of a population, about 98% of a population, or about 99% of a population of cells express CD34. In some embodiments, at least 64% of a population of engineered cells express CD34 at least about 65% of a population, about 70% of a population, about 75% of a population, about 80% of a population, about 85% of a population, about 90% of a population, about 95% of a population, about 96% of a population, about 97% of a population, or about 98% of a population of cells express CD34. In some embodiments, at least 68% of a population of engineered cells express CD34 about 70% of a population, about 75% of a population, about 80% of a population, about 85% of a population, about 90% of a population, about 95% of a population, about 96% of a population, about 97% of a population, about 98% of a population, or about 99% of a population of engineered cells express CD34.

[0073] In some embodiments, at least one cell in the population of cells can express CD43. In some embodiments, a population of cells comprising at least 6% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 17% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 50% of the population of engineered cells express CD43 . In some embodiments, a population of cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 0.5 % of a population of engineered cells can express CD43 ab out 1% of a population to about 20% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 0.5 % of a population of engineered cells can express CD43 about 1% of a population to about 2% of a population, about 1% of a population to about 3% of a population, about 1% of a population to about 4% of a population, about 1% of a population to about 5% of a population, about 1% of a population to about 6% ofa population, about 1% of a population to about 7% of a population, about 1% of a population to about 8% of a population, about 1% of a population to about 9% of a population, about 1% of a population to about 10% of a population, about 1% of a population to about 15% of a population, about 1% of a population to about 20% of a population, about 2% of a population to about 3% of a population, about 2% of a population to about 4% of a population, about 2% of a population to about 5% of a population, about 2% of a population to about 6% of a population, about 2% of a population to about 7% of a population, about 2% of a population to about 8% of a population, about 2% of a population to about 9% of a population, about 2% of a population to about 10% of a population, about 2% of a population to about 15% of a population, about 2% of a population to about 20% of a population, about 3% of a population to about 4% of a population, about 3% of a population to about 5% of a population, about 3% of a population to about 6% of a population, about 3% of a population to about 7% of a population, about 3% of a population to about 8% of a population, about 3% of a population to about 9% of a population, about 3% of a population to about 10% of a population, about 3% of a population to about 15% of a population, about 3% of a population to about 20% of a population, about 4% of a population to about 5% of a population, about 4% of a population to about 6% of a population, about 4% of a population to about 7% of a population, about 4% of a population to about 8% of a population, about 4% of a population to about 9% of a population, about 4% of a population to about 10% of a population, about 4% of a population to about 15% of a population, about 4% of a population to about 20% of a population, about 5% of a population to about 6% of a population, about 5% of a population to about 7% of a population, about 5% of a population to about 8% of a population, about 5% of a population to about 9% of a population, about 5% of a population to about 10% of a population, about 5% of a population to about 15% of a population, about 5% of a population to about 20% of a population, about 6% of a population to about 7% of a population, about 6% of a population to about 8% of a population, about 6% of a population to about 9% of a population, about 6% of a population to about 10% of a population, about 6% of a population to about 15% of a population, about 6% of a population to about 20% of a population, about 7% of a population to about 8% of a population, about 7% of a population to about 9% of a population, about 7% of a population to about 10% of a population, about 7% of a population to about 15% of a population, about 7% of a population to about 20% of a population, about 8% of a population to about 9% of a population, about 8% of a population to about 10% of a population, about 8% of a population to about 15% of a population, about 8% of a population to about 20% of a population, about 9% of a population to about 10% of a population, about 9% of a population to about 15% of a population, about 9% of a population to about 20% of a population, about 10% of a population to about 15% of a population, about 10%of a population to about 20% of a population, or about 15% of a population to about 20% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 0.5% of a population of engineered cells can express CD43 about 1% of a population , about 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, about 7% of a population, about 8% of a population, about 9% of a population, about 10% of a population, about 15% of a population, or about 20% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 0.5 % of a population of engineered cells can express CD43 at least about 1% of a population , about 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, ab out 7% of a population, about 8% of a population, about 9% of a population, about 10% of a population, or about 15% of a population. In some embodiments, a population of cells comprising at least 1% of a population of engineered cells can express CD43 about 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, about 7% of a population, about 8% of a population, about 9% of a population, about 10% of a population, about 15% of a population, or about 20% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 21% of a population of engineered cells can express CD43 about 25% of a population to about 75% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least% of a population of engineered cells can express CD43 about 25% of a population to about 30% of a population, about 25% of a population to about 35% of a population, about 25% of a population to about 40% of a population, about 25% of a population to about 45% of a population, about 25% of a population to about 50% of a population, about 25% of a population to about 55% of a population, about 25% of a population to about 60% of a population, about 25% of a population to about 65% of a population, about 25% of a population to about 70% of a population, about 25% of a population to about 75% of a population, about 30% of a population to about 35% of a population, about 30% of a population to about 40% of a population, about 30% of a population to about 45% of a population, about 30% of a population to about 50% of a population, about 30% of a population to about 55% of a population, about 30% of a population to about 60% of a population, about 30% of a population to about 65% of a population, about 30% of a population to about 70% of a population, about 30% of a population to about 75% of a population, about 35% of a population to about 40% of a population, about 35% of a population to about 45% of a population, about 35% of a population to about 50% of a population, about 35% of a population to about 55% of a population, about 35% of a population to about 60% of a population, about 35% of a population to about 65% of a population, about 35% of a populationto about 70% of a population, about 35% of a population to about 75% of a population, about 40% of a population to about 45% of a population, about 40% of a population to about 50% of a population, about 40% of a population to about 55% of a population, about 40% of a population to about 60% of a population, about 40% of a population to about 65% of a population, about 40% of a population to about 70% of a population, about 40% of a population to about 75% of a population, about 45% of a population to about 50% of a population, about 45% of a population to about 55% of a population, about 45% of a population to about 60% of a population, about 45% of a population to about 65% of a population, about 45% of a population to about 70% of a population, about 45% of a population to about 75% of a population, about 50% of a population to about 55% of a population, about 50% of a population to about 60% of a population, about 50% of a population to about 65% of a population, about 50% of a population to about 70% of a population, about 50% of a population to about 75% of a population, about 55% of a population to about 60% of a population, about 55% of a population to about 65% of a population, about 55% of a population to about 70% of a population, about 55% of a population to about 75% of a population, about 60% of a population to about 65% of a population, about 60% of a population to about 70% of a population, about 60% of a population to about 75% of a population, about 65% of a population to about 70% of a population, about 65% of a population to about 75% of a population, or about 70% of a population to about 75% of a population of engineered cell s express CD43. In some embodiments, a population of cells comprising at least% of a populati on of engineered cells can express CD43 about 25% of a population, about 30% of a population, about 35% of a population, about 40% of a population, about 45% of a population, about 50% of a population, about 55% of a population, about 60% of a population, about 65% of a population, about 70% of a population, or about 75% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least% of a population of engineered cells can express CD43 at least about 25% of a population, about 30% of a population, about 35% of a population, about 40% of a population, about 45% of a population, about 50% of a population, about 55% of a population, about 60% of a population, about 65% of a population, or about 70% of a population of engineered cells express CD43. In some embodiments, a population of cells comprising at least% of a population of engineered cells can express CD43 about 30% of a population, about 35% of a population, about 40% of a population, about 45% of a population, about 50% of a population, about 55% of a population, about 60% of a population, about 65% of a population, about 70% of a population, or about 75% of a population of engineered cells express CD43.

[0074] Provided herein in some embodiments is a population of cells created as described herein and then induced as described herein to generate a population of cells expressing one or more markers of cell specifying lineages. In some embodiments, at least one cell in the population of cells may express CD45 marker. In some embodiments, a population of cells comprising at least 7% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 12% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 25% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising. In some embodiments, a population of cells comprising at least 85% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 90% of a population of engineered cells express CD45. At least one cell in the population of cells may express CD45. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 0.5% of a population of engineered cells can express CD45 about 1% of a population to about 20% of a population of engineered cells can express CD45. In some embodiments, a population of cells comprising at least 0.5% of a population of engineered cells can express CD45 about 1% of a population to about 2% of a population, about 1% of a population to about 3% of a population, about 1% of a population to about 4% of a population, about 1% of a population to about 5% of a population, about 1% of a population to about 6% of a population, about 1% of a population to about 7% of a population, about 1% of a population to about 8% of a population, about 1% of a population to about 9% of a population, about 1% of a population to about 10% of a population, about 1% of a population to about 15% of a population, about 1% of a population to about 20% of a population, about 2% of a population to about 3% of a population, about 2% of a population to about 4% of a population, about 2% of a population to about 5% of a population, about 2% of a population to about 6% of a population, about 2% of a population to about 7% of a population, about 2% of a population to about 8% of a population, about 2% of a population to about 9% of a population, about 2% of a population to about 10% of a population, about 2% of a population to about 15% of a population, about 2% of a population to about 20% of a population, about 3% of a population to about 4% of a population, about 3% of a population to about 5% of a population, about 3% of a population to about 6% of a population, about 3% of a population to about 7% of a population, about 3% of a population to about 8% of a population, about 3% of a population to about 9% of a population, about 3% of a population to about 10% of a population, about 3% of a population to about 15% of a population, about 3% of a population to about 20% of a population, about 4%of a population to about 5% of a population, about 4% of a population to about 6% of a population, about 4% of a population to about 7% of a population, about 4% of a population to about 8% of a population, about 4% of a population to about 9% of a population, about 4% of a population to about 10% of a population, about 4% of a population to about 15% of a population, about 4% of a population to about 20% of a population, about 5% of a population to about 6% of a population, about 5% of a population to about 7% of a population, about 5% of a population to about 8% of a population, about 5% of a population to about 9% of a population, about 5% of a population to about 10% of a population, about 5% of a population to about 15% of a population, about 5% of a population to about 20% of a population, about 6% of a population to about 7% of a population, about 6% of a population to about 8% of a population, about 6% of a population to about 9% of a population, about 6% of a population to about 10% of a population, about 6% of a population to about 15% of a population, about 6% of a population to about 20% of a population, about 7% of a population to about 8% of a population, about 7% of a population to about 9% of a population, about 7% of a population to about 10% of a population, about 7% of a population to about 15% of a population, ab out 7% of a population to about 20% of a population, about 8% of a population to about 9% of a population, about 8% of a population to about 10% of a population, about 8% of a population to about 15% of a population, about 8% of a population to about 20% of a population, about 9% of a population to about 10% of a population, about 9% of a population to about 15% of a population, about 9% of a population to about 20% of a population, about 10% of a population to about 15% of a population, about 10% of a population to about 20% of a population, or about 15% of a population to about 20% of a population of an engineered cells can express CD45. In some embodiments, a population of cells comprising at least% of a population of engineered cells can express CD45 about 1% of a population , about 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, about 7% of a population, about 8% of a population, about 9% of a population, about 10% of a population, about 15% of a population, or about 20% of a population of engineered cells can express CD45. In some embodiments, a population of cells comprising at least 0.5% of a population of engineered cells can express CD45 at least about 1% of a population , ab out 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, about 7% of a population, about 8% of a population, about 9% of a population, about 10% of a population, or about 15% of a population of engineered cells can express CD45. In some embodiments, a population of cells comprising at least% of a population of engineered cells can express CD45 about 2% of a population, about 3% of a population, about 4% of a population, about 5% of a population, about 6% of a population, about 7% of a population,about 8% of a population, about 9% of a population, about 10% of a population, about 15% of a population, or about 20% of a population

[0075] Provided herein some embodiments, a population of engineered cells described herein can be induced to express one or more markers. In some embodiments, a population of cells can express double positives marker expression. Non-limiting examples of double positive expression of markers can include, expressive of both CD34 and CD43, CD34 and CD43, or CD34 and CD43, marker. In some embodiments, a population of cells comprising at least one cell in the population of cells can express CD34 and CD43. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells expressing CD34 and CD43. In some embodiments, a population of cells can comprise at least 3% of the population of engineered cells expressing CD34 and CD43. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45. In some embodiments, a population of cells can comprise at least 3% of the population of engineered cells expressing CD34 and CD45. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45. In some embodiments, a population of cells can express double positive marker expression while presenting expression of another marker e.g., cells expressing triple markers. Non-limiting examples of a cell or population of cells expressive triple positive markers can include, e.g., CD34 / CD43 / CD45 triple expression, where the cell or population of cells describe herein express CD34+, CD43+, CD45+. In some embodiments, a population of cells can comprise about 1% CD34 / CD43 / CD45 of the cells showing a triple expression of these markers. In some embodiments, a population of cells can comprise about 1% CD34 / CD43 / CD45 of the cells showing a triple expression of these markers. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45 -positive cells of the population of engineered cells express CD34 and CD43. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45.

[0076] In some embodiments, a cell e.g., engineered cell described herein may not be induced to express the one or more transcription factors described herein. In some embodiments, an engineered cell that is not induced may expresses one or more markers. In some embodiments,an uninduced engineered cell can express one or more marker comprising one or more stem cell or pluripotency markers. In some embodiments, one or more markers sternness or pluripotency markers can comprise one or more cell lineage-specifying marker. In some embodiments, one or more cell lineage-specifying marker markers can comprise pluripotency or stem-ness markers e.g., a TRA-1-60 marker. In some embodiments, an engineered cell can be analyzed to assess if an engineered cell expresses one or more markers indicative of a cell exiting out of pluripotency e.g., a cell on a differentiation trajectory, a committed differentiation cell lineage, a progenitor of a cell lineage, or any indication that the cell is moving out of sternness. In some embodiments, at most 10%, 9%, 80%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% of a population of engineered cells express TRA-1-60. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI . In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2. In some embodiments, a population of cells can comprise atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express CEBPA. In some embodiments, a population of engineered cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HOXA10.

[0077] Provided in some embodiments herein is a population of engineered cells induced to express one or more transcription factors. In some embodiments, expressing one or more transcription factors can induce a population of engineered cells to differentiate into a different cell lineage(s). In some embodiments, a population of engineered cells can comprise erythroid lineage cells, myeloid lineage cells, granulocyte-macrophage lineage cells, or any combination thereof. In some embodiments, a population of engineered cells can comprise myeloid lineage cells, granulocyte lineage cells, erythrocyte lineage cells, monocyte lineage cells, megakaryocyte lineage cells, or any combination thereof. In some embodiments, a population of engineered cells described herein can comprise at least 5% erythroid lineage cells. In some embodiments, a population of engineered cells described herein can comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of erythroid lineage cells. In someembodiments, a population of engineered cells can comprise at least 50% erythroid lineage cells. In some embodiments, a population of engineered cells can comprise at least 5% myeloid lineage cells. In some embodiments, a population of engineered cells can comprise at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells.In some embodiments, a population of engineered cells can comprise at least 30% myeloid lineage cells. In some embodiments, a population of cells can comprise at least 40% granulocyte-macrophage lineage cells. In some embodiments, a population of engineered cells can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte-macrophage lineage cells. In some embodiments, a population of engineered cells can comprise at least 90% granulocyte-macrophage lineage cells. In some embodiments, a population of engineered cells can comprise erythroid lineage cells expressing CD71, CD235a, or both markers. In some embodiments, a population of engineered cells comprising myeloid lineage cells can express CD13, CD 14, CD 15, or any combination thereof. In some embodiments, a population of engineered cells comprising myeloid lineage cells can express CD14 and CD15.

[0078] Pluripotent stem cells: A pluripotent stem cell (PSC) can be characterized by selfrenewal and potency. In some embodiments, a PSC is capable of dividing indefinitely and producing identical daughter or progeny cells. In some embodiments, providing a signal to an PSC can allow it to differentiate into a different cell lineage or phenotype. Non -limiting examples of a PSC can include an embryonic stem cell (ESC) an induced PSC (iPSC). In some embodiments, a PSCs (e.g., ESCs and iPSCs) can express TRA-1-60. In some embodiments, TRA-1 -60 expression is indicative of the ability of a cell to differentiate to another cell lineage e.g., a hematopoietic stem cell lineage or a hematopoietic -like stem cell differentiation trajectory.

[0079] In some embodiments, induced pluripotent stem cells (iPSCs) comprise a type of pluripotent stem cell derived from adult somatic. In some embodiments the adult somatic cell may have been genetically reprogrammed to an embryonic stem (ES) cell-like state e.g., through the expression of one or more genes or one or more transcription factors important for maintaining the defining properties of an embryonic stem cell (ESCs). In some embodiments, a PSC is differentiated using directed differentiation. In some embodiments, directed differentiation can comprise a bioengineering method that harnesses the potential of a stem cell by constraining the differentiation of the cell toward a specific cell type or tissue of interest. In some embodiments, directed differentiation is primarily applied to a PSC. In some embodiments, cell differentiation can involve a transformation from a proliferative mode toward a differentiation mode. In some embodiments, directed differentiation can comprise mimickingdevelopmental cultures in controlled conditions involving specific substrate or extracellular matrices promoting cell adhesion and differentiation and define culture media compositions. Non- limiting examples of inducing factors or signaling factors, includes growth factors or small molecules which is applied a) sequentially or in a combinatorial manner, at b) varying dosage and c) at different exposure time, to modulate differentiate. In some embodiments, direct reprogramming, also known as trans-differentiation or direct conversion, can comprise overexpressing one or several factors, which is introduced in a cells. In some embodiments, one or several factors can comprise one or more transcription factors. In some embodiments, proper differentiation of the cell type of interest is verified by analyzing cell type by e.g., expression of one or more specific markers. In some embodiments, one or more specific markers comprises general cellular markers. A non-limiting example of expression of one or more specific markers include a specific marker that may be expressed in specific cell types, specific cellular developmental stages, in specific tissues, or under specific signals or conditions. In some embodiments, identifying a differentiated cell can comprise assessing protein expression profile, gene expression profile, functional assays e.g., collecting a profile of one or more metabolic processes, or other biological process, mechanisms, or any combination thereof. General assays carried out to distinguish or identify a cell type are known to a skilled artisan and cannot be listed exhaustively in the disclosure.

[0080] In some embodiments, a PSC disclosed herein can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors. In some embodiments, one or more transcription factors disclosed herein can be exogenous transcription factors. One or more nucleic acid molecules can comprise two or more open reading frames encoding one or more transcription factors. A PSC can comprise one or more transcription factors and additionally one or more open reading frames, or nucleic acid molecules. In some embodiments, a PSC can comprise an activator of transcription of one or more open reading frames encoding one or more transcription factors. In some embodiments, an activator can be a CRISPR activator, a transcription activator, an activator for transcription of one or more transcription factors. In some embodiments, an activator can comprise any other activators known to a skilled artisan. In some embodiments, an activator can comprise a small molecule activator. In some embodiments, a small molecule activator can induce expression of one or more transcription factors. In some embodiments, one or more transcription factors can be exogenous genes or gene fragments thereof. In some embodiments, one or more transcription factors can be endogenous genes or gene fragments thereof. In some embodiments, one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of one ormore open reading frames encoding one or more transcription factors can induce differentiation of a PSC into a hematopoietic stem cell. In some embodiments, one or more open reading frames comprises exogenous nucleic acids, nucleic acid sequences, genes, or gene fragments thereof. In some embodiments, one or more open reading frames comprises endogenous nucleic acids, nucleic acid sequences, genes, or gene fragments thereof. The exogenous genes, gene fragments, sequences described herein can comprise synthetic constructs, recombinant fragments, chimeras, or any combinations thereof as is known by a skilled artisan.

[0081] Hematopoietic stem cells: The ability of the immune system to control the inflammatory response and prevent reactions against self -antigens or harmless environmental molecules can be acquired during fetal life or early after birth. Hematopoietic stem cells (HSCs) can comprise myeloid progenitor cells and lymphoid progenitor cells. In some embodiments, the lymphoid progenitor cells can yield immune cells e.g., B lymphocytes, natural killer (NK cells) cells, or T lymphocytes.

[0082] In some embodiments, a cell of the present disclosure can differentiate into a cell type of the HSCs. In some embodiments, the HSCs can have various characteristics and functions that assist in the development and maintenance of the immune system. In some embodiments, it can be necessary to isolate differentiated cells or to distinguish a differentiated cell from a non- differentiated cell. In some embodiments, one or more cell lineage markers can be evaluated to assess when a cell has differentiated from pluripotency towards e.g., a hematopoietic stem cell or a hematopoietic-like stem cell. Non-limiting examples of markers of a hematopoietic stemcell, progenitor or a hematopoietic-like stem cell can comprise include CD34, CD43 and CD45. In some embodiments, a hematopoietic stem cell or a hematopoietic-like stem cell can comprise a CD71, CD235a, CD13, CD 14, CD15, or any combination thereof.

[0083] In some embodiments, a PSC can be induced to differentiate to a hematopoietic stem cell (HSC) or a hematopoietic-like stem cell. In some embodiments, a PSC can express one or more markers of differentiation when it is induced and exits pluripotency. In some embodiment, the PSC exiting pluripotency can comprise a hematopoietic stem cell or a hematopoietic -like stem cell. In some embodiments, a hematopoietic stem cell or a hematopoietic -like stem cell can express one or more markers suggesting differentiation or marking a developmental trajectory. In some embodiments, the hematopoietic stem cell lineage can comprise a myeloid cell or a lymphoid cell. Non-limiting examples of cellular lineages of a hematopoietic stem cell progenitor cell is disclosed throughout this application, e.g., a myeloid progenitor or common myeloid progenitor, a mast cell, an erythrocyte (e.g., a reticulocyte, an erythrocyte), a megakary oblast (a thrombocyte, a platelet producing megakaryocyte, a platelet), a myeloblast, (e.g., a granulocyte (e.g., a promyelocyte, a neutrophil, an eosinophil, a basophil), a monocyte(e.g., a macrophage, or a dendritic cell), a lymphoid progenitor or a common lymphoid progenitor, a large granular lymphocyte, (e.g., a natural killer cell,), a small lymphocyte (e.g., a B lymphocyte cell, a plasma cell or a T lymphocyte cell), or any combination thereof.

[0084] In some embodiments, the hematopoietic stem cell lineage can express CD34. In some embodiments, CD34 is a cell surface marker that can aid in the attachment of hematopoietic cells in the bone marrow and can identify early hematopoietic lineages. In some embodiments, the hematopoietic stem cell lineage can express CD43. In some embodiments, CD43 can be a negatively charged type I transmembrane sialoglyco-protein expressed by most hem atop oietic- lineage cells. In some embodiments, CD43 is used to select for and as a marker of hematopoietic cells of the progenitors and differentiating hematopoietic stem cells or hematopoietic-like stem cell. In some embodiments, throughout this disclosure, a hematopoietic stem cell can comprise a hematopoietic-like stem cell. In some embodiments, the hematopoietic stem cell lineage can express CD45. In some embodiments, CD45 is a protein tyrosine phosphatase that can be expressed in leukocytes and canbe a marker of committed progenitors. In some embodiments, CD45 can be expressed throughout the progenitor stage or in a mature T cell. In some embodiments, a cell disclosed expresses CD4. In some embodiments, CD4 is a co-receptor for the T Cell Receptor (TCR) that can play a role in the recognition of antigen presenting cell bindingto the Major Histocompatibility Complex II (MHC class II) protein complex. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. In some embodiments, a cell disclosed herein can express CD8. In some embodiments, CD8 can serve as an adhesion molecule for class I MHC molecules. In some embodiments, CD8 can serve as a coreceptor with the TCR for T cell activation.

[0085] Disclosed herein in some embodiments is a cell of the present disclosure that expresses one or more markers. In some embodiments, one or more marker can comprise stem cell markers. In some embodiments, one or more markers can comprise a cell lineage -specifying marker. In some embodiments, one or more markers can comprise pluripotency or stem-ness markers e.g., TRA-1-60. In some embodiments, one or more markers can comprise a differentiating cell -markers e.g., markers indicating a cell is exiting pluripotency or is committed to differentiating to a different cell lineage.

[0086] In some embodiments, at least one hematopoietic stem cell can express one or more markers comprising CD34, CD43, CD45, or any combination thereof. In some embodiments, at least one hematopoietic stem cell can express one or more markers comprising CD34, CD43 or a combination thereof. In some embodiments, at least one hematopoietic stem cell can express one or more markers comprising CD34, CD45 or a combination thereof. In some embodiments, atleast one hematopoietic stem cell can express one or more markers comprising CD43, CD45, or a combination thereof. In some embodiments, at least one hematopoietic stem cell can express one or more cellular markers selected from CD34, CD43, or CD45. In some embodiments, at least one hematopoietic stem cell can express one or more markers selected from the group consisting of CD34, CD43 and CD45. In some embodiments, at least one hematopoietic stem cell can express one or more markers comprising CD34, CD43 and CD45.

[0087] Transcription factors

[0088] The present disclosure provides transcription factors expressed by an engineered cell e.g., a pluripotent stem cell (PSC). In some embodiments, a cell disclosed can differentiate to another cellular phenotype or cell lineage. In some embodiments, the expressed transcription factors can comprise one or more in number. In some embodiments, a cell expresses or is induced to express one or more transcription factors. In some embodiments, expression of transcription factors (TFs) is used to trigger differentiation programs, e.g., the expression, or induction of the one or more transcription factors disclosed can trigger for the cell to differentiate. In some embodiments, a cell expressing one or more transcription factor family member of any transcription factor (TF) disclosed can trigger differentiation of a cell of the present disclosure. Various transcription factors and the respective transcription factor family members of the disclosed transcription factors are here by claimed. In a non -limiting example, a cell e.g., a pluripotent stem cell (PSC) can express one or more transcription factors (TFs) or one or more family members of any transcription factors disclosed. In some embodiments, a PSC expressing or overexpressing one or more TFs can differentiate into another cell lineage. In some embodiments, a PSC expressing one or more TFs can differentiate into different cell or cellular phenotype. In some embodiments, the different cellular phenotype can comprise a hematopoietic stem cell or a hematopoietic -like stem cell.

[0089] In some aspects, combinations of transcription factors can be used to achieve differentiation to a particular cell lineage. In some embodiments, certain transcription factors e.g., certain one or more transcription factor combinations or recipes canbe able to induce stem cells to particular lineages. In some embodiments, the combinations can achieve a cell type or a cell sub-type that is achieved by one or more transcription factors, alone, or in combination. In some embodiments, the combination can achieve the same cell type as one of the transcription factors alone or achieve the cell type more efficiently. In some embodiments, a transcription factor recipe can comprise one or more transcription factor. In some embodiments, a transcription factor recipe can consist of a single transcription factor. In some embodiments, a transcription factor recipe can comprise one transcription factor. In some embodiments, a transcription factor recipe can comprise, 1, 2, 3, 4, 5, 6, 10, 15, 20, 50, 70, or more transcriptionfactors (all integers included). Exemplary transcription factor combination, transcription factor recipes, transcription factors, transcription factor gene(s), or transcription factor family members are disclosed herein e.g., in Tables 1, 2, 3, 4 or disclosed elsewhere throughout. Exemplary transcription factor combination, transcription factor recipes, transcription factors, transcription factor gene(s), or transcription factor family members are disclosed in Nature biotechnology 39.4 (2021): 510-519; PCT Application Number PCT / US2017 / 051122; PCT / US2023 / 069550; and PCT Application Number PCT / US2018 / 030216, all of which are incorporated herein by reference in their entirety.

[0090] The PSC disclosed can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors or one or more transcription factor family members. In some embodiments, one or more nucleic acid or nucleic acid molecules (nucleic acid or nucleic acid molecules are used interchangeably) can comprise two or more open reading frames encoding one or more transcription factors. In some embodiments, a PSC can comprise one or more transcription factors. A PSC can comprise an activator of transcription of one or more open reading frames encoding one or more transcription factors. In some embodiments, the one or more transcription factors can be exogenous transcription factors. In some embodiments, the PSC comprises a hematopoietic stem or progenitor cell. In some embodiments, a hematopoietic stem or progenitor cell can comprise a hematopoietic-like stem cell. Non-limiting examples of cellular lineages of a hematopoietic stem cell progenitor cell is disclosed throughout this application, e.g., a myeloid progenitor or common myeloid progenitor, a mast cell, an erythrocyte (e.g., a reticulocyte, an erythrocyte), a megakary oblast (a thrombocyte, a platelet producing megakaryocyte, a platelet), a myeloblast, (e.g., a granulocyte (e.g., a promyelocyte, a neutrophil, an eosinophil, a basophil), a monocyte (e.g., a macrophage, or a dendritic cell), a lymphoid progenitor or a common lymphoid progenitor, a large granular lymphocyte, (e.g., a natural killer cell,), a small lymphocyte (e.g., a B lymphocyte cell, a plasma cell or a T lymphocyte cell), or any combination thereof.

[0091] In some embodiments, a PSC expressing one or more nucleic acid molecule comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors induced and differentiates into a hematopoietic stem cell.

[0092] In some embodiments, throughout the present disclosure, a hematopoietic stem cell or progenitor cell can comprise a hematopoietic-like stem cell.

[0093] In some embodiments, a PSC disclosed can comprise one or more nucleic acid molecules comprising two or more open reading frames encoding one or more transcription factors. Insome embodiments, the nucleic acid can comprise three or more open reading frames encoding one or more transcription factors. In some embodiments, the nucleic acid can comprise four or more open reading frames encoding one or more transcription factors. In some embodiments, the nucleic acid can comprise five or more open reading frames encoding one or more transcription factors.

[0094] In some embodiments, a cell of the present disclosure can be provided in a media. In some embodiments, the media may not be altered during the differentiation of the PSC e.g., into differentiation into a hematopoietic stem cell. In some embodiments, one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors induces the differentiation of the hematopoietic stem cell from the PSC in 28 days or less. In some embodiments, one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors induces the differentiation of the hematopoietic stem cell from the PSC in 11 days or less, 5 days or less, 4 days or less, 1 day or less. In some embodiments, the one or more transcription factors can comprise any one of the transcription factor combinations in Tables 1 -4 disclosed, or those disclosed in any referenced in this application.

[0095] In some embodiments, one or more transcription factors or transcription factor family members used can require a critical amount of expression to effectively induce differentiation, such as the equivalent of at least 5, 10, 15, 20, 25, or 50 copies of the one or more open reading frames (ORF) per cell. In some embodiments, other factors can require less than a certain threshold of expression due to possible toxicity at high levels, such as less than 20, 10, or 5 copies per cell. In some embodiments, increased levels of expression can also be achieved by increasingthe copy number of the ORF, e.g., by using a higher copy number vector or by using a transposon.

[0096] In some embodiments, PSCs comprising one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors disclosed can have a higher level of expression of the one or more transcription factors compared to PSCs without the one or more nucleic acid molecules comprising the one or more open reading frames encoding the one or more transcription factors, the one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors as disclosed herein. In some embodiments, PSCs comprising one or more nucleic acid molecule comprising the oneor more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors as disclosed herein can have higher level of mRNA expression of one or more transcription factors compared to PSCs without the one or more nucleic acid molecules comprising the one or more open reading frames encoding the one or more transcription factors, the one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors as disclosed herein. PSCs comprising one or more nucleic acid comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding the one or more transcription factors as disclosed herein can have at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold or greater expression of the one or more transcription factors compared to PSCs without the one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors as disclosed herein. In some embodiments, the number of copies of the ORF for one or more TF, one or more groups of TFs or one or more TF family member or members (note any plural and any singular are used interchangeably in the present application) introduced per cell can be about 1 to about 90. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be at least about 1. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be at most about 90. In a non-limiting example, the number of copies of an ORF of interest e.g., for each transcription factor (TF) disclosed (e.g., any one of LM02, TALI, GATA2, HOXAIO, SPI1, NFE2, or CEBPA), that may be introduced per cell can be about 1 to about2, about 1 to about4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0097] In some embodiments, a PSC disclosed can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, the one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors. In non-limiting examples, the one or more transcription factors comprise LM02 and SPI1. In some embodiments, one or more transcription factors can comprise LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further comprise TALI . In some embodiments, the one or more transcription factors described herein further comprise GATA2. In some embodiments, the one or more transcription factors described herein further comprise HOXA10. In some embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXA10, or any combination thereof. In some embodiments, one or more transcription factors can comprise NFE2 and at least one of CEBPA, GATA2, SPI1, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2. In some embodiments, the one or more transcription factors described herein comprise CEBPA. In some embodiments, the one or more transcription factors described herein comprise GATA2. In some embodiments, the one or more transcription factors described herein comprise SPI1. In some embodiments, the one or more transcription factors described herein further comprise LM02, TALI, or a combination thereof. In some embodiments, one or more transcription factors can consist of LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further consist of TALL In some embodiments, the one or more transcription factors described herein further consist of GATA2. In some embodiments, the one or more transcription factors described herein further consist of HOXA10. In some embodiments, the one or more transcription factors described herein consist of LM02, SPI1, TALI, GATA2 and HOXA10, or any combination thereof. In some embodiments, one or more transcription factors consist of NFE2 and at least one of CEBPA, GATA2 and SPI1 , or any combination thereof. In some embodiments, the one or more transcription factors described herein consist of NFE2. In some embodiments, the one or more transcription factors described herein consists of CEBPA. In some embodiments, the one or more transcription factors described herein consist of GATA2. In some embodiments, the one or more transcription factorsdescribed herein consist of SPI1. In some embodiments, the one or more transcription factors described herein further consist of LM02 and TALI, or a combination thereof

[0098] In some embodiments, a PSC disclosed can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, the one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors can induce the differentiation of a PSC. In some embodiments, the PSC comprises one or more transcription factors or transcription factor family members thereof disclosed in the present herein.

[0099] In some embodiments, a PSC disclosed herein can be induced to differentiate into a different cell lineage. In some embodiments, an induced PSC (iPSC) can differentiate into another cellular phenotype or cell lineage in about 30 days, in about 29 days, in about 28 days, in about 27 days, in about 26 days, in about 25 days, in about 24 days, in about 23 days, in about 22 days, in about 21 days, in about 20 days, in about 19 days, in about 18 days, in about 17 days, in about 16 days, in about 15 days, in about 14 days, in about 13 days, in about 12 days, in about 11 days, in about 10 days, in about 9 days, in about 8 days, in about 7 days, in about 6 days, in about 5 days, in about 4 days, in about 3 days, in about 2 days, in about 1 day, or less.

[0100] Hematopoietic stem cell production: In some embodiments, a population of cells comprising one or more induced PSCs can be generated. In some embodiments, a population of cells comprising one or more engineered cells can be generated. In some embodiments, a population of cells comprising one or more hematopoietic stem cells (HSCs), or hematopoietic - like stem cells can be generated. In some embodiments, a population of cells can comprise adherent cells. In some embodiments, a population of cells can comprise suspension cells. In some embodiments, a population of cells can comprise adherent cells and suspension cells. In some embodiments, a population of cells can be provided in a media. In some embodiments, the media may not have to be altered during the differentiation of a cell into a different cellular phenotype or cell lineage (e.g., during when a PSC differentiates into an HSC). In some embodiments, the media may not need any nutrients, growth factors, or microenvironmental or matrix optimizations in order to allow differentiation of a progenitor cell into another cell lineage (e.g., during when a PSC differentiates into an HSC). In some embodiments, a PSC disclosed can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors, the one or more transcription factors, or an activator of transcription of the one or more open reading frames encoding one or more transcription factors and can be induced to differentiate into a hematopoietic stem cell (HSC), hematopoietic-like stem cell, or HSC lineage in 28 days or less, 11 days or less, 4 days or less, 3 days or less, or 1 day or less (including all days between 28 days or less up to the 1 day or less).In some embodiments, the PSC may be provided in a media. In some embodiments, the media may not be altered during the differentiation of the hematopoietic stem cell, hematopoietic -like stem cell, or HSC lineage. In some embodiments, an induced PSC (iPSC) can differentiate into a hematopoietic stem cell (HSC), a hematopoietic progenitor, or a hematopoietic -like stem cell. In some embodiments, the hematopoietic stem cell can comprise one or more nucleic acid molecules comprising one or more open reading frames encoding one or more transcription factors. In some embodiments, the hematopoietic stem cell can comprise an activator of transcription of the open reading frames encoding one or more transcription factors. In some embodiments, an activator of transcription can be a CRISPR activator that induces expression of one or more transcription factor. In some embodiments, an activator of transcription can comprise a small molecule activator that induces expression of the one or more transcription factors. In some embodiments, the hematopoietic stem cell can comprise any cell of the HSC lineage. Non-limiting cells comprising HSC lineage include e.g., a myeloid progenitor or common myeloid progenitor, a mast cell, an erythrocyte (e.g., a reticulocyte, an erythrocyte), a megakary oblast (a thrombocyte, a platelet producing megakaryocyte, a platelet), a myeloblast, (e.g., a granulocyte (e.g., a promyelocyte, a neutrophil, an eosinophil, a basophil), a monocyte (e.g., a macrophage, or a dendritic cell), a lymphoid progenitor or a common lymphoid progenitor, a large granular lymphocyte, (e.g., a natural killer cell,), a small lymphocyte (e.g., a B lymphocyte cell, a plasma cell or a T lymphocyte cell), or any combination thereof. In some embodiments, the hematopoietic stem cell is a hematopoietic stem / progenitor cell. In some embodiments, a hematopoietic stem cell (HSC) can comprise a committed hematopoietic progenitor. In some embodiments, a committed hematopoietic progenitor can comprise an HSC in a state of exiting out of pluripotency. In some embodiments, a cell comprising an HSC exiting out of can be identified or distinguished by the different expression markers on the cell surface. In some embodiments, an HSC exiting out of pluripotency can express protein markers or gene expression profiles indicative of the progress out of pluripotency, e.g., display HSC-associated markers or transcript expression e.g., as disclosed elsewhere herein (exemplary expression of such markers can be seen e g , in FIG. 21A-FIG. 21F; FIG. 23A-FIG. 23D; FIG. 24A-FIG. 24R; FIG. 27A-27B; FIG. 28A-FIG. 28E; FIG. 29A-FIG. 29E; FIG. 30A-FIG. 30D; FIG. 31A- FIG. 31C) In some embodiments, anHSC cell exiting out of pluripotency can be detected by an indication of one or more differences in one or more cellular functions. In some embodiments, an HSC cell exiting out of pluripotency can exhibit an ability to differentiate into colony forming units (CFUs) or colonies of multipotent lineage. In some embodiments, an HSC cell exiting out of pluripotency or undergoing cell lineage differentiation out of pluripotency can be capable of producing final cell types on CFUs e.g., CFUs that produce erythroid lineage cells(BFU-E), granulocyte-macrophage lineage cells (CFU-GM) and myeloid lineage cells (i.e. granulocyte, erythrocyte, monocyte, megakaryocyte lineages (CFU-GEMM) e.g., as disclosed in the example section e.g., in FIG. 25A-FIG. 25B; FIG. 26A-FIG. 26B.

[0101] In some embodiments a hematopoietic stem cell of the present disclosure can comprise an immune cell. In some embodiments, an immune cell can comprise a T cell. In some embodiments, a hematopoietic stem cell is an earlier stage hematopoietic progenitor to a T cell. In some embodiments, the hematopoietic stem cell can comprise a committed progenitor cell. In some embodiments, the hematopoietic stem cell is a mature T cell. In some embodiments, a T cell can comprise a regulatory T cell. In some embodiments, a T cell can comprise a helper (CD4+) T cell. In some embodiments, a T cell can comprise a cytotoxic (CD8+) T cell. In some embodiments, a hematopoietic stem cell can comprise a natural killer (NK) cell.

[0102] In some embodiments, a hematopoietic stem cell (HSC) disclosed herein can express one or more transcription factors described herein. In some embodiments, inducing one or more transcription factors can differentiate a cell into an HSC, a hematopoietic progenitor, or a hematopoietic-like stem cell.

[0103] In some embodiments, the hematopoietic stem cell expresses CD34. In some embodiments, the hematopoietic stem cell expresses CD4. In some embodiments, the hematopoietic stem cell expresses CD8. In some embodiments, the nucleic acid comprising one or more open reading frames encoding one or more transcription factors, the one or more transcription factors, or the activator of transcription of the open reading frames encoding one or more transcription factors is expressed to induce the differentiation of a PSC into the hematopoietic stem cell. In some embodiments, the hematopoietic stem cell is provided in a media. The media cannot have to be altered during the differentiation of the PSC into the hematopoietic stem cell. In some embodiments, the nucleic acid comprising the open reading frames encodingthe one or more transcription factors, the one or more transcription factors, or the activator of transcription of the open reading frames encoding one or more transcription factors induces the differentiation of the hematopoietic stem cell from the PSC in 28 days or less. In some embodiments, the nucleic acid comprising the open reading frames encoding the one or more transcription factors, the one or more transcription factors, or the activator of transcription of the open reading frames encoding one or more transcription factors induces the differentiation of the hematopoietic stem cell from the PSC in 11 days or less. In some embodiments, the nucleic acid comprising the open reading frames encoding the one or more transcription factors, the one or more transcription factors, or the activator of transcription of the open reading frames encoding one or more transcription factors induces the differentiation of the hematopoietic stem cell from the PSC in 4 days or less, or 1 day or less.

[0104] In some embodiments, a PSC can further comprise at least one exogenous expression cassette comprising one or more transcription factors, or molecules that increase transcription or increase activation of transcription factors. In some embodiments, one or more transcription factors may be used. In some embodiments, a PSC can comprise at least one or more exogenous expression cassette comprising one or more transcription factors (TFs), one or more transcription factor family members, or one or more transcription factor recipes or cocktails disclosed. In some embodiments, a PSC comprising at least one or more exogenous expression cassette comprising one or more transcription factors (TFs), one or more transcription factor family members, or any combination thereof, comprises induced to express the one or more TFs, and / or transcription factor family members. In some embodiments, induction of the one or more TFs and / or TF family member can trigger a PSC to differentiate into a different cell lineage. In some embodiments, a PSC comprising at least one exogenous expression cassette can comprise one or more transcription factors described here can trigger differentiation of a PSC into a blood stem cell or a hematopoietic stem cell described herein.

[0105] comprising at least one exogenous expression cassette can comprise one or more transcription factors comprising CEBPA. In some embodiments, a PSC comprising at least one exogenous expression cassette can comprise one or more transcription factors consisting of CEBPA.

[0106] In some embodiments, one or more transcription factors (TFs) disclosed herein can comprise a codon optimization. In non -limiting examples, a transcription factor of the present disclosure comprises a codon optimized transcription factor. In some embodiments a transcription factor of disclosed can lack codon-optimization e.g., a transcription factor that may not be codon optimized. In some embodiments, codon optimization can occur at the DNA level for any transcription factor disclosed herein or a transcription factor belonging to a family of transcription factor disclosed herein. In some embodiments, a combination of one or more transcription factors, or transcription factor (TF) family members may be expressed in a PSC. In some embodiments, any combination of TF or TF families may be combined. In some embodiments, any combination of TF or TF families comprises introduced into a PSC and can be induced and expressed in a PSC or population of PSCs (singular or plural). In some embodiments, introduction of at least one exogenous expression cassette comprising one or more TFs into a PSC may induce differentiation of a PSC. In some embodiments, a PSC is induced to a pluripotent stem cell (iPSC). In some embodiments, an induced PSC (iPSC) differentiates into a cell phenotype comprising a different cell lineage e.g., a hematopoietic stem cell or progenitor, or a hematopoietic-like stem In some embodiments, one or more transcription factors that induces differentiation of one or more PSCs into a hematopoietic stem cells orprogenitors, or hematopoietic-like stems can comprise, one or more TFs or one or more members of a transcription factor (TF) family from among the TFs that are disclosed herein. The TF family members are known to a person of skill in the art (a skilled artisan), and TF family members are listed herein in various sections and references and applications disclosed herein. Where the TF family members are not listed in this application, but the one or more transcription factor is disclosed, is hereby claimed as though added in the application because such TF family members are known in the art or to a person of skill in the art (a skilled artisan).

[0107] Transcription factors as disclosed herein can comprise one or more exogenous transcription factors, or fragments thereof. In some embodiments the one or more transcription factors can comprise isoforms.

[0108] Disclosed herein in some embodiments, a transcription factor expressed in a cell of the present disclosure can take any form. In some embodiments, a transcription factor (TF) of the present disclosure can comprise a variant form of a TF. In some embodiments a variant form of a TF can comprise a TF having a mutation. Non-limiting examples of a mutation can comprise a deletion, a SNP, an indel, or any other genetic mutation. In some embodiments, a mutation can comprise more than one mutation in a TF. In some embodiments, a mutation can be engineered e.g., engineered by an artisan. In some embodiments, a mutation may notbe engineered. In some embodiments, a mutation can comprise as a change in amino acid or nucleic acid sequence. In some embodiments, a mutation can be created by addition of a genetic fragment or sequence. In some embodiments, a mutation can comprise point mutations. In some embodiments, a point mutation or mutations, (by additions or deletions) can comprise a mutation in a synthetic domain. In some embodiments, a synthetic domain or domains can comprise an activation domain or domains. In some embodiments, an activation domain(s) can boost or enhance transcription. In some embodiments, synthetic domain can be a repression domain(s). In some embodiments, a repression domain can be used to repress transcription. In some embodiments, a synthetic domain can comprise a conditional binding domain(s). In some embodiments, a conditional binding domain can be used so that its use can be regulated e.g., by activation upon addition of a binder. In some embodiments, a binder can comprise a small molecule or any other binder. In some embodiments, synthetic mutations, synthetic domains, genetic fragments, or sequences can be added to a TF, nearby in a regulatory region of a TF, a protein, a gene sequence, or in a target site in an engineered cell. In some embodiments, any additions or deletions of synthetic nucleic acid or amino acid sequences can generate one or more truncations. In some embodiments, one or more truncations can be added in an internal region or at the end of a protein. In some embodiments, an isoform of a gene, gene product, TF or TFrecipe can be generated for various uses of the present disclosure. In some embodiments, an isoform can comprise a variant e.g. splice variant.

[0109] Methods of treatment: Disclosed herein are further methods of using a cell a PSC, an engineered cell, a composition, a kit, a cell therapeutic or a pharmaceutical composition described herein. In some embodiments, the method described herein can provide a treatment. In some embodiments, a treatment can comprise a composition in any format or form as desired. In some embodiments, a method of treatment using a cell described herein can be used to treat at least one disease characterized by a blood dysfunction in a patient suffering therefrom. In some embodiments, a method of treatment can comprise administering to a subject, or a patient cell described herein e.g., a cell of a lineage comprising hematopoietic progenitor or hematopoietic stem cells.

[0110] In some embodiments, a method for treating a subject with a blood dysfunction or blood disorder can comprise administering to a subject in need thereof a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcriptionfactors, or an activator of transcription of the openreadingframe encodingthe one or more transcription factors described herein. In some embodiments, a method comprising a cell described herein can differentiate to a cell lineage comprising a blood stem cell e.g., a hematopoietic progenitor, a hematopoietic -like stem cell, or a hematopoietic stem cell (HSC).

[0111] In some embodiments, at least a method comprisestreating a disease in the subject by providing or administeringto a subject an engineered cell describedherein.In some embodiments, one or more diseases that can be treated by the disclosed methods can comprise any blood dysfunction diseases disclosed here. In other cases, one or more diseases that can be treated by the disclosed methods including another disease or disorder that may not be disclosed by the present disclosure, but as long as the present disclosure can lead to treatment of any disease or disorder, then the present disclosure herein claims that indication, disease, disorder, ailment, or condition. In some embodiments, a method of treating a disease in a subject in need thereof may comprise administering an engineered cell or composition of the present disclosure. In some embodiments, administering a treatment can be conducted using an in vivo or ex -vivo method e.g., an in vivo or ex-vivo infusion, an injection, a transplant of a cell therapy of cell described herein. In some embodiments, a method may comprise a cell described in addition to another treatment, e.g., a chemotherapy or a gene therapy, or any other additional therapeutic can be provided in addition to, prior to, or after provisional of a cell or population of cells described herein. In some embodiments, a cell described, a therapeutic of a PSC described or a gene therapy therapeutic mayb e delivered to treat a blood dysfunction disorder or disease in a subj ect in need thereof. The terms “blood dysfunction”, “blood disorder”, “blood disease”, “blood condition” and the like as would be known by a skilled artisan or a standard medical or non -medical dictionary, are used interchangeably throughout the present disclosure without any limitations; various blood diseases or dysfunctions are disclosed herein.

[0112] Provided herein in some embodiments, is a method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof. In some embodiments, a dysfunctional bone marrow can give rise to many blood disorders or diseases described herein. In some embodiments, a method of treating a disease can comprise comprising: administering an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors described herein, to the subject in need thereof. In non-limiting examples, the one or more transcription factors comprise LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further comprise TALI . In some embodiments, the one or more transcription factors described herein further comprise GATA2. In some embodiments, the one or more transcription factors described herein further comprise HOXAIO. In some embodiments, the one or more transcription factors described herein comprise LM02, SPI1, TALI, GATA2, HOXAIO, or any combination thereof. In some embodiments, one or more transcription factors can comprise NFE2 and at least one of CEBPA, GATA2, SPI1, or any combination thereof. In some embodiments, the one or more transcription factors described herein comprise NFE2. In some embodiments, the one or more transcription factors described herein comprise CEBPA. In some embodiments, the one or more transcription factors described herein comprise GATA2. In some embodiments, the one or more transcription factors described herein comprise SPI1. In some embodiments, the one or more transcription factors described herein further comprise LM02, TALI, or a combination thereof. In some embodiments, one or more transcription factors can consist of LM02 and SPI1. In some embodiments, the one or more transcription factors described herein further consist of TALL In some embodiments, the one or more transcription factors described herein further consist of GATA2. In some embodiments, the one or more transcription factors described herein further consist of HOXAIO. In some embodiments, the one or more transcription factors described herein consist of LM02, SPH, TALI, GATA2 and HOXAIO, or any combination thereof. In some embodiments, one or more transcription factors consist of NFE2 and at least one of CEBPA, GATA2 and SPH , or any combination thereof. In some embodiments, the one or more transcription factors described herein consist of NFE2. In some embodiments, the one or more transcription factors describedherein consists of CEBPA. In some embodiments, the one or more transcription factors described herein consist of GATA2. In some embodiments, the one or more transcription factors described herein consist of SPI1. In some embodiments, the one or more transcription factors described herein further consist of LM02 and TALI, or a combination thereof.

[0113] In some embodiments, a method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, can comprise administering an engineered cell described herein to a subject in need thereof, thereby treating a disease associated with a blood stem cell dysfunction, a dysfunctional bone marrow condition, or a hematopoietic stem cell dysfunction.

[0114] In some embodiments, an engineered cell described herein can comprise a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic-like stem cell. In some embodiments, a hematopoietic progenitor, a hematopoietic stem cell, or a hematopoietic -like stem cell can comprise an immune cell or immune-cell-like. In some embodiments, an engineered cell described herein can comprise an erythroid lineage cell, myeloid lineage cell, or granulocyte-macrophage lineage cell. In some embodiments, an engineered cell described herein can comprise is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

[0115] In some embodiments, a method provided is useful to treat a disease such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or acute lymphocytic leukemia. In some embodiments, a method provided is useful to treat a disease such as blood disorder or blood dysfunction such as sickle cell anemia. In some embodiments, a method provided is useful to treat a disease such as multiple sclerosis, systemic lupus erythematosus, or another autoimmune disorder.

[0116] In some embodiments, provided herein is a method of treating a disease comprising providing an engineered cell described herein. In some embodiments, an engineered cell can comprise a human stem cell. In some embodiments, an engineered cell can comprise a human induced pluripotent stem cell. In some embodiments, an engineered cell can comprise a human progenitor cell. In some embodiments, an engineered cell can comprise a human fetal stem cell. In some embodiments, an engineered cell can comprise an embryonic stem cell. In some embodiments, an engineered cell can comprise cell derived from a human stem cell. In some embodiments, an engineered cell inducedto express an expression cassette. In some embodiments, a method for treating a disease described herein can involve inducing an expression cassette which induces expression of one or more transcription factors. In some embodiments, inducing one or more transcription factors can trigger or induce differentiation of a PSC.

[0117] In some embodiments, aPSC or an engineered cell expresses one ormore immune cell or immune-cell-like markers in 3 days or less, 2 days or less, or 1 day or less. In some embodiments, the one or more immune cell or immune-cell-like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, or PECAM1. In some embodiments, a PSC or an engineered cell expresses CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45. In some embodiments, a PSC is derived from a patient. In some embodiments, a PSC is not derived from a patient.

[0118] In some embodiments, a method of treatment can comprise a population of cells can comprise CD34-expressing cells described herein. In some embodiments, at least 25% of a population of engineered cells express CD34. In some embodiments, at least 30% of a population of engineered cells express CD34. In some embodiments, at least 95% of a population of engineered cells express CD34. In some embodiments, at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99%, of a population of engineered cells express CD34. In some embodiments, at least one cell in the population of cells can express CD43. In some embodiments, a population of cells comprising at least 6% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 17% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 50% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the population of engineered cells express CD43. In some embodiments, at least one cell in the population of cells may express CD45 marker. In some embodiments, a population of cells comprising at least 7% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 12% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 25% of the population of engineered cells express CD43. In some embodiments, a population of cells comprising. In some embodiments, a population of cells comprising at least 85% of the population of engineered cells express CD45. In some embodiments, a population of cells comprising at least 90% of a population of engineered cells express CD45. At least one cell in the population of cells may express CD45. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of engineered cells express CD45.

[0119] In some embodiments, a method of treatment can comprise a population of engineered cells described herein can be induced to express one or more markers. In some embodiments, a population of cells can express double positives marker expression. Non-limiting examples of double positive expression of markers can include, expressive of both CD34 and CD43, CD34 and CD43, or CD34 and CD43, marker. In some embodiments, a population of cells comprising at least one cell in the population of cells can express CD34 and CD43. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells expressing CD34 and CD43. In some embodiments, a population of cells can comprise at least 3% of the population of engineered cells expressing CD34 and CD43. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45. In some embodiments, a population of cells can comprise at least 3% of the population of engineered cells expressing CD34 and CD45. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45. In some embodiments, a population of cells can express double positive marker expression while presenting expression of another marker e.g., cells expressing triple markers. Non -limiting examples of a cell or population of cells expressive triple positive markers can include, e.g., CD34 / CD43 / CD45 triple expression, where the cell or population of cells describe herein express CD34+, CD43+, CD45+. In some embodiments, a population of cells can comprise about 1% CD34 / CD43 / CD45 of the cells showing a triple expression of these markers. In some embodiments, a population of cells can comprise about 1% CD34 / CD43 / CD45 of the cells showing a triple expression of these markers. In some embodiments, a population of cells can comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45 -positive cells of the population of engineered cells express CD34 and CD43. In some embodiments, a population of cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45.

[0120] In some embodiments, a method of treatment can comprise a cell e.g., engineered cell described herein. In some embodiments, an engineered cell may not be induced to express the one or more transcription factors described herein. In some embodiments, an engineered cell that is not induced may expresses one or more markers. In some embodiments, an uninduced engineered cell can express one or more marker comprising one or more stem cell orpluripotency markers. In some embodiments, one or more markers sternness or pluripotency markers can comprise one or more cell lineage-specifying marker. In some embodiments, one or more cell lineage-specifying marker markers can comprise pluripotency or stem-ness markers e.g., a TRA-1-60 marker. In some embodiments, an engineered cell can be analyzed to assess if an engineered cell expresses one or more markers indicative of a cell exiting out of pluripotency e.g., a cell on a differentiation trajectory, a committed differentiation cell lineage, a progenitor of a cell lineage, or any indication that the cell is moving out of sternness. In some embodiments, at most 10%, 9%, 80%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% of a population of engineered cells express TRA-1-60. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI . In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2. In some embodiments, a population of cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express CEBPA. In some embodiments, a population of engineered cells can comprise at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HOXA10.

[0121] In some embodiments, a method of treatment can comprise a population of engineered cells. In some embodiments, a population of cells comprising a treatment can require induction to express one or more transcription factors. In some embodiments, expressing one or more transcription factors can induce a population of engineered cells to differentiate into a different cell lineage(s) described herein which can be useful as a treatment for various ailments described herein. In some embodiments, a population of engineered cells can be induced to comprise erythroid lineage cells, myeloid lineage cells, granulocyte-macrophage lineage cells, or any combination thereof. In some embodiments, a population of engineered cells can comprise myeloid lineage cells, granulocyte lineage cells, erythrocyte lineage cells, monocyte lineage cells, megakaryocyte lineage cells, or any combination thereof. In some embodiments, a population of engineered cells described herein can comprise at least 5% erythroid lineage cells. In some embodiments, a population of engineered cells described herein can comprise at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of erythroid lineage cells. In some embodiments, a population of engineered cells can comprise at least 50% erythroid lineage cells. In some embodiments, a population of engineered cells can comprise at least 5% myeloid lineage cells. In some embodiments, a population of engineered cells can comprise at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells. In some embodiments, a population of engineered cells can comprise at least 30% myeloid lineage cells. In some embodiments, a population of cells can comprise at least 40% granulocyte -macrophage lineage cells. In some embodiments, a population of engineered cells can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte -macrophage lineage cells. In some embodiments, a population of engineered cells can comprise at least 90% granulocyte - macrophage lineage cells. In some embodiments, a population of engineered cells can comprise erythroid lineage cells expressing CD71, CD235a, or both markers. In some embodiments, a population of engineered cells comprising myeloid lineage cells can express CD13, CD 14, CD15, or any combination thereof. In some embodiments, a population of engineered cells comprising myeloid lineage cells can express CD 14 and CD15.

[0122] Pharmaceutical composition, formulations: Provided herein in some embodiments is a pharmaceutical composition comprising the engineered cell described herein.

[0123] The present disclosure extends to a pharmaceutical composition, or a pharmaceutical formulation, or a pharmaceutical medicament, a drug, or other composition comprising a PSC or an engineered cell (a cell or population of cells, a PSC, an engineered cell, and the like as disclosed are used interchangeably, singular and plural)producedas described herein. In some embodiments, provided herein is a pharmaceutical composition comprising a pluripotent stem cell (PSC) described herein. In some embodiments, a cell, PSC, or engineered cell of the present disclosure is engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors described herein. In some embodiments, a pharmaceutic composition is administered to treat a disease in a subject, e.g. for treatment.

[0124] In some embodiments, herein is a pharmaceutical composition comprising an engineered cell described for various treatments including for example, a prophylactic treatment, a preventative treatment, a curative treatment, or any treatment to reduce symptoms, pain, or ameliorate a blood disorder or a blood disease or a blood dysfunction in a subject in need thereof. In some embodiments, a pharmaceutical composition described herein can require admixing the present disclosure e.g., a cell population described, or a population of PSCs described herein witha pharmaceutically acceptable excipient, vehicle, or carrier, and optionally one or more other ingredients. In some embodiments disclosed herein are pharmaceutical compositions comprising: (a) compositions or formulations of the present disclosure and (b) a pharmaceutically acceptable excipient. The components of making up the formulations or compositions have been described in detail through out this application. The compositions may for example, comprise the engineered cells comprising engineered or differentiated PSCs described herein e.g., hematopoietic progenitors, hematopoietic stem cells, or hematopoietic-like stem cells. The present compositions or engineered cells or differentiated cells with functional activities described in this application that indicate the engineered differentiated cell is a blood stem cell e.g., a hematopoietic stem cell or hematopoietic progenitor. These compositions may be carried by at least one or more expression cassette which can be expressed by an engineered cell (described in details elsewhere). All forms of the present pharmaceutical composition, or engineered PSC described herein can be envisioned or found in the present disclosure and are herein claimed as suitable for use as a pharmaceutical compositing against any blood disease, disorder, dysfunction of condition disclosed or known to a skilled artisan, as long as the present disclosure can treat, ameliorate, improve, prevent such a condition or pain from the conditions described herein.

[0125] Provided herein is a pharmaceutical formulation or composition for administration to treat at least one disease in a subject, the composition as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, a pharmaceutical composition can comprise, in addition to the composition comprising engineered cells described herein, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabilizer, anti-oxidant or other material that are nontoxic and should not interfere with the activity of the compositions of engineered cell described herein. The precise nature of the carrier or other material will depend on the route of administration. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. In some embodiments, physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. In some embodiments, a pharmaceutical composition can be in the form of a parenterally acceptable aqueous solution, which is pyrogen -free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection. A composition may be prepared using artificial cerebrospinal fluid. As described throughout the term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that doesnot interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known by a skilled artisan and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. In some embodiments, such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. In some embodiments, the compositions are sterile. In some embodiments, a pharmaceutical composition can contain adjuvants such as preservative, emulsifying agents and dispersing agents. In some embodiments, prevention of actions of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents. In some embodiments, a pharmaceutical composition may be in any suitable form, (depending upon a desired method of administration). In some embodiments, a pharmaceutical composition can be provided in unit dosage form, can be provided in a sealed container, and can be provided as part of a kit. In some embodiments, such a kit can include instructions for use. In some embodiments, a kit can include a plurality of the unit dosage forms. In some embodiments, a pharmaceutical composition can be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions can be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

[0126] Administration: In some embodiments, provided herein is a composition for administering to a subject in need thereof. In some embodiments a composition is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. A composition described herein comprises administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0127] In some embodiments, the present composition described herein can comprise engineered cells or population of cells comprising engineered stem cells e.g., hematopoietic progenitors, hematopoietic stem cells, or hematopoietic-like stem cells disclosed can display anormal phenotype of a functional blood stem cell or functional hematopoietic stem cell. In some embodiments, the normal healthy engineered cell described can be implanted, infused, transplanted, or administered by injection or any other method described herein, to a subject in need thereof.

[0128] In other embodiments, cells may be used to generate iPSCs which may then be generated to differentiate into the target engineered a population of PSCs or iPSCs as described herein. In some embodiments, PSCs, cells or a iPSCs cells may be a healthy cell or a cell that may contain a mutation or genetic defect and this mutation or defect may be corrected using conventional recombinant techniques to produce iPSCs with a normal phenotype. This may be a relevant step when generating patient-specific cells e.g., from a blood dysfunction cells of a patient or subject. This procedure of correcting mutated iPSCs may be useful when needed for other targeted studies, e.g., for drug studies, disease-specific iPSCs and the like. The engineered cell, PSCs or iPSCs described herein of a normal phenotype may be produced from these iPSCs, or stem cells as described herein and implanted into the patient to repair or ameliorate a blood disease, disorder, or blood dysfunction.

[0129] In other embodiments, an engineered cell or population of engineered cells produced as described herein can display one or more disease phenotypes. In some embodiments, an engineered cell that may display one or more diseases may be a cell for an autologous cell therapy program or cell therapies. In some embodiments, an engineered cell that may display one or more diseases may be a cell for an allogenic or allogeneic cell therapies or allogenic cell therapy program. In some embodiments, an engineered cell that may display one or more diseases may be a cell for production of master cell bank or not for production of master cell bank. In some embodiments, an engineered cell or population of cells can then be treated to restore a normal phenotype. In some embodiments an engineered cell showing the one or more disease phenotypes can comprise, an engineered cell of choice. Non-limiting examples of engineered cell with the one or more disease phenotypes can comprise engineered iPSC, engineered stem cell, engineered adult stem cell, engineered fetal stem cell, engineered hematopoietic stem cell, engineered PSCs, engineered cells, engineered hematopoietic progenitor or hematopoietic-like stem cell or any other engineered cell comprising the present disclosure. The source of the engineered cell can comprise various cell sources e.g., cells from a tissue, an organ or blood / systemic. There are various examples of the sources of the engineered cells as disclosed herein or as known in the art. In a non -limiting example, the genetic mutation or defect which is responsible for the disease phenotype may be corrected in vitro. Various techniques are available to correct genetic mutations or defects in isolated mammalian cells.Once the defect or mutation is corrected and the normal phenotype restored, the HSC progenitors may be implanted into a subject or patient to repair or ameliorate the blood dysfunction or damaged, diseased hematopoietic stem cells.

[0130] Accordingto some embodiments, provided are methods comprising administering an effective amount of the present compositions comprising e.g., a population of PSCs described herein to an individual in need thereof. For example, the present compositions comprising e.g, a population of PSCs described herein can be used for the treatment of a subject for a condition where administration of an effective amount of the cells will have a desired therapeutic effect. In some instances, the desired therapeutic effect will be a result of one or more endogenous functions of the administered PSC e.g., endogenous functions of a healthy, normal hematopoietic stem cell or hematopoietic progenitor cell - such functions are known to a skilled artisan and targets for use of the present engineered cells have been disclosed herein. In some instances, the desired therapeutic effect will be a result of one or more heterologous functions of the administered blood stem cell or hematopoietic stem cell e.g., restoring a function of a gene product encoded by a functionally integrated transgene of the present disclosure.

[0131] Provided herein are cell populations which can be used for the treatment and / or prevention of any blood dysfunction disease or disorder. For example, reconstitution of blood dysfunction in a patient by the introduction of the present cells can comprise infusing or transplanting cells e.g., hematopoietic stem cells as a potential therapeutic option for a subject. In some embodiments, a subject is suffering from a disease e.g., any blood dysfunction conditions) described herein, such as for example, acute blood dysfunction failure, chronic blood dysfunction disease and / or monogenic disease e.g., sickle cell disease or any otherblooddysfunction disclosed herein). In some embodiments, a cell can be provided for e.g., a permanent treatment for a conditionby persistence of transplanted hematopoietic stem cells and / orrepopulatingthe subject’s blood dysfunction with isolated expanded human hematopoietic stem cells or progenitors as described herein.

[0132] In some embodiments, administering can comprise transplantation, including e.g., orthotopic transplantation, of a cell population e.g., hematopoietic stem cell populations comprising engineered cells described herein into a subject in need thereof. In some embodiments, human cell populations comprising engineered cells described herein produced accordingto the methods described herein can be further purified, cryopreserved, and / or extensively characterized prior to transplantation or infusion. In some embodiments, the present cells or compositions produced accordingto the methods described herein can provide on -demand therapy for subjectsor patients with one or more severe blood dysfunction diseases and who may respond to provision of hematopoietic stem cells described herein.

[0133] In some embodiments, the cell populations comprising engineered cells described herein comprises administered to subjects by any suitable means and to any part, organ, or tissue of the body of a subject in need thereof, without any limitation as to the route of administration or dosages, as long as it is determined by a skilled artisan, qualified medical personnel e.g., a qualified medical doctor or physician. Non-limiting examples of administration means include infusion, umbilical vein infusion, direct capsule injection, artery infusion, infusion into any part of the body via injection (infusion, transplantation). In certain embodiments, the compositions comprise encapsulated cells described herein can be transplanted by infusion using any suitable administration route into a subject in need thereof. The administration route may also be dependent on the type of formulation of the composition disclosed herein.

[0134] Application: In some embodiments, provided herein is a population of PSCs or engineered cells e.gt., hematopoietic stem cells. In some embodiments, the hematopoietic stem cells or progenitors can be transplanted into a subject in need thereof. In some embodiments, such a transplant can be termed a hematopoietic stem cell transplant (HSCT). In some embodiments, an HSCT can involve an injection of hematopoietic stem cells into a subject in need thereof or a patient with a dysfunctional bone marrow condition. In some embodiments, a dysfunctional bone marrow can be indicated for a variety of malignant diseases including, in non-limiting examples, multiple myeloma, Hodgkin and non-Hodgkin lymphoma, acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) and non-malignant diseases (e.g. sickle cell anemia, autoimmune disorders such as Multiple Sclerosis, SLE, and others). In some embodiments over 80,000 allogeneic HSCTs can be performed annually. In some embodiments, HSCTs can require donor and cell availability butthis can be one of the largest limiting factors. In some embodiments, HSCT can involve use of intra-venous (IV) infusion of frozen or fresh cells into a subject or a patient. In some embodiments, HSCT can be provided after chemotherapy with or without total body irradiation. In some embodiments, it may be required that a minimal dose of 2 million cells per kg body weight be used. In some embodiments, it may be required that a dose of 11 million cells per kg body weight be used. In some embodiments, it may be required that a target cell dose of 6-8 million CD34+ cells per kg bodyweight be used. In some embodiments, a dose that is provided can been associated with best overall survival and engraftment. In some embodiments, a dose can comprise infusion or administering a dose of 480 - 640 million cells for the average adult patient. In some embodiments, a dose may be determined by condition or factors specific for a subject in need thereof or can be determined empirically. In some embodiments, HSCT canrequire availability or production of large amounts of cells e.g., the present described cells herein. In some embodiments, large amounts of HSCs may be required to reduce or alleviate the need for deadly conditioning regimens.

[0135] Indications: Hematopoietic stem cells produced (e.g., expanded, enriched, or maintained in a multi-potent state) through the use of the compositions and methods disclosed herein can be used to treat a variety of diseases (e.g., animal diseases (human diseases and nonhuman diseases)). In some embodiments, hematopoietic stem cells, hematopoietic -like cells (HSCs), or progeny thereof administered to a subject can comprise autologous, syngeneic, or allogeneic HSCs. In some embodiments, such HSCs can be administered to a subject using any methods that are known or useful for the treatment. In some embodiments, HSCs administration can be provided in conjunction with one or more agents e.g., an agent that can promote the expansion of a hematopoietic stem cell in vivo. In non-limiting examples, hematopoietic stem cells, hematopoietic-like stem cells, or progeny thereof can be administered to a subject (e.g., a human subject who is a patient) in order to treat such diseases as genetic blood disorder, e.g., a transplant disorder involving the blood system (e.g., HLA incompatibility -associated disorders requiring transplanting of HSCs into a subject in need thereof), treatment of genetic blood disorders e.g., Sickle cell anemia, Alpha thalassemia, Beta thalassemia, Delta thalassemia, Hemoglobin E / thalassemia, Hemoglobin S / thalassemia, Hemoglobin C / thalassemia, Hemoglobin D / thalassemia, Chronic granulomatous disease (X-linked Chronic granulomatous disease, autosomal recessive (AR) chronic granulomatous disease, chronic granulomatous disease AR I NCF1„ Chronic granulomatous disease AR CYBA, Chronic granulomatous disease ARIINCF2, Chronic granulomatous disease AR III NCF4), X-linked Severe Combined Immune Deficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID, Ragl / Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, Congenital agranulocytosis,, Congenital agranulocytosis -congenital neutropenia- SCN1, Congenital agranulocytosis- congenital neutropenia-SCN2, Familial hemophagocytic lymphohistiocystosis (FHL), Familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforin mutation), Agammaglobulinemia (X-linked Agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak -Higashi syndrome, Hemolytic anemia due to red cell pyruvate kinase deficiency, Paroxysmal nocturnal hemoglobinuria, X-linked Adrenoleukodystrophy (X-ALD), X- linked lymphoproliferative disease, Unicentric Castleman's Disease, Multicentric Castleman's Disease, Congenital amegakaryocytic thrombocytopenia (CAMT) type I, Reticular dysgenesis, Fanconi anemia, Acquired idiopathic sideroblastic anemia, Systemic mastocytosis, Von Willebrand disease (VWD), Congenital dyserythropoietic anemia type 2, Cartilage -hair hypoplasia syndrome, Hereditary spherocytosis, Blackfan -Diamond syndrome, Shwachman-Diamond syndrome, Thrombocytopenia-absent radius syndrome, Osteopetrosis, Infantile osteopetrosis, Mucopolysaccharidoses, Lesch-Nyhan syndrome, Glycogen storage disease, Congenital mastocytosis, Omenn syndrome, X-linked Immuno-dysregulation, poly- endocrinopathy, and enteropathy (IPEX), IPEX characterized by mutations in FOXP3, X-linked syndrome of poly-endocrinopathy, immune dysfunction, and diarrhea (XPID), X-Linked Autoimmunity -Allergic Dysregulation Syndrome (XLAAD), IPEX-like syndrome, Hyper IgM type 1, Hyper IgM type 2, Hyper IgM type 3, Hyper IgM type 4, Hyper IgM type 5, X linked hyperimmunoglobulin M, Bare lymphocyte Syndrome type I, and Bare lymphocyte Syndrome type II (Bare lymphocyte Syndrome type II, MHC class I deficiency; Bare lymphocyte Syndrome type II, complementation group A; Bare lymphocyte Syndrome type II, complementation group C; Bare lymphocyte Syndrome type II complementation group D; Bare lymphocyte Syndrome type II, complementation group E). Populations of hematopoietic stem cells expanded, enriched, or maintained by the compositions and / or methods of the invention, as well as progeny thereof, can also be used to treat a patient suffering from a hematolymphoid malignancy, a non- hematolymphoid malignancy, or a protein deficiency. In other embodiments, the patient may be tissue or cell transplantation recipient, and the hematopoietic stem cells or progeny thereof are administered in order to induce tolerance to the transplanted tissue or cells, or as therapy or part of therapy to treat Acute Lymphoblastic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), Hodgkin Lymphoma (HL), Non -Hodgkin Lymphoma(NHL), Myelodysplastic Syndrome (MDS), Multiple myeloma, Aplastic anemia, Bone marrow failure, Myeloproliferative disorders such as Myelofibrosis, Essential thrombocytopenia or Polycythemia vera, Fanconi anemia, Dyskeratosis congenita, Common variable immune deficiency (CVID, such as CVID 1, CVID 2, CVID 3, CVID 4, CVID 5, and CVID 6), Hemophagocytic lymphohistiocystosis, solid tumors such as Neuroblastoma, Germ cell tumors, Breast cancer, Wilms' tumor, Medulloblastoma, and Neuroectodermal tumors, Autoimmune diseases such as rheumatoid arthritis (RA), psoriasis, Scleroderma, Multiple sclerosis, Ulcerative colitis, Systemic lupus erythematosus or Type I diabetes, Human immunodeficiency virus (HIV), or protein deficiencies such as Adrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MED), Hemophilia A & B, Hurler syndrome, Hunter syndrome, Fabry disease, Gaucher disease, Epidermolysis bullosa, Amyloidosis, Globoid Cell Leukodystrophy, Sanfilippo syndrome, and Moquino syndrome. In some embodiments, the present disclosure targets another indication, diseases, disorder, ailment, or condition.

[0136] In some embodiments, another target indication, disease, ailment, condition, disorder, comprises systemic lupus erythematous. In some embodiments, systemic lupus erythematous comprises lupus. Having systemic lupus erythematous (SLE) can increase risk for developing diabetes mellitus (DM). In some embodiments, another target indication, disease, disorder, ailment, or condition comprises DM. In some embodiments, DM comprises gestational diabetes. In some embodiments, SLE and DM are risk factors for cardiovascular disease, kidney damage, or inflammatory diseases or conditions. In some embodiments, another target indication, disease, disorder, ailment, or condition comprises a cardiovascular disease. In some embodiments, another target indication, disease, disorder, ailment, or condition comprises kidney damage. In some embodiments, another target indication, disease, disorder, ailment, or condition comprises inflammatory disease or condition.

[0137] Selectable markers: In some embodiments, a cell comprising the engineered cell with or without the at least one expression cassette of the present disclosure may comprise a selectable biomarker. In some embodiments, the selectable marker that is expressed by the engineered cell may be used to select and characterized the engineered cell. The cell may comprise a population of engineered cells.

[0138] In some embodiments, the cells may not comprise a selectable marker. In some embodiments, the cells may comprise a selectable biomarker encoding a fluorescent biomarker. In some embodiments, the selectable biomarker may be an antibiotic cassette. In some embodiments, the selectable marker may be a vector, an expression cassette, or molecule that produces a morphological change, wherein the morphological change denotes integration of the recombinant polynucleotide and expression of exogenous protein in the cell or expression cassette bearing the recombinant protein. In some embodiments, the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a gene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker. Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, m Scarlet fluorescent protein gene, red fluorescent p rotein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry / texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art. Non -limiting examples of antibiotic selectable resistance marker gene include, kanamycingene, ampicillin gene, streptomycin gene, neomycin gene, puromycin gene gentamycin gene, erythromycin gene, Blasticidin S gene, hygromycin Bgene among many others useful in the art. In some embodiments, the complementing cells may further comprise or bear small interfering RNA or miRNA wherein the siRNA or miRNA maybe short hairpin transcripts, or the short hairpins may be made from a selectable DNA vector.

[0139] A Kit: In another aspect of the present disclosure is provided a kit for inducing differentiation of a pluripotent stem cell (PSC) into hematopoietic stem cell or progenitor, or a hematopoietic-like stem cell, the kit comprising: a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1. In some embodiments, the one or more transcription factors comprise CEBPA. In some embodiments, the one or more transcription factors comprise GATA2. In some embodiments, the one or more transcription factors comprise SPI1. In some embodiments, the one or more transcription factors comprise at least one of LM02 or TALI. In some embodiments, expression of the one or more transcription factors induces differentiation of a PSC into an engineered cell disclosed, e.g., hematopoietic stem cell or progenitor, or a hematopoietic-like stem cell. In some embodiments, the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1. In some embodiments, the one or more transcription factors comprises NFE2. In some embodiments, the one or more transcription factors comprises HOXAIO.

[0140] In another aspect of the present disclosure is provided a kit for inducing differentiation of a pluripotent stem cell (PSC) into hematopoietic stem cell or progenitor, or a hematopoietic-like stem cell, the kit comprising: a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors consist of NFE2 and at least one of CEBPA, GATA2, and SPI1, or any combination thereof. In some embodiments, the one or more transcription factors consist of CEBPA. In some embodiments, the one or more transcription factors consist of GATA2. In some embodiments, the one or more transcription factors comprise SPI1. In some embodiments, the one or more transcription factors consist of at least one of LM02 and TALI, or a combination thereof. In some embodiments, expression of the one or more transcription factors induces differentiation of a PSC into an engineered cell disclosed, e.g., hematopoietic stem cell or progenitor, or a hematopoietic-like stem cell. In some embodiments, the one or more transcription factors consist of NFE2 and at least one of CEBPA, GATA2, and SPI1, or any combination thereof. In someembodiments, the one or more transcription factors consists ofNFE2. In some embodiments, the one or more transcription factors consists of HOXAIO.

[0141] In some embodiments, the kit provided herein comprises a PSC or engineered cell expressing one or more immune cell or immune-cell-like markers in 3 days or less, 2 days or less, or 1 day or less. In some embodiments, the kit provided herein comprises a PSC or engineered cell expressing one or more immune cell or immune -cell-like markers comprising CD34, CD43, CD45, THY1, ITGA6, CD33, or PECAM1. In some embodiments, the kit provided herein comprises a PSC or engineered cell expressing CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45. In some embodiments, the kit provided herein comprises a PSC or engineered cell suitable for a blood disorder disclosed herein

[0142] Articles of Manufacture

[0143] In another aspect of the disclosure, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition which can comprise a population or selection of modified or engineered immune cell or immune-like cell or immune cell or immune-like cell immune cell or immune-like cell cells of the present disclosure.

[0144] The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the modified or engineered immune cell or immune-like cell or immune cell or immune-like cell immune cell or immune- like cell cells of the present disclosure; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent such as a small molecule. The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the compositions can be used to treat a particular condition.

[0145] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically -acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution.It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0146] Methods as described herein may be used to generate a population of one or more immune cells. The population of cells may comprise adherent cells. The population of cells may comprise suspension cells. The population of cells may comprise adherent cells and suspension cells. The population of cells may be provided in a media. The media may not have to be altered during the differentiation of the PSCs into immune cells. The media may not need any nutrients, growth factors, or microenvironmental or matrix optimizations.

[0147] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. A transcription factor may be expressed or have increased expression in a cell through various methods. This may involve delivery of either a nucleic acid comprising an open reading frame encoding the transcription factor, delivery of the transcription factor itself, or delivery of an activator of the transcription factor or its expression. Any technique known in the art, for such delivery may be used. For example, for delivery of a cDNA, a viral or plasmid vector may be used. The open reading frame (encoding any isoform of the TF) may be inducible or repressible for control, to achieve a suitable level of expression. The nucleic acid comprising the open reading frame maybe a complementary DNA (cDNA), a messenger RNA (mRNA), or a synthetic or engineered nucleic acid. Some transcription factors may require a critical amount of expression to effectively induce differentiation, such as the equivalent of at least 5, 10, 15, 20, 25, or 50 copies of the ORF per cell. Other factors may require less than a certain threshold of expression due to possible toxicity at high levels, such as less than 20, 10, or 5 copies per cell. Increased levels of expression may also be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon. Modified RNAs, RNAs that encode the transcription factor but use synthetic nucleotides that improve stability and reduce degradation, may be used. Use of culture media adapted for a particular cell type may increase the expression of the ORF that induces expression of that cell type. Expression of an ORF can be increased from a non-expressed gene, from a gene expressed at a low level, or from a gene expressed at a robust level. The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor as described in Ng, Alex HM, et al. "A comprehensive library of human transcription factors for cell fate engineering." Nature biotechnology 39.4 (2021): 510-519; PCT Application Number PCT / US2017 / 051122;PCT / US2023 / 069550; and PCT Application Number PCT / US2018 / 030216, which are entirely incorporated herein by reference.

[0148] It will be understood that methods for increasing the expression of the transcription factors in the cells to be programmed into immune cells may include any method known in the art, for example, by induction of expression of one or more expression cassettes previously introduced into the cells, or by introduction of nucleic acids (such as DNA or RNA), polypeptides, or small molecules to the cells. Increasing the expression of certain endogenous but transcriptionally repressed genes may also reverse the silencing or inhibitory effect on the expression of these genes by regulating the upstream transcription factor expression or epigenetic modulation. Therefore, methods of the invention may involve culturing the cell population under conditions to artificially increase the expression level of one or more of the transcription factors described herein.

[0149] Host cells: In some embodiments, a PSC or engineered cell disclosed can comprise an allogeneic cell therapy for cell therapy in subjects or patients in need thereof. In some embodiments, a PSC or engineered cell disclosed can be a homologous cell. In some embodiments, a PSC or engineered cell disclosed can comprise an autologous therapeutic for cell therapies. In some embodiments, a PSC or engineered cell disclosed can comprise a heterologous cell. In some embodiments, a PSC or engineered cell disclosed can be derived from a human cell. Non-limiting examples of a PSC include e.g., a human stem cell, an induced PSC e.g., a human iPSC, a human embryonic stem cell or infant stem cell, and any others known by a skilled artisan. In some cases, a PSC or engineered cell disclosed cannot be derived from a human cell. In a non-limiting example, a PSC or engineered cell disclosed can be derived from any mammal or non -mammal cell. In some embodiments, non-mammalian cells can include for example and without limitations, avian cells, insect tissue culture cells, plant cells, or non- mammalian vertebrate cell. Non-limiting examples of mammalian cells can include a mouse cell, a rat cell, a rabbit cell, a hamster cell, a cat cell, a dog cell, a guinea pig cell, a bat cell, a human cell, or a non-human primate cell. In some embodiments, the cell can be a fish cell. In some embodiments, the cell can be a transgenic fly or insect cell. In some embodiments, the cell can be a bird cell. In some embodiments, the cell can be a Chinese hamster ovary cell.

[0150] Transgene delivery: The present disclosure provides methods for increased expression of one or more transgene, increased expression of one or more transcription factors, increased expression of one or more exogenous sequence comprising a recombinant construct. In some embodiments, the methods provided can decrease or diminish expression of one or more gene, transcriptional factors, gene fragment, functional polypeptide, or exogenous nucleic acid. In some embodiments, expression of the transcription factors may be increasedby directly delivering one or more nucleic acids or nucleic acid molecules to a cell. In some embodiments, the nucleicacids or nucleic acid molecules can be a DNA or RNA (or nucleic acid in any form, e.g., single stranded, double stranded, etc.). There are numerous technologies to modify gene expression in stem cells or iPSC each varying in complexity, efficiency, reliability, and safety. In some embodiments, the one or more gene, ORF, or recombinant construct delivery can comprise transient (gene) delivery. In some embodiments, the one or more genes, ORFs, or recombinant constructs delivery can comprise stable gene delivery. In some embodiments, the one or more genes, ORFs, or recombinant constructs can be transduced into stem cell or iPSCs using at least one delivery method disclosed herein or known to an artisan. In some embodiments, one or more genes, ORFs, or recombinant constructs delivery can comprise stable (gene) delivery. In some embodiments, one or more genes, ORFs, or recombinant constructs can be transduced into stem cell or iPSCs using at least a non-viral delivery method or a non-viral gene delivery. The one or more genes, ORF, recombinant constructs, or nucleic acids may be delivered into cells using any suitable methods for nucleic acid delivery for transformation of a cell. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome mediated transfection, by receptor-mediatedtransfection, by microprojectile bombardment, by agitation with silicon carbide fibers, by Agrobacterium-mediated transformation, and any combination of such methods. The delivery methods covered in this application can comprise delivery of viral and non-viral recombinant constructs or an expression cassette disclosed using any chemical or physical (gene) delivery methods disclosed in this application. The viral gene delivery system can be an RNA- based orDNA-based viral vector. The present disclosure can apply a viral delivery which exploits the backbone of a viral genome with insertion of a gene, gene fragments, portions, or genes of interest to be expressed in a stem cell, iPSC, or an engineered cell disclosed. In some embodiments, viral delivery systems can comprise retroviral, lentiviral, adenoviral, adeno- associated viral (AAV), baculoviral transductions or any other viral system known in the art in order to modify the disclosed cell. In other instances, the present disclosure can apply a non-viral delivery technique for delivery of recombinant constructs, or an expression cassette disclosed herein. Non-limiting examples of non-viral delivery methods can include electroporation, lipofection and nucleofection of plasmid or vector encoding a gene of interest. In some embodiments, non-viral methods applied comprises ultrasound, microinjection, and molecular- vibration-mediated delivery. In some cases, use of a non-viral gene delivery system can provide decreased immunogenicity and less insertional mutagenesis.

[0151] Gene / genome editing strategies: any suitable technique for insertion of a nucleic acid sequence into a specific sequence can be used including a method that is known by a person of skill in the art or skilled artisan. Suitable techniques can include a method which introduces a break at the desired location and permits recombination of a vector into the gap. Thus, one step for targeted site-specific genomic modification can be the creation of a double-strand DNA break (DSB) at a genomic locus to be modified. Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, e.g., use of non-homologous end joining repair (NHEJ), which can be more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, that can be used to insert inducible cassettes. In some embodiments, a PSC or an engineered cell disclosed can comprise recombinant constructs, or further engineered to comprise at least one expression cassette which can undergo gene or genome editing or modification. In some embodiments, genetic engineering or modifications can comprise gene or genome editing strategies. In some embodiments, gene or genome editing strategies can be based on custom -engineered or site-specific nuclease systems. Non-limiting examples of site-specific nuclease systems include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR). In some embodiments, any methods applying the CRISPR / Cas9 system for gene editing can be utilized. In some embodiments, the ZFN can comprise hybrid proteins and catalytic domains of Fokl endonuclease. In some embodiments, the TALEN can be hybrid proteins composed of TAL effector DNA-binding domain arrays as well as a catalytic domain of e.g., Fokl endonuclease. In some cases, the CRISPR / Cas9 may comprise Cas9 endonuclease and the single-guide RNA (sgRNA) molecule. In some embodiments, a sgRNA can comprise targets of interest which can include targets (e.g., transcription factors, family members, genes, activators and the like) disclosed herein. In this fashion, the present disclosure can utilize genome editing tools to generate iPSC cell lines or engineered cells bearing overexpressed transcription or reduced transcription (on diminished transcription) of any target herein disclosed in order to generate engineered cells disclosed. The gene delivery strategies that can be applied when stem cells or iPSCs for examples are subjected to gene or genome editing techniques to create modification of endogenous sequences. In some embodiments, a genetic modification may target at least one polynucleotide. In some embodiments, at least one polynucleotide can comprise at least one full length gene, at least one gene fragment, or at least one portion of a gene. In some embodiments, a modification can target at least one polypeptide. In some embodiments, at least one polypeptide can comprise at least one amino acid sequence, at least one protein, one peptide, fragment, or portion thereof. The PSC or engineered cell provided herein may further comprise at least one expression cassette. In some embodiments, at least oneexpression cassette can comprise one or more transcription factors, genes, sequences coding for genes or fragments thereof. In some embodiments, at least one expression cassette can comprise one or more polynucleotides. In some embodiments, one or more polynucleotides can encode one or more functional polypeptides. In some embodiments, one or more polynucleotides encode one or more non-functional polypeptides. In some embodiments, one or more polynucleotides can comprise a wild-type sequence, a native sequence, or a recombinant sequence.

[0152] Methods as described herein comprise providing one or more pluripotent stem cells (PSCs) or engineered cells. In some embodiments, the method can comprise delivering one or more PSCs with at least one exogenous expression cassette. In some embodiments, the at least one exogenous expression cassette can comprise a transcription factor. In some embodiments, the transcription factor can induce differentiation of the one or more PSCs into the population of hematopoietic progenitors, hematopoietic stem cells or hematopoietic -like stem cells. In some embodiments, the method can comprise inducing expression of at least one exogenous expression cassette comprising one or more transcription factors in the PSCs in 96 hours or less. In some embodiments, the method can comprise generating a population of induced PSCs or engineered cells from the PSCs. The population of cells may comprise adherent cells. In some embodiments, the population of cells may comprise suspension cells. In some embodiments, the population of cells may comprise adherent cells and suspension cells. In some embodiments, the population of cells may be provided in a media. In some embodiments, the media may not need any nutrients, growth factors, or microenvironmental or matrix optimizations. In some embodiments, at least 5% of the cells may express one or more hematopoietic stem cell, progenitor, or hematopoietic-like stem cell markers. In some embodiments, at least about 1%, at least about 3%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% of the cells may express one or more hematopoietic stem cell, progenitor, or hematopoietic-like stem cell markers.

[0153] The present disclosure provides methods for expressing, or for increasing expression of, one or more transcription factors. In some embodiments, one or more transcription factors can be expressed at increased expression in a cell (e.g., a PSC or engineered cell disclosed) through various methods. In some embodiments, increased expression can be delivered of one or morenucleic acid molecules comprising an open reading frame encoding one or more transcription factor, or by delivery of one or more transcription factors alone, or delivery of an activator of one or more transcription factors or expression of the activator itself. In some embodiments, any technique known in the art, for such delivery can be used. Non-limiting examples for delivery of a cDNA, DNA, transcript, a synthetic molecule, synthetic nucleic acid, or a recombinant construct to a viral vector, non-viral vector, plasmid vector, nanoparticle delivery or a delivery currently in use by a skilled artisan can be used. In some embodiments, an open reading frame (encoding any isoform, any variant of any transcription factor, fragment, gene, or gene fragment thereof) can be inducible or repressible for control, to achieve a suitable or desired level of transgene expression. In some embodiments, the nucleic acid comprising an open reading frame can be a complementary DNA (cDNA), a messenger RNA (mRNA), or a synthetic or engineered nucleic acid, or any kind of construct disclosed. In some embodiments, In some embodiments, some transcription factors can require a critical amount of expression to effectively induce differentiation, such as the equivalent of atleast 2, 3, 5, 10, 15, 20, 25, 50, or more copies of the ORF per cell (includes all integers). In some embodiments, other factors can require less than a certain threshold of expression due to possible toxicity at high levels, such as less than 20, 10, 5 or less copies per cell. In some embodiments, increased levels of expression can also be achieved by increasing the copy number of an open reading frame (ORF). Nonlimiting example of increasing copy number of ORF can include using a higher copy number vector or by using a transposon.

[0154] Modified RNAs: In some embodiments, RNAs that encode a transcription factor but use synthetic nucleotides that improve stability and reduce degradation, can be used. In some embodiments, use of culture media adapted for a particular cell type can increase the expression of the ORF that induces expression of that cell type. In some embodiments, expression of an ORF can be increased from a non -expressed gene, from a gene expressed at a low level, or from a gene expressed at a robust level. In some embodiments, the present disclosure provides methods for expressing, or for increasing expression of, a transcription factor as described in Ng Alex HM, et al. "A comprehensive library of human transcription factors for cell fate engineering." Nature biotechnology 39.4 (2021): 510-519; PCT Application Number PCT / US2017 / 051122; PCT Application Number PCT / US2018 / 030216 and PCT / US2023 / 068544 each of which is entirely incorporated herein by reference.

[0155] It will be understood that methods for increasing expression of any genes, transcripts, proteins, transcription factors, fragments or portions thereof disclosed herein in a disclosed cells can be induced, differentiated, or programmed into a different cell lineage using any method known in the art. Non-limiting example may include induction of expression of one or moreexpression cassettes (comprising one or more transcription factors) previously introduced into the cells, or by introduction of nucleic acids (such as DNA or RNA), polypeptides, or small molecules to the cells. In some embodiments, increasing the expression of certain endogenous but transcriptionally repressed genes or transcripts can also reverse the silencing or inhibitory effect on an expression of one or more genes by regulating an upstream transcription factor expression or epigenetic modulation. Therefore, methods of the present disclosure can involve culturing a cell disclosed under desired conditions to artificially increase an expression level of one or more of transcription factors described herein.

[0156] Contact with transcription factors or transcription factor family members

[0157] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. The expression of the transcription factors may be increased by contacting at least one cell with one or more proteins encoding transcription factors. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). Delivery of the transcription factors may occur using direct electroporation of transcription factor proteins to the cells.

[0158] Direct contact with agents

[0159] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. The expression of the transcription factors may be increased by contacting at least one cell with one or more agents that activate or increase the expression or amount of the transcription factors. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). Delivery of the transcription factors may occur using direct electroporation of transcription factor proteins to the cells. The agent may be a nucleic acid (i.e., polynucleotide, e.g., messenger RNA (mRNA), coding DNA sequence), a protein, an aptamer and small molecule, ribosome, RNAi agent, guide RNA (gRNA) and peptide-nucleic acid (PNA) and analogues or variants thereof. In one embodiment, the agent is a transcriptional activation system (e.g., a gRNA for use in a gene activation system such as CRISPR / Cas9 or TALEN) for increasing the expression of the one or more endogenous transcription factors.

[0160] Open reading frame

[0161] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. The expression of the transcription factors may be increased by inducing differentiation of at least one cell by delivering to the at least one cell a nucleic acid comprising an open reading frame encoding one or more of the transcription factors, the transcription factor protein, or an activator of transcription of the open reading frame encoding one or more transcription factors. The one or more transcription factors may be delivered in an exogenousexpression cassette. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC).

[0162] Exogenous expression cassette

[0163] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. The expression of the transcription factors may be increased by delivering one or more nucleic acids encoding a transcription factor or an activator of transcription of the open reading frame encoding one or more transcription factors. The nucleic acid may comprise a recombinant or exogenous expression cassette encoding one or more transcription factors. The recombinant or exogenous expression cassette may induce the cell to differentiate into a hematopoietic lineage. The recombinant or exogenous expression cassette may induce the cell to differentiate into an immune cell for example, a regulator T cell. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). One or more recombinant or exogenous expression cassettes may be used. The exogenous expression cassette may comprise an externally inducible transcriptional regulatory element for inducible expression of the one or more transcription factors, such as an inducible promoter, e.g., comprising a tetracycline response element or variant thereof . Any suitable system for delivering the sequence may be used. The gene delivery system may be a transposon system or a viral gene delivery system or an episomal gene delivery system or a homologous recombination system, such as, for example, utilizing a zinc finger nuclease, a transcription activator-like effector nuclease (TALENs), or a meganuclease, or a CRISPR / Cas9, or the like.

[0164] The exogenous expression cassette may include cleavable sequences. Such cleavable sequences are sequences that are recognized by an entity capable of specifically cutting DNA, and include restriction sites, which are the target sequences for restriction enzymes or sequences for recognition by other DNA cleaving entities, such as nucleases, recombinases, ribozymes, or artificial constructs. At least one cleavable sequence may be included, but preferably two or more may be present. These cleavable sequences may be at any suitable point in the cassette, such that a selected portion of the cassette, or the entire cassette, can be selectively removed if desired. The cleavable sites may thus flank the part or may flank all of the genetic sequence that it may be desired to remove. The method may therefore also comprise removal of the expression cassette and / or the genetic material.

[0165] Direct delivery of DNA

[0166] The present disclosure provides methods for expressing, or for increasing expression of, a gene product, such as a transcription factor. The expression of the transcription factors may be increased by directly delivering one or more nucleic acids to a cell. Various methods are used todeliver DNA or any nucleic acid molecule directly into a cell. Various methods are disclosed herein and also known to a skilled artisan. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). The nucleic acid may be a DNA or RNA. The one or more nucleic acids may be delivered into cells using any suitable methods for nucleic acid delivery for transformation of a cell known in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome mediated transfection, by receptor -mediated transfection, by microprojectile bombardment, by agitation with silicon carbide fibers, by Agrobacterium - mediated transformation, and any combination of such methods.

[0167] Expression viral vectors

[0168] The present disclosure provides methods for expressing, or for increasing expression of a gene product, such as for example, a transcription factor. The expression of the transcription factors may be increased by delivering into a cell a nucleic acid comprising an open reading frame encoding one or more of the transcription factors, the transcription factor protein, or an activator of transcription of the open reading frame encoding one or more transcription factors to at least one cell. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). The nucleic acids may be introduced into the cell using a vector. A vector can be constructed through any standard techniques known in the art. Vectors include but are not limited to plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs) among other vectors known in molecular cloning techniques.

[0169] The vector may be a viral vector. The viral gene delivery system may be an RNA-based orDNA-based viral vector. For example, the viral vectors include retroviral vectors, lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), gammaretroviral vectors, adenoviral (Ad) vectors (including replication competent, replication deficient and gutless forms thereof), adeno-associated virus-derived (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Ep stein -Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors and Sendai virus vectors. The viral vector may be selected from a lentiviral vector, an adeno-associated virus vector or a Sendai virus vector. In a yet further embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is used at a high multiplicity of infection (MOI). A high MOI helps to ensure that more than onetranscription factor is introduced into the source cell. In one embodiment, the MOI is greater than 0.5, such as 1.0 or above.

[0170] Plasmids vectors

[0171] The present disclosure provides methods for expressing, or for increasing expression of, a transcription factor. The expression of the transcription factors may be increased by delivering cell a nucleic acid comprising an open reading frame encoding one or more of the transcription factors, the transcription factor protein, or an activator of transcription of the open reading frame encoding one or more transcription factors to at least one cell. The at least one cell may be a stem cell. The stem cell may be a pluripotent stem cell. The pluripotent stem cell may be an induced pluripotent stem cell (iPSC). The nucleic acids may be introduced into the cell using a plasmid.

[0172] The plasmid may be episomal. Episomal vectors are able to introduce large fragments of DNA into a cell but are maintained extra-chromosomally, replicated once per cell cycle, partitioned to daughter cells efficiently, and elicit substantially no immune response. In alternative embodiments, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, or a bovine papilloma virus (BPV)-based vector may be used.

[0173] Site-Specific Delivery

[0174] Any suitable technique for insertion of a nucleic acid sequence into a specific sequence may be used, and several are described in the art. Suitable techniques may include any method which introduces a break at the desired location and permits recombination of the vector into the gap. Thus, a crucial first step for targeted site-specific genomic modification is the creation of a double-strand DNA break (DSB) at the genomic locus to be modified. Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non-homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, that can be used to insert inducible cassettes.

[0175] Several techniques exist to allow customized site-specific generation of DSB in the genome. Many of these involve the use of customized endonucleases, such as zinc finger nucleases, TALENs or the clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas9) system.

[0176] Zinc finger nucleases are artificial enzymes which are generated by fusion of a zinc- finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain which must dimerize in order to cleave DNA. This means that two zinc finger nuclease monomers are required to allow dimerization of the Fokl domainsand to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest, is a tandem array of Cys2His2 zinc fingers, each of which recognizes three contiguous nucleotides in the target sequence. The two binding sites are separated by 5 -7 bp to allow optimal dimerization of the FokI domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break.

[0177] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factor / nucleases. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a repeated highly conserved 33 -34 amino acid sequence with divergent 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing appropriate residues at the two variable positions. TALENs are thus builtfrom arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of the modules, almost any sequence may be targeted. Again, the nuclease used may be FokI or a derivative thereof.

[0178] Three types of CRISPR mechanisms have been identified, of which type II is the most studied. The CRISPR / Cas9 system (type II) utilizes the Cas9 nuclease to make a double - stranded break in DNA at a site determined by a short guide RNA. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “proto spacer DNA” from previous exposures to foreign genetic elements. CRISPR spacers recognize and cut the exogenous genetic elements using RNA interference. The CRISPR immune response occurs through two steps: CRISPR -RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISPR repeat. Each crRNA molecule then hybridizes with a second RNA, known as the trans -activating CRISPR RNA (tracrRNA) and together these two eventually form a complex with the nuclease Cas9. The protospacer DNA encoded section of the crRNA directs Cas9 to cleave complementary target DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). This natural system has been engineered and exploited to introduce DSB breaks in specific sites in genomic DNA, amongst many other applications. In particular, the CRISPRtype II system from Streptococcus pyogenes may be used. At its simplest, the CRISPR / Cas9 system comprises two components that are delivered to the cell to provide genome editing: the Cas9 nuclease itself and a gRNA. The gRNA is a fusion of a customized, site -specific crRNA (directed to the target sequence) and a standardized tracrRNA.

[0179] Once a DSB has been made, a donor template with homology to the targeted locus is supplied; the DSB may be repaired by the homology -directed repair (HDR) pathway allowing for precise insertions to be made.

[0180] Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only. Cas9D10A may be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous.

[0181] The elements for making the double-strand DNA break may be introduced in one or more vectors, such as plasmids, for expression in the cell.

[0182] Thus, any method of making specific, targeted double strand breaks in the genome in order to insert a gene / inducible cassette may be used in the method of the invention. It may be preferred that the method for inserting the gene / inducible cassette utilizes any one or more of zinc finger nucleases, TALENs and / or CRISPR / Cas9 systems or any derivative thereof.

[0183] Once the DSB has been made by any appropriate means, the gene / inducible cassette for insertion may be supplied in any suitable fashion as described below. The gene / inducible cassette and associated genetic material form the donor DNA for repair of the DNA at the DSB and are inserted using standard cellular repair machinery / pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above.

[0184] Controlled Expression

[0185] In one embodiment, expression of the transcription factors is under controlled transcription. In this aspect of the invention, the transcription and translation (expression) of the transcription factors may be controlled within the cell. This permits overexpression of the transcription factor(s), if required.

[0186] An exogenous expression cassette carrying the transcription factors may comprise an externally inducible transcriptional regulatory element (i.e., an inducible promoter) for inducible expression of the transcription factors. The inducible expression cassette may be controlled by addition of an exogenous substance. Whatever culturing conditions are used, the exogenous substance will control expression of the genetic sequence within the inducible expressioncassette; and may either be supplied continuously and then withdrawn in order to induce transcription or supplied as transcription is required, dependent upon its mode of action.

[0187] Expression of the transcription factors described herein may be increased using a dual cassette expression system. This system targets genetic safe harbor (GSH) sites which provides a reduced risk of epigenetic silencing of the inserted genetic material.

[0188] A GSH site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighboring genes and expression of the inducible cassette minimizes interference with the endogenous transcription activity. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al., (2011)) These criteria include a site that is (i) 50 kb or more from the 5' end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA (miRNA), (iv) located outside a transcription unit and (v) located outside ultraconserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is thought that a suitable GSH will satisfy at least 2, 3, 4 or all of these criteria. Any suitable GSH site may be used in the method of the invention, on the basis that the site allows insertion of genetic material without deleterious effects to the cell and permits transcription of the inserted genetic material. Those skilled in the art may use these simplified criteria to identify a suitable GSH, and / or the more formal criteria set out above.

[0189] Specific insertion of genetic material into the particular GSH based upon customized site-specific generation of DNA double-strand breaks at the GSH may be achieved. The genetic material may then be introduced using any suitable mechanism, such as homologous recombination. Any method of making a specific DSB in the genome may be used, but preferred systems include CRISPR / Cas9 and modified versions thereof, zinc finger nucleases and the TALEN system.

[0190] One or more genetic sequences may be controllab ly transcribed from within the second and / or further GSH. Indeed, the inducible cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetic sequences (e.g., transcription factor sequences) which it is desired to insert into the GSH and the transcription of which be controllably induced. Therefore, the transcription factors required by the present invention may be included within the same cassette introduced into the second genetic safe harbor site. For example, the three or more transcription factors may be included in, for example, three mono-cistronic constructs, one mono-cistronic and one bi-cistronic construct or one tri-cistronic construct. It will be understood that similar combinations of constructs may be used to achieve higher orders of transcription factor expression.

[0191] Alternatively, if a combination of transcription factors is used, the individual transcription factors may be introduced into separate GSHs and / or under the control of different inducible promoters. Therefore, in one embodiment, the at least three or more transcription factors are introduced into separate GSHs. This may be achieved by utilizing three or more different GSH sites for the three or more transcription factors (i.e., wherein the transcription factors are introduced as mono-cistronic cassettes). Alternatively, this may be achieved by utilizing the fact that a GSH exists at the same genetic loci on both chromosomes of diploid organisms, e.g., introducing one transcription factor into the GSH on one chromosome and a different transcription factor into the same GSH on the other chromosome. This embodiment is advantageous if different expression levels or timing of expression of the transcription factors is desired. In one embodiment, the method comprises targeted insertion of the at least three or more transcription factors, each operably linked to an inducible promoter into a second, third and fourth genetic safe harbor site of the source cell. The inducible promoter may be the same of each transcription factor and therefore are all regulated by the transcriptional regulator protein.

[0192] A transcriptional regulator protein is a protein that binds to DNA, preferably sequence - specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor).

[0193] The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence. Transcriptional activator proteins bind to the response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression. Transcriptional repressor proteins bind to the response element and prevent gene expression.

[0194] Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins, phosphorylation, and / or methylation. The transcriptional regulator protein may be controlled by activation or deactivation.Any suitable transcriptional regulator protein maybe used, preferably one that may be activated or deactivated. It is preferred that an exogenous substance may be supplied to control the transcriptional regulator protein. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.

[0195] Tetracycline-Controlled Transcriptional Activation

[0196] Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline which is more stable). In this system, the transcriptional activator protein is tetracycline — responsive transcriptional activator protein (rtTa) or a derivative thereof. The rtTA protein is able to bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE). There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rtTA protein.

[0197] In a Tet-Off system, tetracycline or a derivative thereof binds rtTA and deactivates the rtTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE-controlled genes. This system was first described in Gossen et al., (1992).

[0198] The Tet-On system is composed of two components; (1) the constitutively expressed tetracycline — responsive transcriptional activator protein (rtTa) and the rtTa-sensitive inducible promoter (Tet Responsive Element, TRE). This may b e bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes. The use of this may be preferred in the method of the invention.

[0199] Thus, the transcriptional regulator protein may thus be tetracycline — responsive transcriptional activator protein (rtTa) protein, which can be activated or deactivated by the antibiotic tetracycline or one of its derivatives, which are supplied exogenously. If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the second GSH site includes the tetracycline response element (TRE). The exogenously supplied substance is the antibiotic tetracycline or one of its derivatives. Variants and modified rtTa proteins may also be used in the methods of the invention, these include Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-52).

[0200] The tetracycline response element (TRE) generally consists of 7 repeats of the 19 bp bacterial TetO sequence separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible since the minimal promoter can be any suitable promoter. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter inserted into the second GSH may thus comprise a TRE.

[0201] A modified system based upon tetracycline control is the T-REX System (Thermo-Fisher Scientific), in which the transcriptional regulator protein is a transcriptional repressor protein, TetR. The components of this system include (i) an inducible promoter comprising a stronghuman cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites, and a Tet repressor (TetR). In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the inducible promoter and prevent transcription from the promoter. Once added, tetracycline binds with high affinity to each Tet repressor homodimer rendering it unable to bind to the Tet operator. The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of expression. In this instance, the transcriptional regulator protein is TetR, and the inducible promoter comprises two TetO2 sites. The exogenously supplied substance is tetracycline or a derivative thereof. Other inducible expression systems are known and can be used in the method of the invention. These include the Complete Control Inducible system from Agilent Technologies. This is based upon the insect hormone ecdysone or its analogue ponasterone A (ponA) which can activate transcription in mammalian cells which are transfected with both the gene for the Drosophila melanogaster ecdysone receptor (EcR) and an inducible promoter comprising a binding site for the ecdysone receptor. The EcR is a member of the retinoid -X- receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR that binds to the ecdysone-responsive element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0202] Applications

[0203] The differentiated cells that can be produced using the methods described in the present disclosure may have various applications. They can be used for regenerative medicine, such as transplanting the cells into a recipient in need of a certain type of cell. They can be used for drug testing, both in cell culture as well as after transplantation. The cells may be used to deliver a product to a part of a body, for example, if they naturally produce or are engineered to produce and secrete the product.

[0204] Drug testing in the cells may use substances that are known or unknown to have a certain biological activity. The substances may be elements, compounds, or mixtures, whether natural or synthetic. The cells may be used to determine a desirable activity of a potential drug or conversely to determine undesirable effects of a substance or lack of such effects. The contacting of the substance with the cells may be in culture or in a human or animal body. The activity or side effects of the substance may be determined in vitro or in vivo, irrespective of where the contacting occurred.

[0205] The cells can be observed for effects on cell growth, differentiation, apoptosis, secreted products, expression of particular products, etc. The genome of these cells may be edited to match mutations found in patients with disease. Any type of assay known in the art for such changes may be used, including but not limited to immunological assays, morphologicalobservations, histochemical stains, reverse transcription polymerase chain reaction, protein blots, mass spectrometry, hybridization assays, electrophysiology, etc.

[0206] Stem cells of the present disclosure may be obtained from any source. One useful source may be human induced pluripotent stem cells. Mouse induced pluripotent stem cells and mouse embryonic stem cells may also be used, as well as such cells from other animals. The use of human embryonic stem cells may be regulated or ethically undesirable, but these maybe used as well. Differentiated cells may be identified by any property or set of properties that is characteristic or defining of that type of differentiated cell. For example, different cell types have a unique transcriptome. The transcriptome may be used as a means of matching and identifying an unknown cell type to a known cell type. The transcriptome may be used qualitatively or quantitatively. Similarly, a proteome may be used as a means of identifying an unknown differentiated cell type. Some cell types may be identifiable based on morphology, growth habit, secretion products, enzymatic activity, cellular function, and the like. Any means known in the art for identifying cells may be used.

[0207] Definitions

[0208] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. In this application, the use of the singular includes the plural unless specifically stated otherwise. It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” or “for example” are not limiting in any way. As such, use of the term “for example” as well as other forms, such as “for instance”, “e.g.,” and “such as,” and use of other similar words, is not limiting. Use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0209] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1 , 2, or 3 is equivalent to greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 3.

[0210] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1 .

[0211] “Overexpression” is expression at a level that is higher than the level that is expressed before induction from a gene that is expressed at a low, medium, or high level. An exogenous open reading frame (ORF) is typically an open reading frame that may differ from the similar gene or mRNA in the cell. For example, an exogenous ORF may be engineered to have a different control sequence or sequences, such as promoter, operator, enhancer, terminator, etc. It may be engineered to have no introns. It may be engineered to be fused to a second open reading frame to which it is not fused in the human genome.

[0212] As used herein, an “immune cell” refers to a cell associated with the immune system that is at any stage of development. Examples of an immune cell may include a hematopoietic progenitor cell comprising cells of the myeloid progenitor or mature cells. In some instances, examples of an immune cell may include a hematopoietic progenitor cell comprising cells of the lymphoid progenitor cells or mature cells. Such representative cells of the hematopoietic stem cell system are disclosed herein and / or known to a skilled artisan. For instance, the lymphoid cell can comprise B cells, T cells or natural killer cells. An example of a T cell may include a regulatory T cell. The immune cells produced by methods as described herein may be at least as functional as the immune cells produced by directed differentiation to date. Immune cells may express FOXP3. Immune cells may express CD45. Immune cells may express CD34. Immune cells may express CD4. Use of the term “immune-cell like” or “immune-like cell” as described herein, throughout, does convey the same thing, meaning each of these two can be used interchangeably.

[0213] In the present disclosure, ranges and amounts can be expressed as “about” a particular value or range. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a numberthatis nearto or approximately the number thatthe term precedes. Therefore, “about 5 pL” means “5 pL and also “about 5 pL.” In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub -ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at leastequal halves, thirds, quarters, fifths, tenths, and so forth. As a non -limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. Generally, the term “about” includes an amount that would be expected to be within experimental error. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters setforth in the present specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. Also, “about” can refer to an amount that is near the stated amount by 10% or less. In some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value. In general, the term “about,” as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass in one example variations of ± 20% or± 10%, in another example ± 5%, in another example ± 1%, and in yet another example ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method. Finally, and as stated above, whenever “about” is used in reference to a value, it can refer to a value that is similar, in context to the referenced value.

[0214] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0215] As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Therefore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth. Whenever the term “no more than,” “less than,” or “less than or equal to” precedesthe first numerical value in a series of two numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of thenumerical values in that series of numerical values. In some examples, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1 .

[0216] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1 , 2, or 3 is equivalent to greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 3.

[0217] Wherever the term “cells” disclosed in the present disclosure e.g., modified cells, expressed cells, engineered cells, genetically modified cells, genetically engineered cells, or in cell lines etcetera, shall be taken to mean non-naturally occurring cells or cell lines.Alternatively, the term “cells” in this disclosure, e.g., modified cells, expressed cells, engineered cells or cell lines (as understood by those of skill in the art) shall be taken to mean isolated cells and / or in addition such cells comprises isolated e.g., from any contaminants or removed from a composition, a cell culture, or cell mixtures, etcetera.

[0218] Whenever the term “deliver,” is disclosed in the methods of the present disclosure e.g. delivering to the one or more PSCs at least one nucleic acid (or polynucleotide) comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding one or more transcription factors, the term “deliver,” shall mean any way of inducing a cell to express the described polynucleotide, open reading frame, nucleic acid or transcription factors e.g. transfection with DNA and / or RNA, viral infection, and supplementing the cell culture media with the one or more transcription factor proteins.

[0219] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1 .

[0220] Whenever the term ooverexpression is used it refers to expression at level that is higher than the level that is expressed before induction from a gene that is expressed at a low, medium, or high level. For example, an exogenous open reading frame is typically an open reading frame that may differ from the similar endogenous gene or mRNA in the cell. For instance, it may be engineered to have a different control sequence or sequences, such as promoter, operator, enhancer, terminator, etc. In some cases, it may be engineered to have no introns. In some cases,it may be engineered to be fused to a second open reading frame to which it is not fused in the human genome.

[0221] Whenever the term a “immune cell or immune-like cell immune cell or immune-like cell cell” is used it refers to a cell that is related to cells of the immune system, immune cell, or immune-like cell. Immune cell or immune-like cell may refer to cells that have one or more characteristic of a mature immune cell. This term includes further cells with the capacity to engraft immune cell spaces when transplanted in vivo or ex -vivo. The immune cell or immune- like cell produced in vivo or ex -vivo methods may be at least as functional as immune cell or immune-like cell produced by directed differentiation to date. The engineered immune cell or immune-like cell may express any markers of the hematopoietic / immunology lineage cells and progenitor states that may be indicative of a differentiated iPSC. The expression of the immune cell or immune-like cell marker or gene (immune cell or immune-like cell marker or immune cell or immune-like cell gene) may be used interchangeably herein. The percentage, quantity, or amount of expression of any of such markers may be comparable or may not be comparable to that expressed by a comparator immune or immune-like cell, which may be a native cell, uninduced cell, a stem cell, or any other control cell etcetera, depending on the particular experiment.

[0222] Whenever the terms “individual(s)”, “subject(s)”, “host”, “recipient” and “patients)” are used they are used interchangeably herein and refer to any mammal including primates (e.g, a human primate and a non-human primate (e.g., a chimpanzee, a cynomologou, a monkey, a spider monkey, a macaque, e.g., a Rhesus), a rodent (a mouse, a rat, a hamster, a ferret, a woodchuck), pigs, as well as fetuses and live bom mammals. I...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered cell comprising a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise LM02 and SPI1.

2. The engineered cell of claim 1, wherein the one or more transcription factors further comprise TALI .

3. The engineered cell of any one of claims 1 or 2, wherein the one or more transcription factors further comprise GATA2.

4. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors further comprise HOXAIO.

5. An engineered cell comprising a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1.

6. The engineered cell of claim 5, wherein the one or more transcription factors comprise CEBPA.

7. The engineered cell of any one of claims 5 or 6, wherein the one or more transcription factors comprise GATA2.

8. The engineered cell of any one of claims 5-7, wherein the one or more transcription factors comprise SPI1.

9. The engineered cell of any one of claims 5-8, wherein the one or more transcription factors comprise at least one of LM02 or TALI .

10. The engineered cell of any one of the preceding claims, wherein the engineered cell is a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell derived from a human stem cell.

11. The engineered cell of any one of the preceding claims, wherein the engineered cell is an erythroid lineage cell, myeloid lineage cell, or granulocyte -macrophage lineage cell.

12. The method of any one of the preceding claims, wherein engineered cell is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

13. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors induces differentiation of the PSC into the engineered cell.

14. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less, 2 days or less, or 1 day or less.

15. The engineered cell of claim 12, wherein the one or more immune cell or immune -cell-like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, or PECAMl .

16. The engineered cell of any one of the preceding claims, wherein engineered cell expresses CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45.

17. The engineered cell of any one of the preceding claims, wherein the engineered cell is derived from a patient.

18. The engineered cell of any one of the preceding claims, wherein the engineered cell is not derived from a patient.

19. A pharmaceutical composition comprising the engineered cell of any one of the preceding claims.

20. The pharmaceutical composition of claim 17, further comprising an excipient.21 . A method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, the method comprising administering the engineered cell of any one of claims 1-16 to the subject in need thereof, thereby treating the disease associated with dysfunctional bone marrow condition.

22. The method of claim 19, wherein the disease is multiple myeloma, Hodgkin lymphoma, nonHodgkin lymphoma, acute myeloid leukemia, or acute lymphocytic leukemia.

23. The method of claim 19, wherein the disease is sickle cell anemia, Multiple Sclerosis, systemic lupus erythematosus, or another autoimmune disorder.

24. A method of generating an engineered cell, wherein the method comprises: a. contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acid molecules encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frameencodingthe one or more transcription factors, wherein the one or more transcription factors comprise LM02 and SPI1 ; b. inducing expression of the expression cassette, thereby generating an engineered cell.

25. The method of claim 22, wherein the one or more transcription factors further comprise TALI .

26. The method of any one of claims 22 or 23, wherein the one or more transcription factors further comprise GATA2.

27. The method of any one of claims 22-24, wherein the one or more transcription factors further comprise HOXAIO.

28. A method of generating an engineered cell, wherein the method comprises: a. contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acid molecules encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1 ; b. inducing expression of the expression cassette, thereby generating an engineered cell.

29. The method of claim 26, wherein the one or more transcription factors comprise CEBPA.

30. The method of any one of claims 26 or 27, wherein the one or more transcription factors comprise GATA2.31 . The method of any one of claims 26-28, wherein the one or more transcription factors comprise SPI1.

32. The method of any one of claims 26-29, wherein the one or more transcription factors comprise at least one of LM02 or TALI .

33. The method of any one of the preceding claims, wherein the engineered cell is a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, an embryonic stem cell derived from a human stem cell.

34. The engineered cell of any one of the preceding claims, wherein the engineered cell is an erythroid lineage cell, myeloid lineage cell, or granulocyte -macrophage lineage cell.

35. The method of any one of the preceding claims, wherein engineered cell is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

36. The method of any one of the preceding claims, wherein inducing expression of the expression cassette induces expression of the one or more transcription factors, wherein the one or more transcription factors induce differentiation of the PSC into the engineered cell.

37. The method of any one of the preceding claims, wherein the engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less, 2 days or less, or 1 day or less.

38. The method of claim 33, wherein the one or more immune cell or immune-cell-like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, or PECAMl .

39. The method of any one of the preceding claims, wherein engineered cell expresses CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45.

40. The method of any one of the preceding claims, wherein engineered cell does not express TRA-1-60.41 . The method of any one of the preceding claims, wherein the PSC is derived from a patient.

42. The method of any one of the preceding claims, wherein the PSC is not derived from a patient.

43. The method of any one of the preceding claims, wherein the engineered cell is an adherent cell.

44. The method of any one of the preceding claims, wherein the engineered cell is a suspension cell.

45. The method of any one of the preceding claims, further comprising generating a population of engineered cells.

46. The method of claim 41, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the population of engineered cells express CD34.

47. The method of any one of claims 41 or 42, wherein at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the population of engineered cells express CD43.

48. The method of any one of claims 41-43, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of engineered cells express CD45.

49. The method of any one of claims 41-44, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD43.

50. The method of any one of claims 41-45, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45.

51. The method of any one of claims 41-46, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45.

52. The method of any one of claims 41-47, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45 -positive cells of the population of engineered cells express CD34 and CD43.

53. The method of any one of claims 41-48, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45.

54. The method of any one of claims 41-49, wherein atmost 10%, 9%, 80%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% of the population of engineered cells express TRA-1-60.

55. The method of any one of claims 41 -50, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02.

56. The method of any one of claims 41 -51, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1.

57. The method of any one of claims 41 -52, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI .

58. The method of any one of claims 41 -53, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2.

59. The method of any one of claims 41 -54, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2.

60. The method of any one of claims 41 -55, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express CEBPA.

61. The method of any one of claims 41 -56, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HOXA10.

62. The method of any one of the preceding claims, wherein the population of engineered cells comprises erythroid lineage cells, myeloid lineage cells, granulocyte -macrophage lineage cells, or any combination thereof.

63. The method of any one of the preceding claims, wherein the myeloid lineage cells comprise granulocyte lineage cells, erythrocyte lineage cells, monocyte lineage cells, megakaryocyte lineage cells, or any combination thereof.

64. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5% erythroid lineage cells.

65. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% erythroid lineage cells.

66. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 50% erythroid lineage cells.

67. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5% myeloid lineage cells.

68. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells.

69. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 30% myeloid lineage cells.

70. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 40% granulocyte-macrophage lineage cells.

71. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte-macrophage lineage cells.

72. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 90% granulocyte-macrophage lineage cells.

73. The method of any one of the preceding claims, wherein the erythroid lineage cells express CD71, CD235a, or both.

74. The method of any one of the preceding claims, wherein the myeloid lineage cells express CD13, CD14, CD15, or any combination thereof.

75. The method of any one of the preceding claims, wherein the myeloid lineage cells express CD14 and CD15.

76. A method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, the method comprising: administering an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise LM02 and SPI1.

77. The method of claim 58, wherein the one or more transcription factors further comprise TALI .

78. The method of any one of claims 58 or 59, wherein the one or more transcription factors further comprise GATA2.

79. The method of any one of claims 58-60, wherein the one or more transcription factors further comprise HOXAIO.

80. A method of treating a disease associated with dysfunctional bone marrow in a subject in need thereof, the method comprising: administering an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encodingthe one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise NFE2 and at least one of CEBPA, GATA2, or SPI1.

81. The method of claim 62, wherein the one or more transcription factors comprise CEBPA.

82. The method of any one of claims 62 or 63, wherein the one or more transcription factors comprise GATA2.

83. The method of any one of claims 62-64, wherein the one or more transcription factors comprise SPI1.

84. The method of any one of claims 62-65, wherein the one or more transcription factors comprise at least one of LM02 or TALI .

85. The method of any one of the preceding claims, wherein the engineered cell is a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, an embryonic stem cell from a human stem cell.

86. The engineered cell of any one of the preceding claims, wherein the engineered cell is an erythroid lineage cell, myeloid lineage cell, or granulocyte-macrophage lineage cell.

87. The method of any one of the preceding claims, wherein engineered cell is a granulocyte lineage cell, erythrocyte lineage cell, monocyte lineage cell, or megakaryocyte lineage cell.

88. The method of any one of the preceding claims, wherein inducing expression of the expression cassette induces expression of the one or more transcription factors, wherein the one or more transcription factors induce differentiation of the PSC into the engineered cell.

89. The method of any one of the preceding claims, wherein the engineered cell expresses one or more immune cell or immune-cell-like markers in 3 days or less, 2 days or less, or 1 day or less.

90. The method of claim 69 wherein the one or more immune cell or immune-cell-like markers comprise CD34, CD43, CD45, THY1, ITGA6, CD33, or PECAMl .

91. The method of any one of the preceding claims, wherein engineered cell expresses CD34 and CD43; CD34 and CD45; CD43 and CD45; or CD34, CD43, and CD45.

92. The method of any one of the preceding claims, wherein the PSC is derived from a patient.

93. The method of any one of the preceding claims, wherein the PSC is not derived from a patient.

94. The method of any one of the preceding claims, wherein the disease is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or acute lymphocytic leukemia.

95. The method of any one of the preceding claims, wherein the disease is sickle cell anemia, Multiple Sclerosis, systemic lupus erythematosus, or another autoimmune disorder.

96. The method of any one of the preceding claims, wherein the engineered cell is not derived from the subject in need thereof.

97. The method of any one of the preceding claims, wherein the engineered cell is derived from the subject in need thereof.

98. The method of any one of the preceding claims, further comprising administering a population of engineered cells.

99. The method of claim 78, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the population of engineered cells express CD34.

100. The method of any one of claims 78 or 79, wherein at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or75% of the population of engineered cells express CD43.

101. The method of any one of claims 78-80, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the population of engineered cells express CD45.

102. The method of any one of claims 78-81, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD43.

103. The method of any one of claims 78-82, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34 and CD45.

104. The method of any one of claims 78-83, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD43 and CD45.

105. The method of any one of claims 78-84, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of CD45 -positive cells of the population of engineered cells express CD34 and CD43.

106. The method of any one of claims 78-85, wherein atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the population of engineered cells express CD34, CD43, and CD45.

107. The method of any one of claims 78-86, wherein at most 10%, 9%, 80%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or 0% of the population of engineered cells express TRA-1-60.

108. The method of any one of claims 78-87, wherein at least 5%, 6%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, or 80% of the population of engineered cells express LM02.

109. The method of any one of claims 78-88, wherein at least 5%, 6%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, or 80% of the population of engineered cells express SPI1.

110. The method of any one of claims 78-89, wherein at least 5%, 6%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, or 80% of the population of engineered cells express TALI .

111. The method of any one of claims 78-90, wherein at least 5%, 6%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, or 80% of the population of engineered cells express GATA2.

112. The method of any one of claims 78-91, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express NFE2.

113. The method of any one of claims 78-92, wherein atleast 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express CEBPA.

114. The method of any one of claims 78-93, wherein at least 5%, 6%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the population of engineered cells express HOXA10.

115. The method of any one of the preceding claims, wherein the population of engineered cells comprises erythroid lineage cells, myeloid lineage cells, granulocyte -macrophage lineage cells, or any combination thereof.

116. The method of any one of the preceding claims, wherein the myeloid lineage cells comprise granulocyte lineage cells, erythrocyte lineage cells, monocyte lineage cells, megakaryocyte lineage cells, or any combination thereof.

117. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5% erythroid lineage cells.

118. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% erythroid lineage cells.

119. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 50% erythroid lineage cells.

120. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5% myeloid lineage cells.

121. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 5%, 10%, 15%, 20%, 25%, or 30% myeloid lineage cells.

122. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 30% myeloid lineage cells.

123. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 40% granulocyte-macrophage lineage cells.

124. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% granulocyte-macrophage lineage cells.

125. The method of any one of the preceding claims, wherein the population of engineered cells comprises at least 90% granulocyte-macrophage lineage cells.

126. The method of any one of the preceding claims, wherein the erythroid lineage cells express CD71, CD235a, or both.

127. The method of any one of the preceding claims, wherein the myeloid lineage cells express CD13, CD 14, CD 15, or any combination thereof.

128. The method of any one of the preceding claims, wherein the myeloid lineage cells express CD 14 and CD15.-ISO-

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