Scalable expansion of CD71+ erythroid progenitor cells for cell therapy

Abstract

Provided herein is a scalable method for producing CD71+ erythroid progenitor cells for use in cell therapy, such as immunomodulatory therapy, in which T cell response is suppressed.This method comprises: induced fetal liver / yolk sac organoid from pluripotent stem cells (for example, induced pluripotent stem cells) by GATA6, and then co-culture with the CD34+ cells from, for example, umbilical cord blood or bone marrow, and the organoid, optionally in the presence of thrombopoietin, stem cell factor, and / or FLT3 ligand.Also provided herein is a bioreactor for producing CD71+ erythroid progenitor cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 377,783, filed September 30, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] CD71+ erythrocytes (CECs) are normally present in the bone marrow and occur at a rate of 2 × 10 per day. 11 CECs are progenitor and precursor cells that generate more than 100 oxygen-carrying red blood cells (RBCs). CECs regulate a variety of phenomena, including fetal tolerance, immune responses in cancer patients, systemic inflammation in colitis, and antiviral responses in human immunodeficiency virus (HIV) infection, as well as SARS-CoV-2-induced disease (COVID-19). CD45+ CECs induced by advanced tumors inhibit the proliferation of CD8+ and CD4+ T cells and impair antimicrobial immunity. CECs from neonates and patients with advanced cancer possess robust immunosuppressive properties. Thus, we demonstrate the role of CECs in regulating immune responses in both mice and humans, and that their immunomodulatory properties are robust but transient and disappear during their maturation. (See, for example, Grzywa TM, et al. Potent but transient immunosuppression of T-cells is a general feature of CD71+ erythroid cells. Commun Biol. 2021 Dec 10;4(1):1384. doi: 10.1038 / s42003-021-02914-4.) Therefore, CD71 + The cells are expected to have great potential in immunomodulation, particularly in immunosuppressive therapy, for example, in the treatment of inflammatory diseases and transplant immunosuppression.

[0003] While showing great potential, there is a lack of robust and scalable methods to produce sufficient quantities of CD71+ cells. Summary of the Invention

[0004] Provided herein is a scalable method for the inexpensive production of CD71+ cells with immunosuppressive activity. Also provided herein is a bioreactor device for culturing and producing CD71+ cells with immunosuppressive activity.

[0005] According to a first aspect or embodiment, CD71 + A method for preparing a population of cells is provided, which comprises culturing pluripotent stem cells to differentiate into a fetal liver / yolk sac phenotype, including enhanced expression of erythroid markers CD36, CD47, EPOR (erythropoietin receptor), and TGFβ1 (transforming growth factor β1); + Hematopoietic stem and progenitor cells (HPSCs) were co-cultured with differentiated pluripotent stem cells to express HPSC-derived CD71 + (CD71 HI ) producing an expanded culture population of cells.

[0006] According to a second aspect or embodiment, there is provided a cell culture device comprising a cell culture vessel (plate, flask, bioreactor, etc.) comprising a cell culture medium and a co-culture of cells having a fetal liver / yolk sac phenotype and CD34+ hematopoietic stem and progenitor cells.

[0007] According to a further aspect or embodiment, there is provided a method of immunosuppression in a patient having an inflammatory disease or allogeneic or xenogeneic tissue transplant in need thereof, the method comprising administering to the patient an immunosuppressive number of CD71+ or CD71HI cells produced by the method of the first aspect or embodiment, thereby reducing a cell-mediated response in the patient.

[0008] The following numbered items outline various exemplary embodiments of the present invention.

[0009] Section 1 CD71 +A method for preparing a population of cells, comprising culturing pluripotent stem cells to differentiate into a fetal liver / yolk sac phenotype, such as enhanced expression of erythroid markers CD36, CD47, EPOR (erythropoietin receptor) and TGFβ1 (transforming growth factor β1); and + Hematopoietic stem and progenitor cells (HPSCs) were co-cultured with differentiated pluripotent stem cells to express HPSC-derived CD71 + (CD71 HI ) producing an expanded culture population of cells.

[0010] Paragraph 2: The method described in paragraph 1, wherein the pluripotent stem cells are differentiated into a fetal liver / yolk sac phenotype by expressing a gene for expressing GATA6 protein in the pluripotent stem cells or by introducing GATA6 protein or mRNA encoding GATA6 protein into the pluripotent stem cells.

[0011] Paragraph 3. The method according to paragraph 2, wherein the pluripotent stem cells are differentiated into a fetal liver / yolk sac phenotype by expressing a gene for expressing GATA6 protein in the pluripotent stem cells.

[0012] Paragraph 4. The method of paragraph 2, wherein the gene for expressing the GATA6 protein is inducible in the pluripotent stem cells.

[0013] Paragraph 5. The method of paragraph 4, wherein the gene for expressing GATA6 protein in the pluripotent stem cells is induced during the first 3 or 4 days of culture of the pluripotent stem cells.

[0014] Paragraph 6: The method according to paragraph 4 or 5, wherein the gene for expressing GATA6 is doxycycline-inducible.

[0015] Paragraph 7. The method according to paragraph 2, comprising introducing a GATA6 protein or mRNA encoding a GATA6 protein into the pluripotent stem cells.

[0016] Paragraph 8. The method of paragraph 7, wherein the GATA6 protein or mRNA encoding the GATA6 protein is introduced into the pluripotent stem cells during the first 3 or 4 days of culture of the pluripotent stem cells.

[0017] Item 9. The method according to any one of items 1 to 8, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0018] Clause 10. The method of any one of clauses 1.1 to 4, wherein the pluripotent stem cells are cultured on a basement membrane matrix, such as MATRIGEL.

[0019] Clause 11. The method of any one of clauses 1 to 10, wherein the HPSCs are obtained from umbilical cord blood or bone marrow, eg, from mobilized peripheral blood.

[0020] 12. The hPSCs are CD71 + 12. The method of any one of paragraphs 1 to 11, wherein the cells are obtained from the patient to be administered.

[0021] Clause 13. The method of any one of clauses 1 to 12, wherein the HPSCs are co-cultured with differentiated pluripotent stem cells in a medium comprising a factor selected from one or more of thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF.

[0022] Clause 14. The method of clause 13, wherein the medium comprises one or more of thrombopoietin, stem cell factor and / or FLT3 ligand.

[0023] Clause 15. The method of clause 13 or clause 14, wherein the culture medium comprising the factors is at least partially replaced daily.

[0024] Clause 16. The method of any one of clauses 1 to 15, wherein the co-cultivation is carried out in a bioreactor, and optionally a continuous flow bioreactor.

[0025] Clause 17. The method of any one of clauses 1 to 16, further comprising separating expanded HPSC cells comprising CD71+ cells from the differentiated pluripotent stem cells after co-culturing the HPSCs with the differentiated pluripotent stem cells for at least 6 days, and recovering the separated expanded HPSC cells comprising CD71+ cells.

[0026] Clause 18. The method of clause 17, further comprising enriching the CD71+ cells of the separated cells of expanded HPSCs comprising CD71+ cells by affinity purification.

[0027] Clause 19. The method of clause 18, wherein the affinity purification method is fluorescence activated cell sorting, magnetic bead separation, panning, or column purification.

[0028] Clause 20. The method of any one of clauses 1 to 19, wherein the stem cells and / or HPSCs are human.

[0029] Item 21. A cell culture device comprising a cell culture vessel (plate, flask, bioreactor, etc.), said cell culture vessel containing cell culture medium and a culture medium for cells having a fetal liver / yolk sac phenotype and CD34 + A cell culture device comprising a cell co-culture of hematopoietic stem cells and progenitor cells.

[0030] 22. Cells having the fetal liver / yolk sac phenotype and CD34 + 22. The device of paragraph 21, wherein the hematopoietic stem and progenitor cells are human.

[0031] Clause 23. The device of clause 20 or clause 21, wherein the cells having a fetal liver / yolk sac phenotype comprise an inducible gene for expressing a GATA6 protein.

[0032] Clause 24. The device of clause 23, wherein the inducible gene is doxycycline-inducible.

[0033] Item 25: The device according to any one of items 20 to 23, wherein the medium comprises a factor selected from one or more of thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF.

[0034] Clause 26. The device of clause 24, wherein the medium comprises thrombopoietin, stem cell factor and / or FLT3 ligand.

[0035] Clause 27. The apparatus of any one of clauses 20 to 25, wherein the bioreactor is a continuous flow bioreactor.

[0036] 28. The method of claim 27, wherein the cells of said co-culture are at least 10%, at least 15%, or at least 20% CD34+, excluding cells having a fetal liver / yolk sac phenotype. - CD71 + 27. The device according to any one of items 20 to 26, comprising cells.

[0037] 29. The method of claim 29, wherein the cells of said co-culture are at least 70%, at least 75%, or at least 80% CD71 phenotype-dependent, excluding cells having a fetal liver / yolk sac phenotype. + 27. The device according to any one of items 20 to 26, comprising cells.

[0038] Item 30. A method for immunosuppression in a patient with an inflammatory disease or an allogeneic or xenogeneic tissue transplant in need thereof, comprising administering to said patient an immunosuppressive dose of CD71 produced by the method of any one of items 1 to 19. + or CD71 HI administering cells, thereby reducing a cell-mediated response in said patient. [Brief explanation of the drawings]

[0039] [Figure 1A] FIG. 1A shows a flow chart illustrating a method for producing CD71+ (eg, CD17HI) cells. [Figure 1B] FIG. 1B shows a schematic representation of a portion of the surface of an exemplary illustrative bioreactor apparatus as described herein. [Figure 2A] 2A-2C show the mRNA (cDNA) sequence (FIGS. 2A-2B, contiguous) and protein sequence (FIG. 2C) of an exemplary human GATA6 gene. [Figure 2B] 2A-2C show the mRNA (cDNA) sequence (FIGS. 2A-2B, contiguous) and protein sequence (FIG. 2C) of an exemplary human GATA6 gene. [Figure 2C] 2A-2C show the mRNA (cDNA) sequence (FIGS. 2A-2B, contiguous) and protein sequence (FIG. 2C) of an exemplary human GATA6 gene. [Figure 3A] 3A and 3B show FACS results and a graph, respectively, demonstrating the expansion of CD71-High cells (CD71+) in FeLO / CD34+ co-cultures compared to CD34+ monocultures, as described in Example 2. [Figure 3B] 3A and 3B show FACS results and a graph, respectively, demonstrating the expansion of CD71-High cells (CD71+) in FeLO / CD34+ co-cultures compared to CD34+ monocultures, as described in Example 2. [Figure 4] Figure 4 shows a graph demonstrating that the relative expansion of CD71 cells in CD34 co-cultures is highest in early FeLO (day 5) compared to late FeLO (day 10) and designer liver organoids (DesLO) engineered for a mature liver phenotype. Y-axis = CD33, X-axis = CD71. [Figure 5] FIG. 5 shows photomicrographs of monocultures and FeLO cocultures stained with Wright-Giemsa stain. [Figure 6] FIG. 6 shows a graph depicting the accumulation of CD71+ cells in co-culture with FeLO versus monoculture. [Figure 7] Figure 7 is a plot showing single-cell RNA sequencing and the expression levels of erythroid markers in erythroid cluster 4 generated after culture with organoids. [Figure 8] FIG. 8 shows a graph depicting T cell proliferation in the presence of CD71+ cells prepared as described in the Examples. [Figure 9] Figure 9. Flow cytometry gating strategy and scatter plot showing that addition of CD71+ cells to the culture enhances retention of CFSE staining. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description Except in the illustrative examples, or unless otherwise noted, the use of numerical values ​​in the various ranges specified in this application is stated as approximations, as if the word "about" were used to refer to both the minimum and maximum values ​​within the stated range. In this manner, small variations above and below the stated ranges can be used to achieve substantially the same results as values ​​within the range. Also, unless otherwise noted, the disclosure of ranges is intended as a continuous range, including every value between the minimum and maximum values.

[0041] As used herein, "a" and "an" refer to one or more.

[0042] The term "comprising" is open-ended and may be synonymous with "including," "containing," or "characterized by." The term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The term "consisting of" excludes elements, steps, or ingredients not specified in the claim. As used herein, embodiments that "comprise" one or more specified elements or steps also include, but are not limited to, embodiments that "consist essentially of" and "consist of" the recited elements or steps. For definitions provided herein, these definitions refer to forms, synonyms, and grammatical variations of those words or phrases.

[0043] As used herein, the term "patient" or "subject" refers to a member of the animal kingdom, including but not limited to humans, and "mammal" refers to all mammals, including but not limited to humans.

[0044] As used herein, "treatment" of a wound or defect means the administration of a composition, device, or structure to a patient by any suitable regimen, procedure, and / or route of administration for the purpose of achieving a desired clinical / medical endpoint, including, for example, attracting progenitor cells, healing the wound, correcting the defect, inducing neurite outgrowth, or nerve repair.

[0045] As used herein, the term "cell" refers to any type of cell of any animal, including, but not limited to, rat, mouse, monkey, and human. For example, but not limited to, a cell can be a progenitor cell, e.g., a pluripotent cell, including a stem cell, an induced pluripotent stem cell, a multipotent cell, or a differentiated cell, e.g., an endothelial cell and a smooth muscle cell. "Cell" also includes cell populations, e.g., a cell population produced by culturing CD34+ HPSCs. In certain embodiments, cells for medical treatment can be obtained from the patient for autologous treatment, or from another donor for allogeneic treatment, or from a xenogeneic source.

[0046] Stem cells, for example, cell populations comprising pluripotent stem cells and hematopoietic stem cells, can be used in the methods described herein. Stem cells are defined as totipotent, pluripotent, or multipotent cells of a multicellular organism, which can differentiate to give rise to other specific types of cells. Stem cells are generally capable of infinitely propagating the same type of cells in cell culture. Depending on their source, stem cells can be classified as totipotent, pluripotent, or multipotent. Stem cells can be engineered or induced to produce GATA6 and / or other factors, for example, by introducing genes for the expression of the factors into the cells. Stem cells can be induced to differentiate into, for example, fetal liver / yolk sac phenotypes by introducing GATA6 or genes for expressing GATA6, as described herein. Although stem cells can be obtained from many tissue sources, non-limiting examples of tissue sources for cell populations comprising stem cells include umbilical cord stem cells (including umbilical cord blood, umbilical cord matrix, Wharton's gel, etc. (see, e.g., Weiss ML, et al., Stem cells in the umbilical cord. Stem Cell Rev. 2006;2(2):155-162)), adipose tissue, bone marrow, perivascular cells, e.g., pericytes (see, e.g., Avolio E, Alvino VV, Ghorbel MT, Campagnolo P. Perivascular cells and tissue engineering: Current applications and untapped potential. Pharmacol Ther. 2017;171:83-92), and induced pluripotent stem cells (iPSCs, see, e.g., Yamanaka S. Induced pluripotent stem cells: past, present, and future. Cell Stem Cell. 2012 Jun 14;10(6):678-684 and Shi Y, et al. Induced (See Pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017 Feb;16(2):115-130).The cell population useful for the present method and device can be enriched for stem cells by any useful method, including cell separation and sorting techniques and cell culture techniques that are widely known in the stem cell field.Methods for producing useful iPSCs are also widely known.Cell populations comprising stem cells can be, for example, cryopreserved in the patient's (autologous) tissue or in a tissue bank where stem cells are stored for later recovery.

[0047] Fetal liver / yolk sac organoids (niches) can be generated from induced pluripotent stem cells (iPSCs) genetically engineered to contain the doxycycline-inducible GATA6 gene. When iPSCs are cultured in doxycycline-containing pluripotency medium, the cells self-organize and dedifferentiate into mesodermal, endodermal, and ectodermal lineages. The medium is then switched to basic differentiation medium. This tissue further differentiates to acquire a fetal liver / yolk sac phenotype genetic signature, express a range of hematopoietic cytokines, and contain multiple cell populations derived from different lineages (endothelial, pericyte, and hepatic) known to be present in the fetal liver. CD34+ hematopoietic stem and progenitor cells (HPSCs), such as those derived from umbilical cord blood, can be seeded and cocultured with the synthetic fetal liver / yolk sac tissue. HSPCs expand in number during coculture and can be extracted at any time during the coculture. A prominent subpopulation of co-cultured HSPCs is the CD71+ population, which expands in the fetal liver niche, whereas CD34+ HSPCs do not expand when cultured alone.

[0048] CD71 + An exemplary method for producing (e.g., CD17HI) CEC cells is shown in the flowchart of Figure 1A. In the first step 10, stem cells are cultured. Stem cells, e.g., human induced pluripotent stem cells (hiPSCs), can be engineered to express GATA6, e.g., human GATA6. Expression of GATA6 can be under the control of an inducible promoter, such as a doxycycline-inducible promoter (e.g., TET-ON). Stem cells are cultured at a density of 20,000-40,000 cells / cm. 2The cells can be cultured in stem cell growth medium on a surface such as a tissue culture plate, flask, or bioreactor until they reach a density of approximately 1000 ng / cm. The surface on which the cells are cultured can comprise an hESC-qualified growth substrate, e.g., an extracellular matrix material such as hESC-qualified MATRIGEL or a basement membrane. HI or CD71 + Cells and CD71 LO or CD71 - The determination of cells is somewhat arbitrary depending on the applicable cutoff and staining / non-staining, but CD71 HI or CD71 + CD71 cells LO or CD71 - To differentiate from the cells, they were co-cultured (CD71 HI or CD71 + ) cells and monocultured with (CD71 LO or CD71 - Comparisons can be made between CD71 and CD81 cells. HI or CD71 + Cells can be identified by, for example, FACS or bead assays, by CD71 LO or CD71 - This can produce a staining signal at least 50-100 times greater than that of the cells.

[0049] Once cell culture is initiated, GATA6 expression is induced and maintained until fetal liver / yolk sac organoid tissue is formed, which can take 7–14 days. Alternatively, GATA6 mRNA, e.g., mRNA in solid lipid nanoparticles or lipid vesicles, can be added to the cells (see, e.g., Melamed JR, et al. Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery. J Control Release. 2022 Jan;341:206-214; Hou X, et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078-1094; and Yang L, et al. Recent Advances in Lipid Nanoparticles for Delivery of mRNA. Pharmaceutics. 2022 Dec 1;14(12):2682). GATA6 can be added directly to cells as a protein, for example, in vesicles for endocytosis, or by other protein delivery methods (see, for example, Ray M, et al. Intracellular delivery of proteins by nanocarriers. Nanomedicine (Lond). 2017 Apr;12(8):941-952). Then, CD34+ cells, such as CD34+ umbilical cord blood, bone marrow, or mobilized peripheral blood, are added to fetal liver / yolk sac organoids and co-cultured with, for example, an appropriate amount of stem cell factor (SCF, for example, human recombinant SCF), thrombopoietin (TPO, for example, human recombinant TPO), and / or FMS-like tyrosine kinase 3 ligand (FLT3LG, for example, human recombinant FLT3LG), which are effective for producing CD71+ cells. All cytokines are commercially available, and / or their amino acid sequences and cDNA sequences are widely known, and those skilled in the art can synthesize or obtain sufficient amounts of each cytokine.The amount of cytokine added is sufficient and effective when co-cultured with fetal liver / yolk sac organoid as described herein, to induce CD34+ HPSC to produce CD71+ cells.It is noteworthy that compared with the sole culture of CD34+ HPSC, the amount of cytokine required is very small, and furthermore, for example, less than 500ng / μL, at least 25ng / μL, for example, for SCF and FLT3LG less than 200ng / μL, for TPO less than 100ng / μL, for example, but not limited to, 100ng / μL rhSCF and rhFLT3LG and 50ng / μL rhTPO, which further reduces the cost of the claimed method.

[0050] Fetal liver / yolk sac organoid is the synthetic tissue prepared by the method described herein.Pluripotent stem cell is cultured in the presence of the GATA6 protein, for example, that is expressed from transgene, that is translated from exogenously introduced mRNA, or that is exogenously introduced as protein.GATA6 protein and optionally introduced cytokines, for example, one or more of thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6 and GM-CSF, for example, thrombopoietin, stem cell factor and FLT3 ligand, make pluripotent stem cell differentiate and form the organoid that is phenotypically identified as fetal liver or yolk sac (the developmental precursor to fetal liver), and can have mesoderm, endoderm and ectoderm layers, elements or parts. After pluripotent stem cells are at least partially differentiated by GATA6 protein, CD34+ HPSCs are added and cultured in the presence of one or more cytokines, such as thrombopoietin, stem cell factor, and / or FLT3 ligand, for a period of time sufficient to produce CD71+ cells, such as 5, 6, 7, 8, 9, or 10 days, for example 6 or 7 days, at which point CD71+ cells can be washed or aspirated.This process can be repeated by seeding additional CD34+ HPSCs on organoids and culturing them.Pluripotent stem cells do not necessarily have to be autologous to the patient who is treated with CD71+ cell product, but it is preferable that CD34+ cells are autologous (the patient's own cells).

[0051] Hematopoietic stem and progenitor cells (HSPCs) are a population of cells in the bone marrow capable of self-renewal and multilineage differentiation into mature blood cell types (see, e.g., Dzierzak E, Bigas A. Blood Development: Hematopoietic Stem Cell Dependence and Independence. Cell Stem Cell. 2018 May 3;22(5):639-651). CD34+ HSPCs can be isolated from mobilized peripheral blood (see, e.g., Pelus LM, Broxmeyer HE. Peripheral blood stem cell mobilization; a look ahead. Curr Stem Cell Rep. 2018 Dec;4(4):273-281), bone marrow, and umbilical cord blood. HSPCs can be obtained from the patient's umbilical cord blood (if available) or from the patient's bone marrow cells, such as mobilized bone marrow cells available in the patient's blood.

[0052] A "cell growth matrix" is a mesh, matrix, particle, surface, hydrogel, porous structure, or other material on or within which cells can be established, maintained in a viable state, and often proliferated in the presence of an appropriate cell growth medium. A cell growth matrix can be made from a single composition or multiple compositions, such as synthetic and / or natural polymer compositions. A cell growth matrix can comprise cells and / or therapeutic agents. A "scaffold-free cell growth matrix" is a natural product of cells and tissues that does not include synthetic polymer compositions. In the context of this invention and disclosure, a scaffold-free cell growth matrix can be a cell-secreted basement membrane, such as MATRIGEL.

[0053] FIG. 1B schematically illustrates a portion of an exemplary bioreactor device 100 having a bioreactor substrate surface 110 with an optional basement membrane 120, such as MATRIGEL, deposited thereon; a layer of differentiated fetal liver / yolk sac organoid cells 130, which may be a continuous or discontinuous layer depending on the growth and differentiation pattern; a layer of CD34+ cells and / or CD71+ cells 140 on top of the organoid cells 130; and an appropriate culture medium 150. The bioreactor may be a dish, multiwell plate, flask, tube, container, or any other suitable configuration for a bioreactor; FIG. 1B illustrates the culture-facing surface of the bioreactor device. As will be appreciated by those skilled in the art, any part of the culture method can be automated using appropriate robotics and fluidics options. Cells, including a population of CD71+ cells, can be washed, then filtered or centrifuged, and recovered by affinity purification methods, such as, but not limited to, FACS or bead-based sorting (e.g., magnetic beads).

[0054] A composition is "free of" a recited component if the component is not present in the composition or is present in a substantial amount that does not interfere with or significantly interfere with the intended use and function of the composition.

[0055] A "cell population" refers to two or more cells. The cells in a cell population can be the same as an enriched, purified, or clonally expanded population of stem cells or CD71+ cells. The cells in a cell population can comprise different cell types, including not only stem cells but also differentiated cells, such as cells obtained directly from a tissue sample.

[0056] CD71+ cells can be used as an immunosuppressant. Thus, the CD71+ cells prepared by the methods described herein can be prepared from CD34+ cells, such as bone marrow stem cells, derived from the patient to be treated, and therefore the CD71+ cells are autologous. In one example, the patient may have undergone a tissue or organ transplant, and in this case, CD71+ cells are administered to the patient in an amount effective to suppress rejection of the transplanted tissue or organ. Those skilled in the art can determine the number of cells and the mode of administration to effectively reduce transplant rejection. Similarly, diseases caused by T cell-mediated inflammation may also prove to be treatable with the described cells.

[0057] GATA6, which stands for GATA-binding protein 6, is a member of the GATA family of developmentally expressed transcriptional regulatory proteins. GATA factors constitute a family of transcriptional regulatory proteins that are expressed in distinct developmental and tissue-specific profiles and are thought to regulate cellularly restricted programs of gene expression. Suzuki et al. (The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals. Genomics. 1996 Dec 15;38(3):283-90) described the molecular cloning, chromosomal location, and transcription of the human GATA6 gene. This cDNA is highly conserved among vertebrates and encodes a putative 449-amino acid protein containing two adjacent zinc finger / basic domains characteristic of the GATA factor family (e.g., mouse, rat, zebrafish, pig, sheep, Xenopus, cattle, and rhesus monkey). For purposes of this specification, human GATA6 is described, but other vertebrate GATA6 genes, including mutants and alleles, are expected to be effective in the methods and systems described herein. Figures 2A-2C show exemplary human GATA6 cDNA and protein sequences.

[0058] "Expression" or "gene expression" refers to the overall flow of information from a gene. A "gene" is a functional hereditary unit or other expression system encoded on a nucleic acid for producing a gene product, such as RNA or protein, in a cell, and generally comprises a promoter and other cis-acting elements, e.g., a transcription control sequence, such as a transcription response element (TRE) and / or enhancer; an expression sequence (called an open reading frame or ORF) that usually encodes a protein or functional / structural RNA; and a polyadenylation sequence. When transcribed, a gene produces a gene product (usually a protein, an optionally post-translationally modified protein, or a functional / structural RNA). "Expression of a gene under the transcriptional control" or alternatively "controlled by" a specified sequence, such as a promoter, refers to gene expression from a gene containing the specified sequence operably linked (operably linked, usually in cis) to that gene. A gene "under the transcriptional control" of a promoter or transcriptional control element is one that is transcribed at detectably different levels in the presence of a transcription factor, e.g., in the case of a dox-responsive promoter such as a tet-inducible promoter, in the presence of an appropriate chemical compound such as doxycycline. A "gene for expression" of a described gene product, such as GATA6, is a gene that is capable of expressing the described gene product when placed in the appropriate environment, i.e., when a cell is transformed, transfected, transduced, etc., and exposed to the appropriate conditions for expression. For constitutive promoters, "appropriate conditions" typically simply mean that the gene is introduced into a host cell. For inducible promoters, such as the tissue-specific promoters described herein, "appropriate conditions" refers to the presence or absence of factors that regulate transcription, such as DNA-binding proteins; e.g., when an effective amount of each inducer to cause gene expression is available to the expression system (e.g., cell) or when factors that cause gene repression are unavailable or replaced.

[0059] Transcriptional control elements, as are widely known, include promoters, enhancers, transcription factor responsive elements (TREs, e.g., transcription factor binding sequences), suppressors, introns, etc. Additional transcriptional control elements, such as WPREs (woodchuck hepatitis virus posttranscriptional regulatory elements), or introns, such as those described below, that can increase expression from certain viral vectors, can be included in the gene.

[0060] Genes can be introduced into cells such as pluripotent stem cells, for example, iPSCs, by any useful method, such as viral transduction (e.g., AAV or lentivirus transduction), PiggyBac transposon, or any other useful transformation or transduction method.Those skilled in the art of molecular biology will be able to introduce the gene for inducible expression of GATA6 into pluripotent stem cells without undue experimentation.Various cloning and transformation / transduction vehicles, such as plasmids that can introduce GATA6, are widely available from Addgene, among many other vendors.

[0061] In addition to the above, CD71+ cells are circulating erythrocytes (CECs) present in the bone marrow but can be enriched in extramedullary organs in response to physiological or pathological stress conditions (e.g., anemia, cancer). In addition to their role in erythropoiesis, CECs have been shown to play a role in regulating the immune system. Neonatal CECs may contribute to vulnerability to infections, but they also suppress immune cell activation in response to the rapid colonization of resident microorganisms after delivery. CECs are abundant in the peripheral blood and placenta of pregnant women and are involved in regulating fetal-maternal tolerance. Recent studies have revealed a role for CECs in HIV and SARS-CoV-2 infection.

[0062] CECs suppress the inflammatory response of myeloid cells and T cell proliferation through the depletion of L-arginine by arginase. CECs also produce reactive oxygen species, which reduce T cell proliferation. CECs secrete cytokines, including transforming growth factor-β (TGF-β), which promotes the differentiation of regulatory T cells.

[0063] The expansion of HSPCs is in high demand due to the need for therapeutic transplantation of HSPCs for hematologic diseases. Previous methods have utilized culture systems that supplement the medium with exogenous factors to promote HSPC expansion, including fetal bovine serum, low-density lipoprotein, and cytokines such as thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF. The fetal liver niche system requires minimal addition of these factors and therefore presents a less expensive method for expanding HSPCs. Furthermore, due to the tissue's adhesive properties, this system is easily scalable to large vessels, bioreactors, bead cultures, and similar systems with large surface areas.

[0064] Specifically, the fetal liver niche has the unique ability to expand a subpopulation of HSPCs, CD71-high cells, which are minimally expandable using current protocols. Due to their immunosuppressive properties, CD71+ cells have potential applications in cell therapy for immunomodulation and suppression of immune responses, such as in organ transplantation or therapies for autoimmune diseases. Expanding this population in an inexpensive and scalable manner offers opportunities for the biomanufacturing of immunosuppressive cell therapies.

[0065] This system offers a scalable and inexpensive alternative to traditional methods for expanding HSPCs, and is capable of expanding a cell population (CD71+) that is poorly expanded using traditional methods.

[0066] Unique features of the technology described herein include: Synthetic fetal tissue established from a single, expandable starting population Expansion of the CD71+ population, which was not possible using conventional methods. Because CECs are known to have immunosuppressive properties, this novel method of expanding this population provides a more economically viable way to develop cell therapies for immunosuppression. Tissue growth is scalable as a function of culture surface area, making it easily scalable to larger culture conditions. Cord blood CD34+ HSPCs can be expanded with minimal addition of exogenous factors, such as fetal bovine serum, low-density lipoprotein, and cytokines such as thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF, as conventionally added.

[0067] Guye et al. (Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6. Nat Commun. 2016 Jan 6;7:10243) describes the generation of fetal liver organoids using Dox-inducible GATA6-transduced human induced pluripotent stem cells (hiPSCs). HiPSCs were transduced with an engineered lentiviral vector containing the human GATA6 gene under the control of a doxycycline-inducible promoter. While the dox(tet) promoter is very common and well-studied, other inducible promoter systems suitable for expression in stem cells, such as iPSCs, may be employed, as known to those skilled in the field of genetic engineering. Suitable hiPSCs are widely available (e.g., ATCC-HYR0103 human induced pluripotent hepatic (IPS) cells, among others).

[0068] Delyea, C. et al. (Delyea, C., Elahi, S., CD71 + Erythroid Suppressor Cells Promote Fetomaternal Tolerance through Arginase-2 and PDL-1. J Immunol. 2018 Jun 15;200(12):4044-4058) provides experimental evidence for the role and mechanism of CD71 + cells. The authors demonstrate that CD71 + erythrocytes expand during pregnancy in both humans and mice at the feto-maternal interface and in the periphery. These cells exhibit immunosuppressive properties, and their abundance has been shown to be associated with a Th2-biased immune response, as their depletion triggers an inflammatory immune response at the feto-maternal interface. In addition to their function in suppressing inflammatory responses in vitro, maternal CD71+ erythrocytes suppress aggressive alloreactivity directed toward the fetus, including reduced TNF-α and IFN-γ production, through arginase-2 activity and PD-1 / programmed death ligand-1 (PDL-1) interactions. Depletion of these cells leads to pregnancy failure due to fetal immunological rejection. Similarly, fetal liver CD71+ erythrocytes also exhibit immunosuppressive activity. Therefore, immunosuppression mediated by both maternal and fetal CD71+ erythrocytes is crucial for fetal tolerance. This study uncovers a previously unrecognized role for CD71+ erythrocytes in pregnancy and demonstrates that these cells mediate homeostatic immunosuppressive / immunomodulatory responses during pregnancy. While this study provides important evidence for the immunomodulatory role of CD71+ cells, it does not provide a biological method for their production. Furthermore, it only focuses on the physiological role of these cells in vivo and does not explore how these cells can be utilized for other therapies.

[0069] To evaluate the biological role of CD71+ cells in systemic inflammation, Kanemasa et al. (Kanemasa, H., et al. The immunoregulatory function of peripheral blood CD71+ erythroid cells in systemic-onset juvenile idiopathic arthritis. Sci Rep. 2021 Jul 13;11(1):14396) investigated gene expression and function in systemic juvenile idiopathic arthritis (SoJIA). Peripheral blood mononuclear cells from SoJIA patients showed upregulated expression of erythropoiesis-related genes. Among other inflammatory diseases, the increase in CECs was greatest in active SoJIA. The number of circulating CECs in inflammatory diseases was positively correlated with the levels of C-reactive protein, IL-6, IL-18, or soluble TNF receptors. Coculture with active SoJIA-driven CECs suppressed the secretion of IL-1β, IL-6, and IL-8 from healthy donor monocytes. During the acute phase of SoJIA, CECs are driven into the periphery at higher levels than in other inflammatory diseases. This study concluded that circulating CECs may control excessive inflammation, in part through an immunomodulatory pathway involving arginase-2. While this study highlighted the role of CD71+ cells in systemic inflammation and provided further evidence of their potential therapeutic value as cell therapy, it only focused on the physiological role of these cells in vivo and did not explore how these cells could be efficiently biomanufactured or utilized for other therapies.

[0070] Previous red blood cell cultures relied on the addition of serum or erythropoietin. Juutistenaho, S., et al. (Juutistenaho, S., Kekomaki, R., Growth of erythroid cells from thawed unseparated cord blood in vitro without exogenous erythropoietin. Transfus Apher Sci. 2013 Oct;49(2):193-9) reported the expansion of red blood cells from thawed unseparated cord blood units in vitro without serum or exogenous erythropoietin. However, this was discovered in a system optimized for megakaryocytic erythroid differentiation, and large numbers of red blood cells were not obtained. Furthermore, the goal of this study was to generate mature red blood cells without immunosuppressive activity.

[0071] Trakarnsanga, K., et al. (Trakarnsanga, K., Frayne, J., An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red blood cells. Nat Commun. 2017 Mar 14;8:14750) created a platform for erythropoiesis by immortalizing early adult erythroblasts to generate stable cell lines for providing a continuous supply of red blood cells. The immortalized cells efficiently differentiated into mature, functional reticulocytes, which could be isolated by filtration. Extensive characterization revealed no functional or molecular differences between these reticulocytes and in vitro-cultured adult reticulocytes, and importantly, no abnormal protein expression. This study demonstrates a feasible approach for producing clinical-grade red blood cells from in vitro culture. However, this study remains focused on an optimized protocol for the generation of mature erythropoiesis and not for the generation of CD71+ red blood cells with immunosuppressive functions. Therefore, this study is a method for erythropoiesis, not for the biomanufacturing of immunosuppressive CD71+ cells for cell therapy applications.

[0072] Elahi, S. et al. (Elahi, S., Oyegbami, O., CD71+ Erythroid Cells in Human Neonates Exhibit Immunosuppressive Properties and Compromise Immune Response Against Systemic Infection in Neonatal Mice. Front Immunol. 2020 Nov 24;11:597433) demonstrated that CD71+ cells (CECs) are abundant in the peripheral blood of human newborns. Their frequency is significantly more variable than that of mice, but declines rapidly by 4 weeks of age. However, their proportion remains significantly higher in neonates up to 6 months of age than in older neonates. The researchers found that human neonatal CECs suppressed cytokine production by CD14+ monocytes and T cells, which was partially suppressed by apocynin in vitro. Furthermore, depletion of CECs in neonatal mice increased the number of activated effector immune cells in the spleen and liver, enhancing resistance to Listeria monocytogenes infection. This study provided further evidence and a mechanism for the immunosuppressive effects of CECs. However, this study also focused only on the physiological role of these cells in vivo and did not explore how these cells could be efficiently biomanufactured or utilized for other therapies.

[0073] Shahbaz, S., et al. (Shahbaz, S., Elahi, S., CD71+VISTA+ erythroid cells promote the development and function of regulatory T cells through TGF-β. PLoS Biol. 2018 Dec 14;16(12):e2006649) demonstrates that neonatal CD71+ erythroid cells express significant levels of V-domain immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and play a pivotal role in promoting naive CD4+ T cells to regulatory T cells (Tregs) through constitutive production of transforming growth factor (TGF)-β. The authors show that CD71+VISTA+ erythrocytes significantly enhance TGF-β-mediated promotion of naive CD4+ T cells into induced Tregs (iTregs) in vitro compared with CD71+VISTA- and CD71+ erythrocytes from VISTA KO mice. They demonstrate that iTreg development by CD71+ erythrocytes is mediated by inhibition of the key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). They also found that depletion of Tregs using forkhead box P3 (FOXP3)-diphtheria toxin receptor (DTR) mice resulted in a significant expansion of CD71+ erythrocyte frequencies in vivo. This provides insight into the crosstalk between CD71+ erythrocytes and Tregs in neonates and highlights the biological role of CD71+ erythrocytes in the neonatal period and possibly beyond. This study further confirms the potential use of CD71+ cells as an immunosuppressive cellular therapy. [Example]

[0074] Generation of CD71+ cells by co-culture of CD34+ cord blood cells with fetal liver organoids (FeLO) material: ·GATA6-inducible engineered hiPSC hESC-Qualified Matrigel (Corning Catalog No. 354277) Human umbilical cord blood-derived CD34+ cells Doxycycline [1 mg / mL stock solution] (StemCell Technologies, catalog number 72742) mTeSR1 (StemCell Technologies, catalog number 85850) IMDM 1x [L-glutamine, 25 mM HEPES] (ThermoFisher, catalog number 12440053) Pen Strep [10k units / mL penicillin, 10k ug / mL streptomycin [100x]] (ThermoFisher, catalog number 15140122) MEM Non-Essential Amino Acid Solution [100x] (ThermoFisher, Cat. No. 11140050) (NEAA) GlutaMAX Supplement [100x] (ThermoFisher, catalog number 35050061) FBS (selected sellers) rhFLT3L (R and D Systems, catalog number 308-FK) rhTPO (R and D Systems, catalog number 288-TP) rhSCF (R and D Systems, catalog number 255-SC) CD71 microbeads (Miltenyi, catalog number 130-046-201)

[0075] procedure: 1. GATA6-inducible hiPSCs were cultured at 30kJ / cm 2 The culture is initiated by seeding at a cell density of 0.1%. 2. The next day, prepare mTesR1 with doxycycline to a final concentration of 1 μg / mL (1:1000). 3. Replace the medium with 1 μg / mL Dox-mTeSR1 medium. Change the medium every day until day 4.5. On day 5.5, Dox-mTeSR1 medium is replaced with IMDM optionally supplemented with 10% FBS, 1× GlutaMAX, 1× Pen Strep, and 1× NEAA. 6. Change every day until the 10th day. 7. On day 10, CD34+ cord blood cells were cultured at 10k / cm in IMDM optionally supplemented with 10% FBS, 1x GlutaMAX, 1x Pen Strep, and 1x NEAA, 100ng / ul rhSCF and rhFLT3LG, and 50ng / ul rhTPO. 2 Sow seeds in. 8. Every day or every other day, manually replace the maximum total volume of medium in each well by aspirating with a micropipette (medium as described in step 7). 9. After up to 20 days of co-culture, harvest the expanded CD34+ cord blood cells from the FeLO by aspirating the medium, washing once with PBS, and combining the PBS wash with the first aspirate. 10. Isolate the CD71+ population by magnetic-based MACS separation using CD71 microbeads (following the manufacturer's protocol) or flow cytometry sorting for CD71.

[0076] Monoculture and coculture CD71+ cells were prepared essentially according to the coculture method described above. For monoculture preparation, CD34+ cells were isolated from umbilical cord blood using the EasySep Human Cord Blood CD34 Positive Selection Kit II (Stem Cell Technologies, Catalog No. 17896) according to the manufacturer's protocol. Cord blood fractionation was performed by diluting the blood 1:1 with PBS containing 2% FBS and 1 mM EDTA, gently layering 30 mL of diluted blood onto 15 mL of Lymphoprep (Stem Cell Technologies, Catalog No. 07801), and centrifuging the tube at 1200 × g for 20 minutes without braking. The buffy coat (enriched mononuclear cell layer) was gently removed and washed with PBS containing 2% FBS and 1 mM EDTA. The CD34+ selection cocktail and RapidSpheres were incubated with mononuclear cells, and CD34+ cells were magnetically separated using "The Big Easy" EasySep magnet (Stem Cell Technologies catalog no. 18001) according to the manufacturer's instructions. Isolated CD34+ cells were used immediately or cryopreserved.

[0077] Cord blood CD34+ control monolayer cultures were cultured in IMDM as the basal medium supplemented with 10% FBS, 1x GlutaMax, MEM non-essential amino acid solution, and penicillin-streptomycin, supplemented with 100 ng / mL human stem cell factor, 100 ng / mL Flt3 ligand, and 50 ng / mL human thrombopoietin. Cord blood CD34+ cells were seeded at 40,000 cells / mL, and half of the medium volume in each well was manually replaced daily.

[0078] The monocultured and cocultured CD71+ cells were evaluated for CD38 and CD71 expression by FACS. As shown in Figures 3A and 3B, the coculture method produced significantly more CD71+ cells than the monoculture method.

[0079] Monoculture and coculture in FeLO and designer LO CD71+ cells were prepared essentially as described above. CD71+ cells were also expanded using designer liver organoids (DesLO) engineered to a mature liver phenotype, essentially as described in Velazquez JJ, et al. Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids. Cell Syst. 2021 Jan 20;12(1):41-55.e11. doi: 10.1016 / j.cels.2020.11.002. Epub 2020 Dec 7. As shown in Figure 4, the FeLO coculture method clearly outperformed both monoculture and coculture with DesLO material, yielding slightly higher CD71+ cell numbers on day 5 of coculture.

[0080] Cells generated from monoculture and FeLO coculture were stained with Wright-Giemsa stain. As shown in Figure 5, CD71 cells expanded in the coculture were stained with Wright-Giemsa stain. + The cells exhibit large nuclei and lack of granules characteristic of CECs.

[0081] Figure 6 shows the cell counts of CD71+ cells in monoculture compared to coculture, demonstrating a significant accumulation of CD71+ cells starting from day 7 of coculture. Single-cell RNA sequencing revealed that cocultured cord blood CD34 + 1 shows the expression levels of erythroid cluster 4 erythroid markers in cells.

[0082] To examine whether in vitro generated CD71+ erythrocytes suppress T cell proliferation after activation, an in vitro mixed lymphocyte reaction assay was performed. Experimental approach: Bead activation 1-Enrich CD3 T cells using MojoSort Human CD3 kit; 2-Enriched CD3 T cells are stained with CFSE (proliferating cells are CFSE-); 3-Stimulate cells with 2.5 ml of anti-CD3 anti-CD28 beads or allogeneic dendritic cells (DC); 4 - Co-culture of CD3 T cells with CD71+ erythroid cells; and After 5-4 days of co-culture, flow cytometry is performed.

[0083] A 29.23% reduction in T cell proliferation was observed in the presence of CD71+ cells prepared using the FeLO co-culture method described (Figure 8).

[0084] For flow cytometry analysis, human CD3+ T cells were cultured with dendritic cells in the presence or absence of CD71+ cells and stained with the CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific, Catalog No. C34554) according to the manufacturer's protocol. Cells were cultured for 24 hours, stained with an antibody against human CD3, and analyzed for the CFSE proliferation marker by flow cytometry.

[0085] In this assay, antigen-presenting cells (dendritic cells) are used to activate and induce proliferation of human CD3+ T cells. Proliferating cells do not retain the CFSE dye, so a higher CFSE signal indicates fewer proliferating cells. Addition of CD71+ cells to T cell cultures inhibits T cell activation and proliferation, as indicated by the higher CFSE signal in the CD71+ co-culture condition compared to the control condition (Figure 9).

[0086] Although several examples and embodiments of the method have been described in detail above, other examples and embodiments will be apparent to and readily made by those skilled in the art without departing from the scope and spirit of the present invention. For example, it should be understood that the present disclosure contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment. Accordingly, the foregoing description is intended to be illustrative and not limiting.

Claims

1. CD71 ＋ 1. A method for preparing a population of cells, comprising: Culturing the pluripotent stem cells to differentiate into a fetal liver / yolk sac phenotype, including enhanced expression of erythroid markers CD36, CD47, EPOR (erythropoietin receptor) and TGFβ1 (transforming growth factor β1); and CD34 ＋ Hematopoietic stem and progenitor cells (HPSCs) were co-cultured with differentiated pluripotent stem cells to induce HPSC-derived CD71 ＋ (CD71 ＨＩ ) producing an expanded culture population of cells. The method comprising:

2. The method of claim 1, wherein the pluripotent stem cells are differentiated into a fetal liver / yolk sac phenotype by expressing a gene for expressing GATA6 protein in the pluripotent stem cells or by introducing GATA6 protein or mRNA encoding GATA6 protein into the pluripotent stem cells.

3. The method of claim 2, wherein the pluripotent stem cells are differentiated into a fetal liver / yolk sac phenotype by expressing a gene for expressing GATA6 protein in the pluripotent stem cells.

4. The method of claim 2, wherein the gene for expressing the GATA6 protein is inducible in the pluripotent stem cells.

5. The method of claim 4, wherein the gene for expressing the GATA6 protein in the pluripotent stem cells is induced during the first 3 or 4 days of culture of the pluripotent stem cells.

6. The method according to claim 4 or 5, wherein the gene for expressing GATA6 is doxycycline-inducible.

7. The method of claim 2, comprising introducing GATA6 protein or mRNA encoding GATA6 protein into the pluripotent stem cells.

8. The method of claim 7, wherein the GATA6 protein or mRNA encoding the GATA6 protein is introduced into the pluripotent stem cells during the first 3 or 4 days of culture of the pluripotent stem cells.

9. The method of any one of claims 1 to 8, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

10. The method of any one of claims 1 to 4, wherein the pluripotent stem cells are cultured on a basement membrane matrix such as MATRIGEL.

11. The method of any one of claims 1 to 10, wherein the HPSCs are obtained from umbilical cord blood or bone marrow, such as from mobilized peripheral blood.

12. The HPSCs are CD71 ＋ The method according to any one of claims 1 to 11, wherein the cells are obtained from the patient to be administered.

13. 13. The method of any one of claims 1 to 12, wherein the HPSCs are co-cultured with differentiated pluripotent stem cells in a medium comprising a factor selected from one or more of thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF.

14. 14. The method of claim 13, wherein the medium comprises one or more of thrombopoietin, stem cell factor, and / or FLT3 ligand.

15. 15. The method of claim 13 or 14, wherein the culture medium comprising the factors is at least partially changed daily.

16. The method according to any one of claims 1 to 15, wherein the co-cultivation is carried out in a bioreactor, and optionally in a continuous flow bioreactor.

17. separating expanded HPSC cells comprising CD71+ cells from the differentiated pluripotent stem cells after co-culturing the HPSCs with the differentiated pluripotent stem cells for at least 6 days; and Recovering isolated cells of expanded HPSCs comprising CD71+ cells. The method of any one of claims 1 to 16, further comprising:

18. 18. The method of claim 17, further comprising enriching the CD71+ cells of the separated cells of expanded HPSCs comprising CD71+ cells by affinity purification.

19. 19. The method of claim 18, wherein the affinity purification method is fluorescence activated cell sorting, magnetic bead separation, panning, or column purification.

20. 20. The method of any one of claims 1 to 19, wherein the pluripotent stem cells and / or HPSCs are human.

21. A cell culture device comprising a cell culture vessel (plate, flask, bioreactor, etc.), The cell culture vessel contains cell culture medium and cells having a fetal liver / yolk sac phenotype and CD34 ＋ A cell culture device comprising a cell co-culture of hematopoietic stem cells and progenitor cells.

22. The cells having a fetal liver / yolk sac phenotype and CD34 ＋ 22. The device of claim 21, wherein the hematopoietic stem and progenitor cells are human.

23. 23. The device of claim 21 or 22, wherein the cells having a fetal liver / yolk sac phenotype comprise an inducible gene for expressing a GATA6 protein.

24. 24. The device of claim 23, wherein the inducible gene is doxycycline-inducible.

25. The device of any one of claims 21 to 24, wherein the medium comprises a factor selected from one or more of thrombopoietin, stem cell factor, FLT3 ligand, IL-3, IL-6, and GM-CSF.

26. 26. The device of claim 25, wherein the medium comprises thrombopoietin, stem cell factor and / or FLT3 ligand.

27. The apparatus of any one of claims 21 to 26, wherein the bioreactor is a continuous flow bioreactor.

28. and wherein the cells of the co-culture are at least 10%, at least 15%, or at least 20% CD34+, excluding cells having a fetal liver / yolk sac phenotype. － CD71 ＋ 28. The device of any one of claims 21 to 27, comprising cells.

29. wherein the cells of the co-culture are at least 70%, at least 75%, or at least 80% CD71 phenotype-exclusive, excluding cells having a fetal liver / yolk sac phenotype. ＋ 28. The device of any one of claims 21 to 27, comprising cells.

30. A method for immunosuppression in a patient with an inflammatory disease or an allogeneic or xenogeneic tissue transplant in need thereof, comprising administering to said patient an immunosuppressive dose of CD71 produced by the method of any one of claims 1 to 19. ＋ or CD71 ＨＩ administering cells, thereby reducing a cell-mediated response in said patient.

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