Modeling developmental hematopoietic niches of human embryo for near-physiological blood stem cell expansion ex vivo

Abstract

Provided herein is a method of producing an embryoid model including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; and culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components.

Description

MODELING DEVELOPMENTAL HEMATOPOIETIC NICHES OF HUMAN EMBRYO FOR NEAR-PHYSIOLOGICAL BLOOD STEM CELL EXPANSION EX VIVOCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Nos. 63 / 668,644, filed July 8, 2024, 63 / 719,449, filed November 12, 2024, 63 / 748,203, filed January 22, 2025, and 63 / 777,826, filed March 26, 2025, the disclosures of which are hereby incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant number HL141805 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the file containing the Sequence Listing is 2502478. xml. The size of the file is 6,522 bytes, and the file was created on July 7, 2025.BACKGROUND OF THE INVENTIONField of the Invention

[0004] Provided herein are methods and systems for regenerative biology, including methods and systems for the production of cells of various lineages and types, production of embryoid models, and for rejuvenation of cells.Description of Related Art

[0005] Stem cell-derived human embryo models (embryoids) at early postimplantation present a unique window into otherwise inaccessible stages of human development, addressing the challenge of direct access to the human embryos of appropriate stages containing organ precursors and developmental niches. Recently, novel embryoid models of the early post-implantation stage were developed. While these models represent significant breakthroughs, their applications for human health remain largely unexplored, particularly their potential to advance regenerative medicine.SUMMARY OF THE INVENTION

[0006] Provided herein is a method of producing an embryoid model including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or s expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include nonengineered stem cells; and culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components.

[0007] Also provided herein is a method of expanding a population of CD34+ cells, including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; and culturing one or more CD34+ cells with the embryoid in stem cell growth medium.

[0008] Also provided herein is a method of rejuvenating a cell, including: co- culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; and culturing one or more third cells with the embryoid or medium in which the embryoid is cultured, wherein the embryoid or the medium prevents exhaustion and aging of the third cells.

[0009] Also provided herein is a method of identifying one or more factors effective to rejuvenate a cell, including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturingthe embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; culturing one or more third cells with the embryoid in stem cell growth medium comprising one or more factors; and identifying one or more of the one or more target factors that are capable of rejuvenating the one or more third cells.

[0010] Also provided herein is a method of producing a macrophage or monocyte progenitor, including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; and culturing the embryoid in a medium including glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage or monocyte progenitor from the culture.

[0011] Also provided herein is method of producing a macrophage progenitor cell, including co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; and culturing the embryoid in a medium including glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage progenitor cell from the culture.

[0012] Also provided herein is a method of producing an embryoid model including: co-culturing one or more first cells and one or more second cells in serum- free stem cell culture medium, thereby generating an embryoid structure, wherein: thefirst cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components; and isolating one or more regions of the heterogenous tissue, wherein the one or more isolated regions are subsequently used as a feeder layer, for expansion of one or more cells, for the production of a target factor, production of an exosome, production of extracellular matrix (ECM), and / or for rejuvenation of one or more cells.

[0013] Additional non-limiting embodiments are set forth in the following numbered clauses:

[0014] 1. A method of producing an embryoid model comprising: coculturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; and culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components.

[0015] 2. The method of clause 1 , further comprising co-culturing one or more third cells with the one or more first cells and the one or more second cells.

[0016] 3. The method of clause 1 or clause 2, wherein the one or more third cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0017] 4. The method of any of clauses 1 -3, wherein expression of GATA6 is under the control of an inducible system, optionally a Dox-inducible system.

[0018] 5. The method of any of clauses 1 -4, wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

[0019] 6. The method of any of clauses 1 -5, wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

[0020] 7. The method of any of clauses 1 -6, wherein the GATA6 is endogenous to the cell.

[0021] 8. The method of any of clauses 1 -7, wherein the GATA6 is exogenous to the cell.

[0022] 9. The method of any of clauses 1 -8, wherein the GAT A6 is humanGATA6 or mouse GATA6.

[0023] 10. The method of any of clauses 1 -9, wherein the GATA6 is transiently expressed in the cell.

[0024] 1 1. The method of any of clauses 1 -10, wherein the embryoid secretes one or more of secreting stem cell growth factor (hSCF), thrombopoietin (TPO), and / or Fms-like tyrosine kinase 3 (FLT3L).

[0025] 12. A method of expanding a population of CD34+ cells, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing one or more CD34+ cells with the embryoid in stem cell growth medium.

[0026] 13. The method of clause 12, further comprising co-culturing one or more third cells with the one or more first cells and the one or more second cells.

[0027] 14. The method of clause 12 or clause 13, wherein the one or more third cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0028] 15. The method of any of clauses 12-14, wherein expression ofGATA6 is under the control of an inducible system, optionally a Dox-inducible system.

[0029] 16. The method of any of clauses 12-15, wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

[0030] 17. The method of any of clauses 12-16, wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

[0031] 18. The method of any of clauses 12-17, wherein the GATA6 is endogenous to the cell.

[0032] 19. The method of any of clauses 12-18, wherein the GATA6 is exogenous to the cell.

[0033] 20. The method of any of clauses 12-19, wherein the GATA6 is human GATA6 or mouse GATA 6.

[0034] 21. The method of any of clauses 12-20, wherein the GATA6 is transiently expressed in the cell.

[0035] 22. The method of any of clauses 12-21 , wherein the embryoid is capable of expanding CD34+ cells without the addition of a cytokine to the embryoid or the stem cell culture medium.

[0036] 23. The method of any of clauses 12-22, wherein the CD34+ cells are autologous or allogenic stem cells expressing one or more markers of a hematopoietic stem cell, for example, a CD34+ stem cell.

[0037] 24. The method of any of clauses 12-23, wherein the CD34+ cells are human cells.

[0038] 25. The method of any of clauses 12-24, further comprising delivering the expanded CD34+ cells to an immunocompromised mouse, wherein the expanded CD34+ cells engraft into bone marrow of the immunocompromised mouse.

[0039] 26. The method of any of clauses 12-25, wherein the engrafted human hematopoietic stem cells differentiate into leukocytes which generate immune chimerism in the lung, liver, spleen, kidney, brain, gonads, and / or peripheral blood of the transplanted mouse.

[0040] 27. The method of any of clauses 12-26, wherein the immunocompromised mouse is of NSG strain or NBSGW.

[0041] 28. A method of rejuvenating a cell, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise nonengineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing one or more third cells with the embryoid or medium inwhich the embryoid is cultured, wherein the embryoid or the medium prevents exhaustion and aging of the third cells.

[0042] 29. The method of clause 28, further comprising co-culturing one or more fourth cells with the one or more first cells and the one or more second cells.

[0043] 30. The method of clause 28 or clause 29, wherein the one or more fourth cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0044] 31. The method of any of clauses 28-30, wherein expression ofGATA6 is under the control of an inducible system, optionally a Dox-inducible system.

[0045] 32. The method of any of clauses 28-31 , wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

[0046] 33. The method of any of clauses 28-32, wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

[0047] 34. The method of any of clauses 28-33, wherein the GATA6 is endogenous to the cell.

[0048] 35. The method of any of clauses 28-34, wherein the GATA6 is exogenous to the cell.

[0049] 36. The method of any of clauses 28-35, wherein the GATA6 is human GATA6 or mouse GATA6.

[0050] 37. The method of any of clauses 28-36, wherein the GATA6 is transiently expressed in the cell.

[0051] 38. The method of any of clauses 28-37, wherein the CD34+ cells are autologous or allogenic stem cells expressing one or more markers of a hematopoietic stem cell, for example, a CD34+ stem cell.

[0052] 39. The method of any of clauses 28-38, wherein the one or more third cells are egg cells.

[0053] 40. The method of any of clauses 28-39, wherein the one or more third cells are sperm cells.

[0054] 41 . The method of any of clauses 28-40, wherein the one or more third cells are obtained from a patient.

[0055] 42. The method of any of clauses 28-41 , wherein the one or more third cells comprise a hematopoietic stem cell, a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglial cell, a blood cell and / or a blood cell derivative, a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased cell, and / or modified stem cell.

[0056] 43. The method of any of clauses 28-42, wherein the third cells are cultured with the embryoid in the presence of a target factor.

[0057] 44. The method of any of clauses 28-43, wherein the target factor is one or more of an exosome, an extracellular component, MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN, LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 , SELPLG, SIGLEC1 ,HGF, IGBP2, IGBP6, MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF1 1 , IGFBP4, PLTP, PROS1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IL1 1 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA, DHEA-S, CCL23, WNT3, LTE4, FGF2, KITLG, VTN, DKK1 , WNT1 1 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

[0058] 45. A method of identifying one or more factors effective to rejuvenate a cell, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; culturing one or more third cells with the embryoid in stem cell growth medium comprising one or more factors; and identifying one or more of the one or more target factors that are capable of rejuvenating the one or more third cells.

[0059] 46. The method of clause 45, wherein the one or more target factors are one or more of an exosome, an extracellular component, MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN, LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 ,SELPLG, SIGLEC1 , MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF1 1 , IGFBP4, PLTP, PR0S1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IGFBP2, IGFBP6, IL1 1 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA, DHEA-S, CCL23, WNT3, LTE4, FGF2, VTN, DKK1 , WNT11 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

[0060] 47. The method of clause 45 or clause 46, wherein the one or more third cells comprise a hematopoietic stem cell, a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglial cell, a blood cell and / or a blood cell derivative, a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased cell, and / or a modified stem cell.

[0061] 48. A method of rejuvenating a cell, comprising administering to a cell in need of rejuvenation one or more target factors identified according to the method of any of clauses 45-47.

[0062] 49. A method of producing a macrophage or monocyte progenitor, comprising: co-culturing one or more first cells and one or more second cells in serum- free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing the embryoid in a medium comprising glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage or monocyte progenitor from the culture.

[0063] 50. The method of clause 49, wherein the macrophage or monocyte progenitors express CD68, CD14, and / or IBA1.

[0064] 51 . A method of generating extracellular vesicles (EVs) or exosomes with immunomodulatory or regenerative activity, comprising: culturing one or moremacrophages generated according to clause 49 or clause 50 in a culture medium, thereby generating a conditioned medium; collecting the conditioned medium from the culture; and isolating EVs or exosomes from the conditioned medium with centrifugation, size-exclusion chromatography, or affinity-based capture.

[0065] 52. The method of clause 51 , further comprising enriching the EVs or exosomes for those containing a cargo.

[0066] 53. The method of clause 51 or clause 52, wherein cargo comprises mRNA, miRNA, and / or an anti-inflammatory or regenerative protein.

[0067] 54. An embryoid model prepared according to any of clauses 1 -1 1.

[0068] 55. A culture of expanded CD34+ cells prepared according to any of clauses 12-27.

[0069] 56. A rejuvenated cell prepared according to any of clauses 28-44.

[0070] 57. A macrophage prepared according to any of clauses 49-50.

[0071] 58. An EV or exosome prepared according to any of clauses 51 -53.

[0072] 59. A method of treating a disease or condition in a patient, comprising administering to the patient an amount of CD34+ cells prepared according to any of clauses 12-27 effective to treat the disease or condition.

[0073] 60. A method of treating a disease or condition in a patient, comprising administering to the patient an amount of a rejuvenated cells prepared according to any of clauses 28-44 effective to treat the disease or condition.

[0074] 61 . A method of preparing a patient for in vitro fertilization, comprising administering to the patient one or more rejuvenated cells prepared according to any of clauses 28-44.

[0075] 62. A method of generating an organ bud for transplantation, comprising expanding one or more rejuvenated cells prepared according to any of clauses 28-44.

[0076] 63. A method of producing a macrophage progenitor cell, comprising:

[0077] co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing the embryoid in a mediumcomprising glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage progenitor cell from the culture.

[0078] 64. A method of producing an embryoid model comprising: coculturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and isolating one or more regions of the heterogenous tissue, wherein the one or more isolated regions are subsequently used as a feeder layer, for expansion of one or more cells, for the production of a target factor, production of an exosome, production of extracellular matrix (ECM), and / or for rejuvenation of one or more cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 shows a schematic representation of a non-limiting embodiment of a method as described herein;

[0080] FIG. 2 shows a schematic representation of a non-limiting embodiment of a method as described herein;

[0081] FIG. 3 shows a schematic representation of a non-limiting embodiment of a method as described herein;

[0082] FIG. 4 shows a schematic representation of a non-limiting embodiment of a method as described herein;

[0083] FIG. 5 shows a schematic representation of a non-limiting embodiment of a method as described herein

[0084] FIG. 6 shows a schematic demonstrating mixing, sorting and selforganization of wild type and iGATA6 hiPSCs resulting in the development of GAT AG- derived yolk sac-like compartment of heXembryoid and its close relationship with the fetal liver. (EXE, Extra embryonic)

[0085] FIG. 7 shows immunofluorescence staining for HNF4a (endoderm / hepatic), DLK1 , NES (pericytes), DES (fibroblast / stellate cells), and CD31 (endothelial) at day 14 of culture; Scale bars: 200 pm.

[0086] FIG. 8 shows immunofluorescence staining for CD31 , NES, and DLK1 cells at day 10, day 14, day 17, and day 20 of heX-embryoid culture utilized for area quantification. Total percent area of CD31 +, NES+, and DLK1 + cells was calculated at each time point of heX-embryoid culture, (mean + SEM, n=X).

[0087] FIG. 9 shows fold change in total, Lin-CD34+CD38-, and Lin- CD34+CD38-CD45RA-CD49f cell numbers compared to corresponding populations in the initial cord blood cells (Mean + SEM of three independent cultures).

[0088] FIG. 10 shows fold change in total, Lin-CD34+CD38-, and Lin- CD34+CD38-CD45RA-CD49f cell numbers compared to corresponding populations in the initial cord blood cells (Mean + SEM of three independent cultures).

[0089] FIG. 11 shows flow cytometry data showing the human hematopoietic reconstitution out of total CD45+ cells in peripheral blood, bone marrow, and spleen of mice transplanted with the expanded human CD34+ cells cultured with heX- embryoid compared to cytokine-only condition after 20 weeks (*p< 0.05; **p<0.01 ).

[0090] FIG. 12 shows chimerism of different human blood lineages in peripheral blood, bone marrow, and spleen of mice transplanted with the expanded human CD34+ cells cultured with heXembryoid compared to cytokine-only condition after 20 weeks.

[0091] FIG. 13 shows a schematic demonstrating mixing, sorting, and selforganization of wild-type and iGATA6 hiPSCs resulting in the development of a GATA6-derived YS-like compartment of heX-embryoids along with wild-type-derived cells. EXE, Extra embryonic.

[0092] FIGS. 14-15 show scRNA-seq UMAP of day 16 heX-embryoids showing clusters and general cell types, and dot plots depicting the expression for selected genes specific to in vivo YS lineages.

[0093] FIG. 16 shows IF image of day 14 heX-embryoids showing CD31 + endothelium, HNF4-a+ endoderm, Desmin+ fibroblasts, and Nestin+ pericytes. Scale bars, 50 pm.

[0094] FIG. 17 shows IF staining showing the HOXA10+ cells localization in the proximity of the YS-WT interface shown by the dotted yellow line on day 16. AAT marks YS endodermal cells. Scale bar, 100 pm.

[0095] FIG. 18 shows IF staining showing the HOXA1 1 + HAND1 + pre-umbilical cord mesodermal cells and spirally-oriented HOXA1 1 +CD31 + pre-umbilical cord vasculature. Scale bar, 100 pm.

[0096] FIG. 19 shows (left) Feature plots of the endoderm cluster of the day 16 heX-embryoids scRNA-seq dataset, presented in FIG. 18. The dotted line shows the subcluster of cells with allantois-like signature, (right) IF staining the spatial localization of CST1 + SOX17+ allantois-like endodermal cells localized in the center of a spirally- oriented SOX17+ CD31 + vasculature with showing pre-umbilical cord-like structure. Scale bar, 100 pm.

[0097] FIG. 20 shows a schematic of seeding CB CD34+ cells on day 10 heX- embryoids and the subsequent harvest of expanded cells after 10 days.

[0098] FIG. 21 shows fold change of total cells and % of immunophenotypic HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+) of heX-embryoid-cultured CB CD34+ cells with the addition of 0, 1 , 10, and 100% of cytokines in 3 biological replicates. **p< 0.01 , p-value was measured using an unpaired, two-tailed t-test. Error bar represent mean ± s.e.m.

[0099] FIG. 22 shows fold change of total cells and immunophenotypic HSCs in heX-embryoid cocultured cells (low cytokine) compared to the cytokine-only control (high cytokine).

[0100] FIG. 23 flow cytometry panels of CD34+ and CD34+CD38- cells in heX- embryoids cultured CB CD34+ cells compared to the 100% cytokine only control.

[0101] FIG. 24 shows percentage of CD34+ and CD34+CD38- cells in heX- embryoids cultured CB CD34+ cells compared to the 100% cytokine only control in 3 biological replicates.

[0102] FIG. 25 shows fold change of phenotypic HSCs relative to initial CB-HSC during the 40-days culture (right) and after 10 days in 3 biological replicates (****p< 0.0001 ).

[0103] FIG. 26 shows CFU representing the number of distinct colony types (CFU-E, BFU-E, CFU-GM, CFU-GEMM) across different conditions. An equal number of Lin-CD34+CD38- cells was seeded in duplicate from at least 2 biological replicates. **p< 0.01 , ****p< 0.0001 . p-value was calculated via a one-way ANOVA, using T ukey’s multiple comparisons test. Error bars represent mean ± s.e.m.

[0104] FIG. 27 shows proportion of colony types in the CFU assay, showing the ratio of each colony type (CFU-GEMM, CFU-GM, CFU-G, BFU-E) to the total numberof colonies in equal number of Lin-CD34+CD38- cells from uncultured and heX- embryoids cultured CB cells, ns, not significant, **p< 0.01 . p-value was calculated via unpaired, two-tailed t-test. Error bars represent mean ± s.e.m.

[0105] FIG. 28 shows a schematic illustrating the process of harvesting the human hematopoietic cells after expansion on heX-embryoids tissue, retroorbital injection to mice, and harvesting different hematopoietic organs at the endpoint to assess human hematopoietic cells chimerism using flow cytometry and immunohistochemical analysis.

[0106] FIG. 29 shows percentage of human hematopoietic reconstitution within total CD45+ cells in the peripheral blood, bone marrow, and spleen of mice transplanted with expanded CB CD34+ cells cultured with heX-embryoids compared to cytokine-only condition after 20 weeks. n=14 mice for heX-embryoids and n=8 mice for cytokine-only condition. Replicates from 3 independent experiments. The dotted line represents the median for each group. Medians for the heX-embryoids group: 83.85% (peripheral blood), 94.00% (bone marrow), and 84.15% (spleen). Medians for control group: 1.01 % (peripheral blood), 0.50% (bone marrow), and 0.50% (spleen). ***** p < 0.0001. p-values were calculated via unpaired, two tailed t-test for reconstitution levels in bone marrow, spleen, and peripheral blood between the two groups.

[0107] FIG. 30 shows human myeloid (CD33), B lymphoid (CD19), T lymphoid (CD3), and erythroid (CD235a) reconstitution in the bone marrow of mice, 20 weeks post-transplantation with expanded CB CD34+ cells cultured with heX-embryoids compared to cytokine-only condition. n=6 mice for heX-embryoids and n=6 mice for cytokine-only control from 3 biological replicates. Error bars represent mean ± s.e.m.

[0108] FIG. 31 shows immunohistochemical staining for CD20 and CD3 markers in spleen sections 20 weeks post-transplantation with the expanded CB CD34+ cells cultured on heX-embryoids or low cytokine control. Scale bars, 200pm.

[0109] FIG. 32 shows the flow cytometry panels and percentage of human CD34+ cells in the bone marrow of primary transplanted mouse in 3 biological replicates. *p< 0.05. p-values were calculated via unpaired, two tailed t-test. BM, Bone marrow.

[0110] FIG. 33 shows performing secondary transplantations with the cells extracted from the bone marrow of the primary recipient mice transplanted with expanded CD34+ cells after 20 weeks.

[0111] FIG. 34 shows flow cytometry plot and percentage of human CD45+ chimerism in the bone marrow of secondary recipient mice at 18 weeks posttransplantation. n=5 mice secondary recipients of heX-embryoids cultured CB cells and n=3 mice secondary recipients of low cytokine control. **** p<0.0001. p-value calculated using unpaired, two-tailed t-test.

[0112] FIG. 35 shows hCD45+ peripheral blood chimerism at 8. 18-, and 35- weeks following transplantation with expanded CB CD34+ cells cultured with or without heX-embryoids. n=14 mice at 8 and 18 weeks, and n=3 at 35 weeks for both groups. **** p< 0.0001 , * p< 0.05. p-value calculated using unpaired, two-tailed t-test. Error bars represent mean ± s.e.m.

[0113] FIG. 36 shows percentage of multilineage (hCD19, CD33, CD3) reconstitution at 8, 18-, and 35-weeks post-transplantation out of total hCD45+ cells in peripheral blood. n=14 mice at 8 and 18 weeks, and n=3 at 35 weeks for both groups, ns, not significant, **** p< 0.0001 , * p< 0.05. p-value calculated using unpaired, two-tailed t-test. Error bars represent mean ± s.e.m.

[0114] FIG. 37 shows expression of genes; hematopoietic factors from heX- embryoids (down) and their respective receptor on CB CD34+ cells (top).

[0115] FIG. 38 shows a violin plot showing the expression of MLLT3 and MYCT1 in CD34+HOPX+SPINK2+ population, p-value was calculated via Wilcoxon rank-sum test.

[0116] FIG., 39 shows a violin plot showing CYP1 B1 expression in uncultured, heX-embryoid cocultured and regular cytokine control cultured cells.

[0117] FIG. 40 shows the effect of addition of SR1 (aryl hydrocarbon receptor inhibitor) on the percentage of Lin-CD34+CD38- cells.

[0118] FIG. 41 shows a violin plot showing CDKN1 C expression in uncultured, heX-embryoid cocultured and regular cytokine control cultured cells.

[0119] FIG. 42 shows a cell cycle EdU assay showing different phases of cell cycle among the Lin-CD34+CD38- cells of CB CD34+ cells expanded on heX- embryoids or in cytokine-only condition with high and low concentration of cytokines of 3 biological replicates, ns, not significant, **p< 0.001 , ***p< 0.001 , ****p< 0.0001 . p- values were calculated via one-way ANOVA, using Tukey’s multiple comparisons test. Error bars represent mean ± s.e.m.

[0120] FIG. 43 shows quantification of active mitochondrial content among the Lin-CD34+CD38- cells of CB CD34+ cells expanded on heX-embryoids or in cytokine-only with high and low concentration of cytokines of 3 biological replicates, ns, not significant, *p< 0.05, **p< 0.01 . p-values were calculated via one-way ANOVA, using Tukey’s multiple comparisons test. Error bars represent mean ± s.e.m.

[0121] FIG. 44 shows quantification of MitoSox+ CD34+CD38- cells in heX- embryoid cocultures with the addition fo both low and regular levels of cytokines, and cytokine only controls (low and regular level).

[0122] FIGS. 45A-45B show expression of HSC homing markers assessed via FACS IN heX-embryoid low cytokine and cytokine only controls.

[0123] FIG. 46 shows dot plots sing the expression of HSC markers in Lin- CD34+CD38- population in the combined single cell data of heX-embryoids, cytokine only control, and uncultured cells.

[0124] FIG. 47 shows Immunofluorescence staining for CD34 and CD44 (CB homing marker) on day 20 of heX-embryoids and CB co-culture. Scale bars, 100 pm.

[0125] FIG. 48 shows immunofluorescence staining for CD45 and CD34 on day 20 of heX-embryoids and CB co-culture. Scale bars, 100 pm.

[0126] FIG. 49 shows immunofluorescence staining, histogram, and barplots showing the higher coverage of vasculature around the WT-derived structure.

[0127] FIGS. 50A-D show feature plots showing the markers associated with different endothelium subtypes in the endothelium cluster of day 16 heX-embryoids scRNA-seq dataset.

[0128] FIG. 51 shows immunofluorescence staining (day 14) demonstrating the association of arterial (CXCR4) and sinusoidal (LYVE1 ) niche with WT-derived structure. EGFP-GATA6 indicates the expression of residual EGFP from iGATA6 cells. Scale bar, 100 pm. The area surrounded with white dotted lines shows the WT-derived structure as indicated by the lack of EGFP expression.

[0129] FIG. 52 shows immunofluorescence staining shows the CD34+ cord blood cells in the vicinity of CXCR4+ arterial endothelial cells on day 20 of heX- embryoids cocultured with CB-CD34+ cells.

[0130] FIG. 53 shows quantification of endothelial association of expanding CD34+ CB clusters with different vascular niches from 3 biological replicates. Error bars represent mean ± s.e.m.

[0131] FIG. 54 shows the dot plot showing the expression of hematopoietic factors from endothelial subtypes within heX-embryoids at day 16.DESCRIPTION OF THE INVENTION

[0132] Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0133] As used herein, “a” and “an” refer to one or more.

[0134] 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 characteristic(s) of the claimed invention. The term “consisting of” excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments “comprising” one or more stated elements or steps also include, but are not limited to embodiments “consisting essentially of” and “consisting of” those stated elements or steps. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases.

[0135] As used herein, the terms “patient” or “subject” refer to members of the animal kingdom including but not limited to human beings and “mammal” refers to all mammals, including, but not limited to human beings.

[0136] As used herein, “treatment” or “treating” of a wound or defect means administration to a patient by any suitable dosage regimen, procedure and / or administration route of a composition, device or structure with the object of achieving a desirable clinical / medical end-point, including, for example, attracting progenitor cells, healing a wound, correcting a defect, causing neurite outgrowth, or repairing a nerve.

[0137] As used herein, the terms “cell” and “cells” refer to any types of cells from any animal, such as, without limitation, rat, mouse, monkey, canine, and human. For example and without limitation, cells can be progenitor cells, e.g., pluripotent cells, including stem cells, induced pluripotent stem cells, multipotent cells, or differentiated cells, such as endothelial cells and smooth muscle cells. “Cells” also includespopulations of cells, such as, for example, a population of cells produced by culturing CD34+ HPSCs. In certain aspects, cells for medical procedures can be obtained from the patient for autologous procedures, or from other donors for allogeneic procedures, or from xenogeneic sources.

[0138] Cell populations comprising stem cells, e.g., pluripotent and hematopoietic stem cells, may be used in the methods described herein. A stem cell may be defined as a totipotent, pluripotent, or multipotent cell of a multicellular organism from which certain other kinds of cells arise by differentiation. Stem cells are generally capable of giving rise to indefinitely more cells of the same type in cell culture. Stem cells may be characterized as totipotent, pluripotent, or multipotent, depending on their source. Stem cells may be genetically engineered. Engineered stem cells may be modified to upregulate or downregulate native or exogenous gene expression or to express genes in an inducible manner. Examples of inducible gene expression systems include doxycycline-inducible systems, though others are known and fall within the scope of this disclosure, as described below. Stem cells may be manipulated or induced to produce GATA6 and / or other factors, e.g., by introducing a gene into the cells for expression the factors. Stem cells may be induced to differentiate, e.g. to a fetal liver / yolk sac phenotype by introduction of GATA6 or a gene for expressing GATA6, as described herein. Stem cells may be obtained from many sources, including from a patient, and may be autologous and / or allogenic to a given patient to whom a product of the methods described herein may be administered. Although obtainable from many tissue sources, non-limiting examples of tissue sources for cell populations comprising stem cells include umbilical cord (including umbilical cord blood, umbilical cord matrix, Wharton’s jelly, 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 pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017 Feb;16(2):1 15-130). Cell populations useful in the present method and device may be enriched for stem cells by any useful method, including cell separation and cell sorting techniques and cell culturing techniques, as are broadly-known in the stemcell field. Methods of generation of useful iPSCs are also broadly-known. Cell populations comprising stem cells may be cryogenically preserved, e.g., in a tissue bank in which a patient’s (autologous) tissue or stem cells are stored for later retrieval. Cells may be screened for and isolated based on any suitable marker, for CD34, CD68, CD14, and / or IBA1 ), markers for gametes (e.g., egg and sperm cells), and / or markers for immune cells, such as macrophages and / or monocytes, including their progenitors.

[0139] As used herein, “embryoid” means a three-dimensional structure of pluripotent cells that mimics early aspects of embryonic development, for example the blastocyst or gastrula stage of development. “Embryoids” as described herein may include a mixed population of cells, including stem cells (such as, without limitation, embryonic pluripotent stem cells or induced pluripotent stem cells).

[0140] Provided herein are methods and compositions, which may include cells, cells exposed to a target environment (e.g., co-cultured with an embryoid as described herein cells exposed to a conditioned medium (e.g., from a cultured embryoid as described herein), factors isolated from medium in which the embryoid is cultured, and the like. Such methods and compositions may be used to expand cell populations, treat conditions (e.g., rejuvenate cells, such as egg cells and / or sperm cells), generate new cell types (such as macrophage precursors or macrophages), and as an investigative tool to identify factors that are useful rejuvenation of various cell types. As used herein, the term “rejuvenation” means the phenotypic and / or genetic reprogramming of cells, restoring them to a more youthful, robust, and functional state. This process is typically characterized by an enhancement of cellular division capacity, the ability to generate diverse, differentiated progenitor cells, and the reversal of age- related molecular damage. Key mechanisms driving this rejuvenation include the activation of telomerase, the repair and maintenance of DNA integrity, and the restoration of cellular metabolic and mitochondrial function. In the context of stem cells or germ cells, rejuvenation can be assessed by their improved proliferative potential and their ability to give rise to a broader range of specialized cell types. In animals, the effects of cellular rejuvenation manifest as an extension of healthspan, a potential increase in lifespan, the regeneration of healthier, more functional tissues, and a reduction in the apparent age of tissues, leading to overall improvements in physiological function and resilience.

[0141] Accordingly, in non-limiting embodiments, a method of generating an embryoid is provided herein. The method may include co-culturing one or more first cells and one or more second cells. Any cell cultured as described herein may be cultured with a cell growth matrix. A “cell growth matrix” is a mesh, matrix, particle, surface, hydrogel, porous structure, or other material upon which or into which a cell can be deposited and can be maintained in a living state, and often propagates (multiplies) in the presence of suitable cell growth media. A cell growth matrix can be manufactured from a single composition, or multiple compositions, such as synthetic and / or natural polymer compositions. A cell growth matrix may comprise cells and / or therapeutic agents. A “scaffold-free cell growth matrix” contains no synthetic polymeric compositions and is a natural product of cells and tissues. In the context of the present invention and disclosure, a scaffold-free cell growth matrix may be a basement membrane secreted from cells, such as MATRIGEL.

[0142] Any cells cultured as described herein may be cultured in cell medium, such as stem cell culture medium, such as serum-free stem cell culture medium, thereby generating an embryoid structure. Useful media are known to those of skill in the art and are commercially available including from Sartorius, Thermo-Fisher Scientific, STEMCELL Technologies, Neuromic, and others known to those of skill in the art. The first and second cells (and, optionally as described below, third cells) may be cultured together for any suitable time, including about 1 -10 days, about 2-9 days, about 3-8 days, about 4-7 days, and / or about 5-6 days, all values and subranges therebetween inclusive. In non-limiting embodiments the cells are cultured for about 2-7 days, all values and subranges therebetween inclusive. The first and second cells may be from any useful source (e.g., human, mouse, canine, etc.). In non-limiting embodiments the first cells may be pluripotent stem cells such as induced pluripotent stem cells (iPSCs), multipotent stem cells, and / or embryonic stem cells (ESCs) expressing a factor of interest, such as GATA binding protein 6 (GATA6) or a variation or derivative thereof. The GATA6 may be endogenous to the cell, exogenous to the cell, may be human GATA6, and / or may be mouse GATA6. Methods for expressing a gene, such as GATA6, in a cell are known to those of skill in the art. In non-limiting embodiments the second cells may be non-engineered stem cells. In non-limiting embodiments, the method may include culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that includes embryonic and / or extraembryonic components. In non-limiting embodiments, embryonic componentsinclude epiblast, ectoderm, definitive endoderm, embryonic mesoderm, lateral plate mesoderm, paraxial mesoderm, gut tube, neural plate, neural tube, forebrain, midbrain, and / or hindbrain. In non-limiting embodiments, extraembryonic components include yolk sac endoderm, yolk sac mesoderm, yolk sac endothelium, yolk sac mesenchyme, allantois, connecting stalk, and / or umbilical cord. Useful media are known to those of skill in the art and are commercially available including from Sigma- Aldrich, Thermo-Fisher Scientific, and others known to those of skill in the art. In nonlimiting embodiments, the embryoid is cultured for about 10-100 days, all values and subranges therebetween inclusive. In non-limiting embodiments, the embryoid is cultured for about 10-40 days, all values and subranges therebetween inclusive.

[0143] In non-limiting embodiments, the method may include co-culturing one or more third cells with the one or more first cells and the one or more second cells. As with the first and second cells, the third cells may be from any source, and may be stem or pluripotent cells. In non-limiting embodiments, the third cells may be nonengineered cells or may be engineered to express a factor of interest, such as ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0144] In non-limiting embodiments, the factors of interest (e.g., GATA6 and / or ETV2) are under the control of an inducible system, such as a Dox-inducible system, (e.g., Tet-On / Tet-Off systems), cumate-inducible systems, temperature-inducible systems, and those based on other chemicals such as dexamethasone, beta-estradiol, and ethanol are known to those of skill in the art. In non-limiting embodiments, for example with an inducible system, the factor of interest (e.g., GATA6 and / or ETV2) may be transiently expressed in the cell.

[0145] The cell culture medium in which the first and second (and, optionally, third) cells are cultured and / or in which the embryoid is cultured may be any suitable cell culture medium, as described above. In non-limiting embodiments, the cell may include 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO), all values and subranges therebetween inclusive.

[0146] The embryoid, once formed, may secrete one or more factors (which may result in formation of a conditioned medium that may be useful for purposes described herein). In non-limiting embodiments, the embryoid secretes one or moreof secreting stem cell growth factor (hSCF), thrombopoietin (TPO), and / or Fms-like tyrosine kinase 3 (FLT3L).

[0147] Also provided herein is method of expanding a population of CD34+ cells, including co-culturing one or more first cells and one or more second cells in medium, for example stem cell medium, for example serum-free stem cell culture medium, thereby generating an embryoid structure. The first and second cells (and, optionally as described below, third cells) may be cultured together for any suitable time, including about 1 -10 days, about 2-9 days, about 3-8 days, about 4-7 days, and / or about 5-6 days, all values and subranges therebetween inclusive. In nonlimiting embodiments the cells are cultured for about 2-7 days, all values and subranges therebetween inclusive. Cells useful in this non-limiting embodiment may be, as described above, obtained from any useful source (e.g., human, mice, canine). In non-limiting embodiments, the first cells may be pluripotent cells (e.g., iPSCs), multipotent stem cells, and / or embryonic stem cells expressing a factor of interest, such as GATA6 or a variation or derivative thereof. The GATA6 may be endogenous to the cell, exogenous to the cell, may be human GATA6, and / or may be mouse GATA6. The second cells may be non-engineered stem cells. In non-limiting embodiments the method may further include culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components and culturing one or more CD34+ cells with the embryoid in stem cell growth medium. In non-limiting embodiments, the embryoid is cultured (alone and / or with the one or more CD34+ cells) for about 10-100 days, all values and subranges therebetween inclusive. In non-limiting embodiments, the embryoid is cultured (alone and / or with the one or more CD34+ cells) for about 10-40 days, all values and subranges therebetween inclusive. In non-limiting embodiments, the CD34+ cells may be of human, mouse, or canine origin, and / or may be autologous or allogenic stem cells (to the cells utilized to generate the embryoid and / or to an individual to which an expanded population of cells may be delivered) expressing, or not expressing, one or more markers that define a hematopoietic stem cell collectively, including, but not limited to, CD34 (e.g., a CD34+ stem cell), CD38, CD45RA, CD90 (Thy1 ), Sca-1 , CD150, and / or CD48.

[0148] In non-limiting embodiments, the method may include co-culturing one or more third cells with the one or more first cells and the one or more second cells. As with the first and second cells, the third cells may be from any source, and may bestem or pluripotent cells. In non-limiting embodiments, the third cells may be nonengineered cells or may be engineered to express a factor of interest, such as ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0149] In non-limiting embodiments, the factors of interest (e.g., GATA6 and / or ETV2) are under the control of an inducible system, such as Dox-inducible systems (e.g., Tet-On / Tet-Off systems), cumate-inducible systems, temperature-inducible systems, and those based on other chemicals such as dexamethasone, beta-estradiol, and ethanol are known to those of skill in the art. In non-limiting embodiments, for example with an inducible system, the factor of interest (e.g., GATA6 and / or ETV2) may be transiently expressed in the cell.

[0150] The cell culture medium in which the first and second (and, optionally, third) cells are cultured and / or in which the embryoid is cultured may be any suitable cell culture medium, as described above. In non-limiting embodiments, the cell may include 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO), all values and subranges therebetween inclusive.

[0151] Embryoids generated from the methods disclosed herein may, among other properties, be capable of expanding CD34+ cells cultured therewith without the addition of a cytokine to the embryoid or the stem cell culture medium.

[0152] In non-limiting embodiments, the method may further include delivering the expanded CD34+ cells to an animal. In non-limiting embodiments, the animal is a human. In non-limiting embodiments, the animal is a non-human mammal. In nonlimiting embodiments, the animal has a condition for which the CD34+ cells may be useful, for example the animal may be immunocompromised. In non-limiting embodiments, the animal is an immunocompromised mouse (e.g., an NSG strain or NBSGW strain) and the CD34+ cells are human cells. In non-limiting embodiments, when the expanded CD34+ cells engraft into bone marrow of the immunocompromised animal (such as, without limitation, a mouse).

[0153] In non-limiting embodiments, the engrafted CD34+ cells differentiate into a different cell type, for example a leukocyte, and, as a result, an immune chimerism is generated. In non-limiting embodiments, the immune chimerism is generated in the lung, liver, spleen, kidney, brain, gonads, and / or peripheral blood.

[0154] Also provided herein is a method of rejuvenating a cell. In non-limiting embodiments, the method includes generating an embryoid as described herein. Then one or more third cells, which may be any cell type of any organism (e.g., human, mouse, canine) as described herein, are cultured with the embryoid, or medium in which the embryoid was cultured. In non-limiting embodiments, the embryoid or the medium prevents exhaustion and aging of the third cells.

[0155] In non-limiting embodiments, as described above, the method may include co-culturing one or more fourth cells with the one or more first cells and the one or more second cells. As with the first and second cells, the fourth cells may be from any source, and may be stem or pluripotent cells. In non-limiting embodiments, the third cells may be non-engineered cells or may be engineered to express a factor of interest, such as ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

[0156] In non-limiting embodiments, the factors of interest (e.g., GATA6 and / or ETV2) are under the control of an inducible system, such as Dox-inducible systems (e.g., Tet-On / Tet-Off systems), cumate-inducible systems, temperature-inducible systems, and those based on other chemicals such as dexamethasone, beta-estradiol, and ethanol are known to those of skill in the art. In non-limiting embodiments, for example with an inducible system, the factor of interest (e.g., GATA6 and / or ETV2) may be transiently expressed in the cell.

[0157] The cell culture medium in which the first and second (and, optionally, third and / or fourth) cells are cultured and / or in which the embryoid is cultured may be any suitable cell culture medium, as described above. In non-limiting embodiments, the cell may include 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO), all values and subranges therebetween inclusive.

[0158] As noted above, the first and second cells (and, optionally the fourth cells) may be cultured together for any suitable time, including about 1 -10 days, about 2-9 days, about 3-8 days, about 4-7 days, and / or about 5-6 days, all values and subranges therebetween inclusive. In non-limiting embodiments the cells are cultured for about 2-7 days, all values and subranges therebetween inclusive. In non-limiting embodiments, the embryoid is cultured (alone and / or with the one or more cells to be rejuvenated) for about 10-100 days, all values and subranges therebetween inclusive.In non-limiting embodiments, the embryoid is cultured (alone and / or with the one or more cells to be rejuvenated) for about 10-40 days, all values and subranges therebetween inclusive.

[0159] As also described above, the first cells, which may be modified to express GATA6, may express a human our mouse, endogenous or exogenous, GATA6, may do so under the control of an inducible system (as described herein), and may express GATA6 transiently. Similarly, the fourth cells, which may express ETV2, may also express ETV2 under the control of an inducible system as described herein.

[0160] In non-limiting embodiments, the one or more third cells (e.g., cells to be rejuvenated) may be CD34+ cells may be of human, mouse, or canine origin, and / or may be autologous or allogenic stem cells (to the cells utilized to generate the embryoid and / or to an individual to which a rejuvenated cell may be delivered) expressing one or more hematopoietic stem cell markers, as described herein and known to those of skill in the art.

[0161] In non-limiting embodiments, the one or more third cells may be a, egg cell and / or a sperm cell.

[0162] In non-limiting embodiments, the third cell is obtained from a patient, and, in non-limiting embodiments, the rejuvenated cell is delivered to the same patient.

[0163] In non-limiting embodiments, the one or more third cells include a hematopoietic stem cell. Hematopoietic stem cells may be obtained from any useful source, including, without limitation, from cord blood or adult bone marrow or peripheral blood. In non-limiting embodiments, the one or more third cells include a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglia, a blood cell and / or a blood cell derivative (e.g., monocyte, lymphocyte, neutrophil, eosinophil, basophil, macrophage, erythrocyte, platelet, and / or any suitable blood cell derivative), a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased and / or modified stem cell (e.g., a cell from a subject with sickle cell disease, leukemia, and / or any suitable disease or modification), and / or any suitable cell.

[0164] In non-limiting embodiments, the one or more third cells are cultured with the embryoid in the presence of a target factor. Suitable target factors include, without limitation, an exosome (e.g., an exosome produced by one or more cells of the embryoid), an extracellular component (e.g., ECM, for example produced by one or more cells of the embryoid) MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN,LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 , SELPLG, SIGLEC1 ,HGF, IGBP2, IGBP6, MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF11 , IGFBP4, PLTP, PR0S1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IL11 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA, DHEA-S, CCL23, WNT3, LTE4, FGF2, KITLG, VTN, DKK1 , WNT11 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

[0165] Also provided herein is a method of using an embryoid, generated as described herein, to identify one or more target factors effective to rejuvenate a cell.

[0166] The method includes generating an embryoid as described herein. Then one or more third cells, which may be any cell type of any organism (e.g., human, mouse, canine) as described herein, are cultured with the embryoid in stem cell growth medium including one or more target factors (which may be produced by one or more cells of the embryoid). The method may then include identifying one or more of the one or more target factors that are capable of rejuvenating the one or more third cells. Methods for identifying a target factor in a sample (e.g., a sample from a cell culture) are known to those of skill in the art and may include, without limitation, spectrometry (e.g., mass spectrometry), chromatography (e.g., gas and / or high-pressure liquid chromatography), and like methods known to those of skill in the art. The target factor included in the stem cell growth medium with the embryoid and the one or more third cells may be an exosome (e.g., an exosome produced by one or more cells of the embryoid), an extracellular component (e.g., ECM, for example produced by one or more cells of the embryoid) MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN, LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 , SELPLG, SIGLEC1 ,HGF, IGBP2, IGBP6, MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF1 1 , IGFBP4, PLTP, PROS1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IL11 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA,DHEA-S, CCL23, WNT3, LTE4, FGF2, KITLG, VTN, DKK1 , WNT11 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

[0167] The one or more third cells may be a hematopoietic stem cell (from any useful origin as described herein). In non-limiting embodiments, the one or more third cells include a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglia, a blood cell and / or a blood cell derivative (e.g., monocyte, lymphocyte, neutrophil, eosinophil, basophil, macrophage, erythrocyte, platelet, and / or any suitable blood cell derivative), a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased and / or modified stem cell (e.g., a cell from a subject with sickle cell disease, leukemia, and / or any suitable disease or modification), and / or any suitable cell.

[0168] In non-limiting embodiments, a target factor identified from a method described herein may be administered to a cell in need of rejuvenation (e.g., by adding the target factor to a cell culture including the cells to be rejuvenated).

[0169] Also provided herein is a method of using an embryoid generated as described herein to produce a macrophage, macrophage progenitor, monocyte, or monocyte progenitor. The method includes generating an embryoid as described herein. Then one or more third cells, which may be any cell type of any organism (e.g., human, mouse, canine) as described herein, are cultured with the embryoid under defined conditions to promote hematopoietic emergence. In non-limiting embodiments, the one or more third cells are cultured with the embryoid in a basal medium including glutamine, glucose, and sodium bicarbonate. In non-limiting embodiments, the medium includes about 4 mM l-glutamine, about 4500mg / L glucose, and about 1500 mg / L of sodium bicarbonate. In non-limiting embodiments the medium is supplemented with an l-glutamine alternative, such as an l-alanyl-l-glutamine dipeptide (e.g., 200 mM l-alanyl-l-glutamine). In non-limiting embodiments, the one or more third cells are CD14+ cells and a macrophage progenitor is generated. In nonlimiting embodiments, the medium is Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with GlutaMAX™. Thereafter, the method may include harvesting macrophages, macrophage progenitors, monocytes, or monocyte progenitors from the culture.

[0170] In non-limiting embodiments the macrophage, macrophage precursor, monocyte, or monocyte progenitor expresses CD68, CD14, and / or IBA1 .

[0171] Also provided herein is a method of using an embryoid generated as described herein to produce extracellular vesicles (EVs) or exosomes with immunomodulatory or regenerative activity. The method includes generating an embryoid as described herein. Thereafter, a macrophage may be generated as described herein, and the macrophages may be cultured in a culture medium. By releasing factors into the culture medium, the macrophages may thus generate a conditioned medium, from which EVs or exosomes may be isolated by known methods, including centrifugation, size-exclusion chromatography, and / or affinitybased capture. In non-limiting embodiments, the isolated samples may be further enriched, in non-limiting embodiments for EVs and / or exosomes carrying a cargo (such as, without limitation, mRNA, miRNA, an anti-inflammatory protein (such as an anti-inflammatory cytokine such as, without limitation, Interleukin 1 receptor agonist (IL-1 ra), Interleukin-10 (IL-10), lnterleukin-4 (IL-4), Interleukin 6 (IL-6), Interleukin-13 (IL-13), Interleukin 1 1 (IL-1 1 ), and / or Transforming Growth Factor-beta (TGF-[3)), and / or a regenerative protein (such as telomerase, REG1 A, REG1 B, REG3A, REG3G, and / or REG4). Non-limiting examples of methods for further enrichment may include polymer precipitation, polymer-mediated enrichment, immunoaffinity capture, differential ultracentrifugation, tangential flow filtration, and / or size exclusion chromatography.

[0172] In non-limiting embodiments, expanded CD34+ cells, macrophages, macrophage precursors, monocytes, monocyte progenitors, rejuvenated cells, EVs, and / or exosomes generated as described herein may be delivered to a patient having a condition, including but not limited to BMT failure, BMT for either hematological conditions such as MDS, leukemia, aplastic avenue, neurological conditions: cerebral palsy, ALS, stroke, Alzheimer, MS, liver diseases such as cirrhosis, skin conditions such as wound, burn lesions, and skin aging, joint and cartilage and muscle, and / or cardiovascular conditions in an amount effective to treat the condition.

[0173] In non-limiting embodiments, the condition is infertility and the method includes administered a rejuvenated cell (e.g., an egg cell) to the patient.

[0174] In non-limiting embodiments, the patient is in need of an organ transplant, and the method includes generating an organ bud by expanding one or more rejuvenated cells generated as described herein.

[0175] In non-limiting embodiments, an embryoid generated as described herein may be further processed, for example by isolating one or more regions of theheterogenous tissue. One or more of the isolated regions may then be used as a feeder layer, for example for expansion of one or more cells, for the production of a target factor, production of an exosome, production of extracellular matrix (ECM), and / or for rejuvenation of one or more cells.

[0176] A schematic of an exemplary method is depicted in FIG. 1 . In a first step S100 a target environment (e.g., an embryoid) is generated by, in non-limiting embodiments, culturing one or more first cells, which may be stem cells, with one or more second cells, which may also be stem cells (the same and / or different cells than the first cells).

[0177] The first stem cells, such as human induced pluripotent stem cells (hiPSCs), may be engineered to express GATA6, e.g. human GATA6. In non-limiting embodiments, the second cells do not express GATA. Expression of the GATA6 may be under control of an inducible promoter, such as a doxycycline-inducible promoter (e.g., TET-ON). The stem cells may be cultured on a surface, such as a tissue culture plate, flask, or bioreactor, in stem-cell growth media until reaching a density of 20,000- 40,000 cells / cm2. The surface on which the cells are cultured may comprise an extracellular matrix material or basement membrane, such as an hESC-qualified growth substrate, e.g., hESC-qualified MATRIGEL.

[0178] After the cell culture is initiated, expression of GATA6 may be induced and may be maintained until an embryoid tissue is formed. In non-limiting embodiments, the GATA6 is endogenous to the cell, and in non-limiting embodiments, the GATA6 is exogenous. In non-limiting embodiment, the GATA6 is human GATA6. This development may take from 7-14 days. Alternatively, GATA6 mRNA may be added to the cells, such as mRNA in a solid lipid nanoparticle or lipidic vesicle (see, e.g., Melamed JR, etal. 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). The GATA6 may be added directly to the cells as a protein, for example in a vesicle for endocytosis, or other protein delivery methods (see, e.g., Ray M, et al. Intracellular delivery of proteins by nanocarriers. Nanomedicine (Lond). 2017 Apr;12(8):941 -952). The provision and / or expression of GATA6 may be transient.

[0179] Non-limiting embodiments of step S100 are disclosed in, for example and without limitation, International Patent Application Publication No. 2024 / 073665 and Hislop et al., Modelling post-implantation human development to yolk sac blood emergence. Nature 626, 367-376 (2024), each of which is incorporated herein by reference in its entirety.

[0180] As described above, the co-culture of the first cells and the second cells generates an embryoid. At this juncture, a number of further methods are possible, as described herein and as shown, without limitation, in FIGS. 1-5.

[0181] In non-limiting embodiments, the one or more second cells, which may also be stem cells, CD34+ cells, such as CD34+ cord blood, bone marrow, or mobilized peripheral blood, are added to the fetal liver / yolk sac organoids and are cocultured, e.g., with appropriate amounts of stem cell factor (SCF, e.g., human recombinant SCF), thrombopoietin (TPO, e.g., human recombinant TPO (hTPO)), and / or FMS-like tyrosine kinase 3 ligand (FLT3LG, e.g., human recombinant FLT3LG) effective to produce CD71 + cells. All cytokines are commercially-available and / or their amino acid and cDNA sequences are broadly-known such that a person of ordinary skill in the art can synthesize or otherwise obtain sufficient quantities of each cytokine. The amount of cytokine added is sufficient and effective to induce production of CD71 + cells from CD34+ HPSCs when co-cultured with a fetal liver / yolk sac organoid as described herein. Of note, very small quantities of the cytokines may be necessary as compared to monoculture of CD34+ HPSCs, further adding cost savings to the claimed methods, e.g., less than 500 ng / pL and at least 25 ng / pL, e.g., less than 200 ng / pL for SCF and FLT3LG, and less than 100 ng / pL for TPO, such as, for example and without limitation, rhSCF and rhFLT3LG at 100 ng / pL and rhTPO at 50 ng / pL.

[0182] A fetal liver / yolk sac organoid (e.g., an embryoid) is a synthetic tissue prepared according to methods described herein. Pluripotent stem cells are cultured with GATA6 protein present, for example either expressed from a trans-gene, translated from exogenously-introduced mRNA, or introduced exogenously as a protein. The GATA6 protein, and optionally later-introduced cytokines, such as 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, act to differentiate the pluripotent stem cells to form an organoid that phenotypically may be identified as fetal liver or yolk sac (as a developmental precursor to fetal liver), and may have mesodermal, endodermal, and ectodermal layers, elements, or parts. Once thepluripotent stem cells are at least partially differentiated by the GATA6 protein, CD34+ HPSCs may be added, and cultured in the optional presence of the cytokines, e.g., one or more of thrombopoietin, stem cell factor, and / or FLT3 ligand for a length of time sufficient for production of CD71 + cells, such as 5, 6, 7, 8, 9, or 10 days, e.g., 6 or 7 days, at which time the CD71 + cells can be washed or aspirated from the organoids. The process may be repeated by seeding and culturing additional CD34+ HPSCs on the organoids. The pluripotent stem cells do not necessarily have to be autologous to a patient to be treated with the CD71 + cell product, but the CD34+ cells may be preferably autologous (the patient’s own cells).

[0183] Hematopoietic stem and progenitor cells (HSPCs) are a cell population in the bone marrow capable of self-renewal and multi-lineage 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. CurrStem Cell Rep. 2018 Dec;4(4):273-281 ), bone marrow, and umbilical cord blood. The HPSCs may be obtained from cord blood of the patient (if available), or from the patient’s bone marrow cells, such as obtained from the patient’s bone marrow, or mobilized bone marrow cells available in a patient’s blood.

[0184] As shown in FIG. 1 , the method can include: generating a target environment S100 and extracting cells from the target environment S400. However, the method can additionally or alternatively include any other suitable steps, for example those shown in FIGS. 2-6.

[0185] In variants, the method can function to perform cell rejuvenation, cell expansion, and / or cell production (e.g., cell manufacturing) as described herein. For example, the method can function to rejuvenate, expand, and / or produce cells for a cell therapy.

[0186] As shown in FIG. 1 , the method can include: generating a target environment S100 and extracting cells from the target environment S400. The method can optionally include exposing cells to the target environment S200, producing new cells from the target environment S300, administering a therapy using the extracted cells S500, and / or any other suitable steps.

[0187] Generating a target environment S100 functions to create a platform for cell rejuvenation, cell expansion, and / or cell production. The target environment caninclude cells (e.g., an embryoid model), factors (e.g., target molecules), media, and / or any other components. The target environment (e.g., the composition of the target environment) can optionally be specific to the cell type of the cells exposed to the target environment in S200 and / or the cell type of the cells produced in S300. The target environment can optionally be contained within a vessel (e.g., a well). The target environment can optionally change over time (e.g., while cells are exposed to the target environment during S200 and / or before the cells are exposed to the target environment). For example, the target environment can include a first target environment composition for a first set of days (e.g., day 0 to day 5) and a second target environment composition for a second set of days (e.g., day 5 to day 10).

[0188] In non-limiting embodiments, the target environment includes an embryoid model. The target environment can optionally additionally include media, supplementary molecules, secretome molecules (e.g., molecules released by the embryoid model), and / or any other components. In an example, supplementary molecules can include growth factors, inducer molecules, and / or any other added molecules. In an example, the secretome molecules can include factors (e.g., as described below).

[0189] The embryoid model can include a set of cells configured to simulate all or a portion of embryogenesis. The set of cells in the embryoid model are preferably human cells, but can alternatively be other suitable cells. The set of cells in the embryoid model are preferably stem cells and / or stem cell derivatives, but can alternatively be other suitable cells. The set of cells in the embryoid model can be derived from a subject (e.g., a patient undergoing a cell therapy, the same subject used to derive cells in S200, a donor, etc.). The embryoid model can optionally simulate an embryo at or beyond a threshold stage (e.g., at least day 5, at least day 10, at least day 14, at least day 21 , at least day 28, etc.).

[0190] In an example, the embryoid model can include a first subset of cells simulating epiblast cells and a second subset of cells simulating yolk sac cells. The ratio between the first subset of cells and the second subset of cells can be between 1 :50 - 1 :2 or any range or value therebetween (e.g., 1 :20 - 1 :3), but can alternatively be less than 1 :50 or greater than 1 :2. In a specific example, the ratio can be selected based on the target environment composition. Within the second subset of cells, the number of transgenes (e.g., GATA6) expressed in each cell can optionally vary. For example, at seeding, one or more clones with different transgene expression countscan be used (e.g., wherein the composition can be selected based on the target environment composition). In a specific example, the second subset of cells can include: wildtype cells, low copy number clones, medium copy number clones, and / or high copy number clones. In a specific example, the number of copies of GATA6 in the low copy number clones can be between 1 -5 or any range or value therebetween (e.g., 1 , 2, 3, 4, 5, less than 2, less than 5, etc.), but can alternatively be greater than 5. In another specific example, the number of copies of GATA6 in the medium copy number clones can be between 2-20 or any range or value therebetween (e.g., at least 2, at least 3, 2-4, 2-10, greater than the number of copies of GATA6 in the low copy number clones, less than the number of copies of GATA6 in the high copy number clones, etc.), but can alternatively be greater than 20. In another specific example, the number of copies of GATA6 in the high copy number clones can be between 3- 100 or any range or value therebetween (e.g., at least 3, at least 4, 4-10, etc.), but can alternatively greater than 100. The percent of wildtype cells within the second subset of cells can be between 10%-100% or any range or value therebetween. The percent of low copy number clone cells within the second subset of cells can be between 0%- 50% or any range or value therebetween. The percent of medium copy number clone cells within the second subset of cells can be between 0%-50% or any range or value therebetween. The percent of high copy number clone cells within the second subset of cells can be between 0%-50% or any range or value therebetween.

[0191] The embryoid model can optionally be cultured in media for a period of time (e.g., at least 1 day, at least 5 days, at least 10 days, at least 14 days, etc.) prior to S200 and / or S300. The media composition can optionally change over time during culturing (e.g., embryonic media can be swapped for basal media). The day of the media change can be between day 2-day 10 or any range or value therebetween (e.g., day 5), but can alternatively be before than day 2 or after day 10. The embryoid model can optionally be exposed to an inducer molecule (e.g., doxycycline) during culturing. The concentration of the inducer molecule can be between 0.1 pg / mL -1 Opg / mL or any range or value therebetween (e.g., approximately 1 pg / mL), but can alternatively be less than 0.1 pg / mL or greater than 10pg / mL. In a specific example, the concentration can be selected based on the target environment composition. The period of exposure to the inducer molecule can be between 1 hr - 10 days or any range or value therebetween, but can alternatively be less than 1 hr or greater than 10 days. In aspecific example, the exposure time can be selected based on the target environment composition.

[0192] All or a portion of the cells in the embryoid model can optionally be genetically modified. In a first example, the cells can be genetically modified to upregulate pathway(s) corresponding to one or more factors. In a second example, the cells can be genetically modified to add genetic safety switches (e.g., for removal in S400). In a third example, the cells can be genetically modified to add tags (e.g., for identification in S400).

[0193] In non-limiting embodiments, the target environment includes target factors, such as target molecules (the terms target factors and target molecules are used interchangeably herein). For example, the target environment can include targets molecules within cell media. In an example, the target molecules can be or include (as described elsewhere herein): vesicles, exosomes, proteins, ligands (e.g., hormones, growth factors, chemokines, cytokines, neurotransmitters, etc.), sugars, ions, metabolites, integrins, stimulatory factors, inhibitory factors, any signaling molecule, and / or any other molecule. In a specific example, the target molecules are ligands interacting with receptors of a cell (e.g., the cells exposed to the target environment in S200).

[0194] The target molecules can optionally be secreted by a cell (e.g., by one or more cells in the embryoid model). In a specific example, the target molecules can be all or a subset of the secretome of the embryoid model. The number of target molecules can be between 1 -50 or any range or value therebetween (e.g., 5-50, 20- 35, 10-20, approximately 32, less than 35, less than 10, less than 6), but can alternatively be greater than 50. The concentration of a target molecule in the target environment (e.g., when diluted in media) can be between 0.1 ng / mL-10ng / mL or any range or value therebetween (e.g., 0.3ng / mL-3 ng / mL, at least 0.2ng / mL, at least 0.3ng / mL, at least 0.5ng / mL, at least 1 ng / mL, etc.), but can alternatively be less than 0.1 ng / mL or greater than 10ng / mL. In a specific example, the concentration is above a naturally occurring concentration in the secretome of a human embryo.

[0195] In an example, the target molecules can include one or more molecules (e.g., ligands) in the following ligand-receptor pairings (ligand on the left, receptor on the right): MDK-NCL, MDK-(ITGA4+ITGB1 ), APP-CD74, FN1 -CD44, COL1A1 -CD44, COL1A2-CD44, MIF-(CD74+CD44), MIF-(CD74+CXCR4), IGF2-IGF1 R, PTN-NCL, LAMB1 -CD44, DLK1 -NOTCH1 , JAG1 -NOTCH1 , PRSS3-F2R, F2-F2R, PLG-F2RL3,LGALS9-CD45, LGALS9-CD44, CDH1 -KLRG1 , ITGB2-ICAM2, JAM1 - (ITGAL+ITGB2), EFNB3-EPHB6, PECAM1 -PECAM1 , RETN-CAP1 , ICAM2- (ITGAL+ITGB2), TGFB1 -(TGFBR1 +TGRBR2), GDF15-TGFBR2, THBS1 -CD47, SELPLG-SELL, SIGLEC1 -SPN, and / or SEMA3C-PLXND1 .

[0196] In non-limiting embodiments, target molecules are directly added to cell media. In a second embodiment, cells (e.g., cells in S200 and / or other cells) are engineered to produce the target molecules. In a first example of the second embodiment, the cells are transfected with mRNA encoding the respective sequences for all or a portion of set of target molecules, wherein the mRNA can be translated to produce one or more target molecules. In a specific example, the mRNA can be delivered to the cells via lipid nanoparticles. In non-limiting embodiments, the cells are genetically modified to produce one or more target molecules and / or to produce one or more target molecules at a higher concentration. In non-limiting embodiments, the target molecules are secreted by the embryoid model (e.g., as described in the first variant). In non-limiting embodiments, a combination of the previous embodiments can be used. For example, target molecules secreted by the embryoid model can be supplemented with additional target molecules added to the cell media.

[0197] The method can optionally include exposing cells to the target environment S200, which can function to rejuvenate and / or expand cells. For example, S200 can function to reprogram cells to return to an early cell state (e.g., a fetal cell state). S200 can be performed after S100 and / or at any other time.

[0198] The cells exposed to the target environment can include hematopoietic stem cells (HSCs), skeletal muscle stem cells, mesenchymal stem cells, any other stem cells, germ cells, gametes, microglia, blood cells and / or blood cell derivatives (e.g., monocytes, lymphocytes, neutrophils, eosinophils, basophils, macrophages, erythrocytes, platelets, and / or any suitable blood cell derivatives), megakaryocytes, dendritic cells, pancreatic islet cells, diseased and / or modified stem cells (e.g., cells from a subject with sickle cell disease, leukemia, and / or any suitable disease or modification), and / or any suitable cells. In a specific example, the cells can be or include CD34 cells.

[0199] The cells can be derived from a subject (e.g., the same subject used to derive the embryoid model cells or a different subject). In non-limiting embodiments, the cells can be derived from (e.g., extracted from) a patient, wherein the cells and / or other cells derived therefrom are later transplanted back into the patient in anautologous transplant (e.g., in S500). In non-limiting embodiments, the cells can be derived from a donor, wherein the cells and / or other cells derived therefrom are later transplanted into a patient in an allogeneic transplant (e.g., in S500). In non-limiting embodiments, the cells can be derived from a "universal donor", wherein the cells can optionally be continuously expanded (e.g., using all or parts of the method) from a donor cell line. The cells are preferably cells derived from an adult, but can alternatively be any other suitable cells. For example, the cells can be extracted from a subject over a threshold age (e.g., at least 1 year old, at least 5 years old, at least 10 years old, at least 15 years old, at least 25 years old, and / or any suitable age). After exposure to the target environment, the cells can optionally resemble a target developmental age (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, etc.). The cells preferably have limited or no expansion capabilities prior to exposure to the target environment but can alternatively be able to expand. The cells preferably have increased expansion capabilities after exposure to the target environment but can alternatively not have increased expansion capabilities.

[0200] The cells can be exposed to the target environment for a period between 1 day - 50 days or any range or value therebetween (e.g., 2 days-20 days, approximately 10 days, at least 5 days, and / or any suitable duration), but can alternatively be less than 1 day or greater than 50 days. Media changes performed during this step can optionally be partial media changes (e.g., half media changes) instead of complete media changes, such that target molecules remain present.

[0201] In non-limiting embodiments, exposing the cells to the target environment can include co-culturing (e.g., incubating) the cells with the embryoid model. In a first example, the cells are directly in contact with the embryoid model. In a second example, the cells are separated from the embryoid model via a permeable membrane (e.g., allowing target molecules to pass through). In non-limiting embodiments, exposing the cells to the target environment can include exposing the cells to target molecules by supplying the cells with media that contains the target molecules (e.g., manually adding target molecules to the media). In non-limiting embodiments, exposing the cells to the target environment can include exposing the cells to target molecules by engineering the cells to produce the target molecules, as described in S100. In non-limiting embodiments, exposing the cells to the target environment can include a combination of the previous variants.

[0202] The method can optionally include producing new cells from the target environment S300, which functions to de novo manufacture new cells. S300 can be performed after S200 and / or at any other time.

[0203] The new cells can include blood cells, blood cell derivatives, germ cells, gametes, neuro progenitors (e.g., brain, spinal cord, etc.), intestine progenitors, gallbladder progenitors, bone progenitors, muscle progenitors, cardiac progenitors, skin progenitors, any other organ progenitors, fetal cells, and / or any other cell type. In a specific example, the new cells can include hemoglobin (e.g., hemoglobin F).

[0204] In an example, the new cells are produced by the embryoid model (e.g., the embryoid model generated via S100 methods). For example, after a period of time of culturing the embryoid model (e.g., 1 day, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.), the embryoid model can produce one or more (desired) new cells. The new cells can then be extracted as described in S400. The media composition used to culture the embryoid model can optionally be specific to the cell type being produced.

[0205] Extracting cells from the target environment S400 functions to extract cells that can be used for cell therapy, used as a donor cell line, and / or used for any other downstream use case. S400 can be performed after S200, after S300, and / or at any other time.

[0206] In non-limiting embodiments, the extracted cells can be the rejuvenated and / or expanded cells from S200. In non-limiting embodiments, the extracted cells can be the new cells produced from S300. In non-limiting embodiments, the extracted cells can be a combination of rejuvenated and / or expanded cells from S200 and new cells produced from S300.

[0207] In an example, S400 can include identifying target cells and separating the target cells from other cells (e.g., from the embryoid model cells, from other nontarget cells, etc.). For example, the target cells can be: cells with a target cell type, cells that are not the embryoid model cells, cells that are rejuvenated, and / or any other target cell. S400 can optionally include identifying target cells. S400 can optionally include identifying non-target cells and / or removing non-target cells. In non-limiting embodiments, genetic safety switches (e.g., kill switches) in the embryoid model cells can be activated to remove non-target cells.

[0208] All or a portion of S400 can use cell sorting techniques. For example, the cell sorting techniques can include Fluorescence-activated cell sorting (FACS),Magnetic-activated cell sorting (MACS), Buoyancy Activated Cell Sorting (BACS), and / or any other suitable cell sorting methods.

[0209] The method can optionally include administering a therapy using the extracted cells S500, which functions to treat a disease, to provide a longevity therapy, and / or for any other treatment. For example, the extracted cells can form all or a portion of the therapy. Examples of therapies include: blood cell therapy, oocyte culture and / or regeneration (e.g., prior to in vitro fertilization), generation of organ buds for transplants, and / or any other therapy using the extracted cells. The therapy can be a stem cell therapy or a non-stem cell therapy.

[0210] In non-limiting embodiments, the therapy can be a monotherapy. For example, the therapy can be or include extracted (rejuvenated and / or expanded) CD34 cells. In a second variant, the therapy can be a combination therapy. For example, the therapy can be or include CD34 cells and one or more additional cells (e.g., CD33, CD71 , CD43, and / or any suitable additional cells). In non-limiting embodiments, the additional cells can increase the efficiency of engraftment.

[0211] In non-limiting embodiments, rejuvenated and / or expanded cells can be used as all or a portion of a cell therapy. For example, hematopoietic stem cells (HSCs) can be used as a cell therapy (e.g., for patients experiencing HSC transplantation failure). In non-limiting embodiments: HSCs can be extracted from a patient (e.g., a patient experiencing HSC transplantation failure); the HSCs can be rejuvenated and / or expanded using all or a portion of the method; and the resulting rejuvenated and / or expanded cells can be transplanted back to the patient (e.g., an autologous transplant). In non-limiting embodiments, HSCs can be extracted from a donor; the HSCs can be rejuvenated and / or expanded using all or a portion of the method; and the resulting rejuvenated and / or expanded cells can be transplanted to the patient (e.g., an allogeneic transplant). In non-limiting embodiments, new cells produced using all or a portion of the method can be used as all or a portion of a cell therapy.

[0212] A composition can optionally include a set of cells. The set of cells can optionally be modified (e.g., genetically modified, rejuvenated, etc.) and / or produced using all or parts of the method. The set of cells can include any cells described herein. In a first example, the set of cells can include rejuvenated cells. In non-limiting embodiments, cells derived from an adult are rejuvenated via exposure to the target environment (e.g., as described in S200). In non-limiting embodiments, the set of cellscan include expanded cells. In non-limiting embodiments, cells with limited or no capacity to expand (e.g., cells derived from an adult) are rejuvenated via exposure to the target environment (e.g., as described in S200), leading to cell population expansion. In non-limiting embodiments, the set of cells can include new cells produced using the embryoid model (e.g., as described in S300). The new cells can be cells of a single cell type or a combination of cell types. In non-limiting embodiments, the set of cells can include all or a portion of the embryoid model. In non-limiting embodiments, the set of cells can include a combination of cells in one or more of the previous examples.

[0213] The composition can optionally include a cell therapy that includes or uses the set of cells (e.g., a therapy as described in S500).

[0214] All or a portion of the set of cells can optionally be genetically modified (e.g., genetically engineered, edited, produced, generated, etc.). In non-limiting embodiments, the embryoid model is genetically modified (e.g., as described in S100). In non-limiting embodiments, a set of cells are genetically modified to produce one or more target molecules and / or to produce one or more target molecules at a higher concentration.

[0215] The composition can optionally include a genetically modified sequence. One or more sequences (e.g., DNA sequence, RNA sequence, mRNA sequence, etc.) can optionally be genetically modified using all or parts of the method. In a first example, the sequence can include a nucleic acid sequence (e.g., mRNA sequence, DNA sequence, etc.) encoding the respective sequences for all or a portion of set of target molecules. In non-limiting embodiments, the sequence can be an mRNA sequence delivered to a set of cells, wherein the mRNA sequence can be translated to produce one or more target molecules.

[0216] The composition can optionally include the set of target molecules. For example, the composition can include a media containing the set of target molecules. In non-limiting embodiments, the set of target molecules can be the target molecules (e.g., as described in S100).

[0217] Without wishing to be bound by the theory, it is believed that an embryoid as described herein enables the de novo generation of primitive hematopoietic progenitors within an organized yolk sac-like niche. These progenitors may give rise to a diverse pool of macrophages and monocytes in vitro, which may transcriptionally resemble early embryonic-derived tissue-resident macrophages. Early data reveals,based on single-cell RNA sequencing, gene expression profiles in these cells that are highly similar to human fetal microglia, liver Kupffer cells, and / or yolk sac-derived monocytes.

[0218] The present embryoids and methods of making and using the same allow for scalable and reproducible production of early embryonic-like macrophages from pluripotent stem cells using a structured, self-organizing embryoid platform. These macrophages can be harvested either as functional cells or as a source of secreted factors (e.g., cytokines, growth factors) and extracellular vesicles (EVs), including exosomes. EVs secreted by these cells are enriched in bioactive RNAs, microRNAs, and proteins that may contribute to immunomodulation and tissue repair.

[0219] With regard to uses, direct transplantation of macrophages derived from embryoids described herein into injured or diseased tissues (e.g., brain, liver, skin, lungs) can promote repair, modulate inflammation, and restore homeostasis. Additional uses include treatment of neurodegenerative diseases (e.g., Alzheimer's and / or Parkinson's) to deliver supportive signals or clear pathological aggregates. Liver regeneration in chronic injury or transplant models via Kupffer-like cell integration is also possible with the described embryoids.

[0220] In addition to use of the cells themselves, secretome and vesicle-based therapeutics may be generated from embryoids as described herein, including through generation and isolation of exosomes and other EVs from macrophage-conditioned media for use as acellular therapeutics, delivery of regenerative or immunomodulatory molecules via vesicles to target tissues, and / or development of vesicle-based RNA or protein therapies using the macrophage secretome.

[0221] Furthermore, embryoids, and the methods of making and using the same, may be useful in disease modeling and drug discovery, including in vitro disease modeling using macrophages with yolk sac ontogeny and high-throughput screening for compounds that modulate macrophage activity, migration, or secretion.

[0222] The embryoids, and methods, described herein provide benefits over existing technology, at least in autonomous production of yolk sac-like tissue, enabling a more rapid and scalable approach. The advances described herein further provide access to developmentally primitive macrophage subtypes not easily obtainable from adult hematopoietic differentiation, and combined use of cellular and acellular (EV- based) outputs can enhance therapeutic versatility.

[0223] In addition to the foregoing, the embryoids and methods described herein allow for directing and capturing spatiotemporally distinct hematopoietic lineage differentiation events within embryoids. These embryoids are capable of recapitulating early human hematopoiesis with high fidelity, including the emergence of lymphoid, myeloid, erythroid, and megakaryocytic lineages from distinct anatomical and endothelial niches. Advantages include the identification and use of specialized endothelial subtypes (e.g., sinusoidal, hemogenic) and cytokine-rich microenvironments that orchestrate lineage commitment, enabling applications in blood cell production, disease modeling, and therapeutic development.

[0224] Other benefits include the spatiotemporal compartmentalization of hematopoietic differentiation, as embryoids are capable of exhibiting spatially segregated differentiation of hematopoietic lineages, including:

[0225] CD7+ lymphoid progenitors, which may arise adjacent to or within the embryonic body, specifically within vascular-rich domains and often in close association with endothelial markers associated with developmental niches.

[0226] CD45+ myeloid and Hb+ erythroid cells, which may emerge independently of the embryonic body, distributed across the culture dish, suggesting distinct niche independence and possibly yolk sac-derived hematopoiesis.

[0227] Megakaryocytes may appear de novo in fibrotic or collagen-rich regions, where IL-1 1 expression is locally elevated, identifying a specialized niche for thrombopoiesis.

[0228] Further benefits may be seen in endothelial diversity recapitulation in vitro, including embryoids as described herein recapitulating developmentally relevant endothelial subtypes, including:

[0229] Sinusoidal endothelium (LYVE1 +), Arterial endothelium, Hemogenic endothelium capable of hematopoietic transition, and Von Willebrand Factor (vWF)- expressing endothelium mimicking coagulation-associated vasculature.

[0230] The presence of LYVE1 + sinusoidal endothelial cells surrounding lymphoid clusters can provide evidence of a hematopoietically active vascular niche, functionally analogous to fetal liver and yolk sac sinusoids. In addition, functional microenvironments for hematopoietic specification may be generated, allowing for discovery of IL-11 -producing zones in fibrotic areas, which supports a localized megakaryopoietic microenvironment. These preliminary data suggest that engineered or induced fibrosis within embryoids can serve as a controllable megakaryocytedifferentiation zone. Further vascular and stromal patterning enables mapping and manipulation of lineage emergence using small molecules, growth factors, or genetic engineering.

[0231] In addition to those compositions described herein, the embryoids and methods described herein allow for generation and use of compositions including embryoid-derived LYVE1 + endothelial cells co-localized with CD7+ lymphoid progenitors.

[0232] Also provided herein is a culture system containing hemogenic endothelium, sinusoidal and arterial endothelial subtypes, and fibrotic IL-1 1 + zones for multi-lineage blood cell differentiation, as well as vesicles or secreted factors derived from endothelial niches (e.g., LYVE1 + or hemogenic endothelium) with hematopoietic supportive functions.

[0233] Further applications of the embryoids and methods described herein include scalable production of lineage-specific blood and immune cells (e.g., T / NK progenitors, erythroid precursors, megakaryocytes), modeling congenital hematopoietic or vascular disorders associated with specific endothelial niches (e.g., primary immunodeficiencies, thrombocytopenia), and use of engineered fibrotic zones or endothelial subtype-specific domains for drug screening, regenerative therapy development, or tissue-specific immune cell programming.Example 1Materials and Methods

[0234] Cell Culture

[0235] All cells and tissues were placed in a humidified incubator set at 37°C with 5% CO2 for cultivation. Our hiPSC lines were grown in a sterile environment using mTeSR-1 medium (by Stem Cell Technologies, Vancouver) with daily medium changes. To prepare tissue culture plates, BD ES-qualified Matrigel (BD Biosciences) was applied and allowed to coat the plates at room temperature for 1 hour, diluted following the manufacturer's instructions and using ice-cold DMEM / F-12. For routine passaging, hiPSC colonies were incubated with Accutase (Sigma) up to a 5-minute at 37°C. Subsequently, the suspension was collected, mixed with 5 mL of DMEM / F-12 medium containing 10 pM Y-27632, and centrifuged at 300g for 5 minutes. After centrifugation, the cells were resuspended in DMEM / F-12 supplemented with 10 pM Y-27632 for cell counting purposes. The cells were then seeded at a density of 25,000 cells per cm2 for regular maintenance. Mesenchymal stromal cells were cultured inIMDM as basal media supplemented with FBS at 10% and GlutaMax, MEM Non- essential Amino Acids Solution, and Penicillin-Streptomycin at 1 x (Thermo Fisher Scientific). Cord blood CD34+ cells in control monocultures and co-cultures with heX- embryoid were cultured in IMDM as basal media supplemented with FBS at 10% and GlutaMax, MEM Non-essential Amino Acids Solution, and Penicillin-Streptomycin at 1 x supplemented with 100ng / mL human stem cell factor (SCF), 100ng / mL Flt3 ligand (Flt3L), and 50ng / mL human thrombopoietin (TPO). Cord blood CD34+ cells were seeded at 40,000 cells / mL and half the media volume per well was manually exchanged daily.

[0236] Production of Lentiviral Particles and Titration

[0237] HEK293FT cells (Life Technologies) were cultured following the manufacturer's recommendations in a humidified incubator at 37 °C with 5% CO2. One day before transfection, 8 million HEK293FT cells were seeded onto a collagen I- coated (Gibco A10483-01 ) 15 cm2tissue culture dish. On the day of transfection, cells were co-transfected with 15 pg psPAX2 (Addgene Plasmid 12260), 3.75 pg pCMV- VSV-G (Addgene Plasmid 8454), and 11 .25 pg of the plasmid to be packaged, using 90 pg of linear polyethylenimine (Polysciences, Inc 23966-1 ). The following morning, the medium was changed, and the supernatant was collected after 48 and 72 hours. Pooled supernatant was passed through a 0.45 pm 648 low protein binding filter (Corning) and concentrated in Amicon Ultra 15 filter columns (100 kDa cutoff, Millipore) at 4,000g for 23 minutes. The concentrated virus was then divided into aliquots, snap frozen, and stored at -80 °C. Lentiviral concentrate was diluted 2000- fold in phosphate buffered saline (PBS) and its titer was determined via qRT-PCR using a commercially available kit (ABM LV900). Titers were calculated following the manufacturer's instructions.

[0238] Design and Construction of Vectors

[0239] CDS cDNA for KITLG (Clone ID# HsCD0050821 1 ), THPO (Clone ID# HsCD00510750), and FLT3LG (Clone ID# HsCD00353661 ) were procured from DNASU. These plasmids were individually amplified from their respective vectors and sub-cloned into gateway entry vectors via a golden gate reaction. All four entry vectors were combined with gateway entry vectors for the hEF-1 a or AAT promoter into a lentiviral gateway destination vector using gateway cloning. The AAT promoter was designed through truncation of the endogenous AAT promoter. To create the doxycycline (Dox)-inducible GATA6 vector, pENTR_L1_hGATA6-2A-EGFP_L2, aspreviously published [ref 49 Ryan’s diss], was cloned into an All-in-One PiggyBac transposon destination vector from Addgene (Addgene plasmid ID: 80479) through gateway reaction.

[0240] Lentiviral Transduction

[0241] The pre-aliquoted lentiviral concentrate was rapidly thawed and kept on ice until needed. Subsequently, hiPSCs were transduced while adherently cultured in Matrigel-coated 48-well cell culture plates. They were exposed to either mTeSR-1 containing 8pg / mL polybrene, 10 pM Y-27632, and 1 pg / mL doxycycline or mTeSR-1 containing 8pg / mL polybrene. The medium was replaced the next day. The Multiplicity of Infection (MOI) was calculated by dividing the number of lentiviral infectious units (determined during the lentivirus titration step) by the number of cells seeded with the virus.

[0242] Tissue Harvest from Culture Dishes, RNA Extraction, qRT-PCR

[0243] Tissues were collected and underwent lysis by the addition of 500pLTrizol (Life Technologies) directly into the tissue culture well, followed by storage at -80 °C. For the subsequent extraction process, the lysate was thawed on ice, and 100pL of chloroform was introduced. After vigorous vortexing for 30 seconds, the mixture was then centrifuged at 12,000g for 15 minutes at 4°C. Following centrifugation, the aqueous phase was carefully transferred to a QIAGEN gDNA eliminator column and subjected to centrifugation at 10,000g for 1 minute at room temperature. The resulting flow-through was combined with an equal volume of 70% EtOH and transferred to an RNEasy mini spin column.

[0244] The remainder of the procedure adhered to the manufacturer's protocol for the RNEasy Plus Mini Kit (QIAGEN). Subsequently, cDNA was synthesized using the High-Capacity cDNA reverse transcription kit from Applied Biosystems. For qRT- PCR analysis, the SYBR Green intercalating dye from ThermoFisher Scientific was utilized. Expression data were normalized to 18S ribosomal RNA, and the relative gene expression was calculated using the 2-AACT method. Detailed information regarding the primers used for qRT-PCR are listed in Table 1 , below:Table 1

[0245] Generation of heX-embryoid and heX-embryoid -Cord Blood CD34+ cells Co-culture

[0246] Gata6 / Dox-inducible hiPSCs were initially seeded at a cell density of 100,000 cells per ml (equivalent to 25,000 cells / cm2). Over the next five days, the culture medium was replaced daily700 with mTeSRI supplemented with Dox at a concentration of 1 microgram per milliliter. On the fifth day, the culture medium was transitioned to IMDM as the base medium, supplemented with 10% FBS, GlutaMax, MEM Non-essential Amino Acids Solution, and Penicillin-Streptomycin at a 1 x concentration.

[0247] Upon reaching the tenth day of heX-embryoid culture, cord blood CD34+ cells were introduced to the culture at a cell density of 40,000 cells per milliliter (equivalent to 10,000 cells / cm2). Simultaneously, the medium was enriched with 100 ng / ml of hSCF, 100 ng / ml of h Flt3L, and 50 ng / ml of hTPO. Throughout the co-culture period, half of the medium volume in each well was manually replaced daily, up to a duration of ten days. To harvest the CD34+ cells, the wells were gently pipetted by manually aspirating and expelling the contents, followed by a single wash with PBS.

[0248] To investigate the enduring presence of HSCs within the culture, a passaging procedure was employed. Following ten days of co-culture and the subsequent harvest of cord blood cells, cell counting was performed. These harvestedcells were then re-seeded onto fresh heX-embryoid D10 cultures, maintaining a consistent cell density of 10,000 cells per milliliter, up to how many wells that was possible based on the harvested cells counts. These co-cultures were sustained for an additional ten days before another harvest and subsequent seeding onto fresh heX- embryoid D10 niches. The cumulative cell counts for each well in each passage was recorded. This iterative process continued until the point was reached where the initial cell population from the monoculture controls was exhausted and could no longer be introduced into the culture. At this juncture, the experiment was terminated.

[0249] Enzyme-linked Immunosorbent Assays (ELISA)

[0250] Samples were assayed for SCF, FLT3L, and TPO according to manufacturer’s instructions, using R&D Systems DuoSet ELISA kits. Sample dilutions were optimized to attain detection in the linear range of the standard curves for each individual assay.

[0251] Cord Blood Processing and CD34+ cells Isolation

[0252] Human cord blood samples were sourced either from the University of Arizona Biorepository in Tuscon, AZ or obtained via Vitalant in Pittsburgh, PA. The isolation of CD34+ cells was conducted following the guidelines provided by the manufacturer for the EasySep Human Cord Blood CD34 Positive Selection Kit II (Stem Cell Technologies Cat# 17896).

[0253] To fractionate the cord blood, it was initially diluted at a 1 :1 ratio with PBS containing 2% FBS and 1 mM EDTA. Subsequently, 30mL of the diluted blood was gently layered over 15mL of Lymphoprep (Stem Cell Technologies Cat# 07801 ). The tubes were then centrifuged at 1200x g for 20 minutes with the brake off. The buffy coat, which represents the enriched mononuclear cell layer, was carefully separated and washed with PBS containing 2% FBS and 1 mM EDTA.

[0254] The isolation of CD34+ cells involved incubating the mononuclear cells with a CD34+ selection cocktail and RapidSpheres. The CD34+ cells were magnetically isolated using "The Big Easy" EasySep magnet (Stem Cell Technologies Cat# 18001 ), following the manufacturer's provided instructions. These isolated CD34+ cells were either utilized immediately or preserved through cryopreservation.

[0255] Colony Forming Unit Assays

[0256] Colony forming unit assays were conducted using methylcellulose- based media (specifically, MethoCult H4434 Enriched and MethoCult SF H4636 from StemCell Technologies) following the manufacturer's guidelines. Cord blood CD34+cells, which had been expanded either in monoculture or coculture, were plated at either 1 ,000 or 2,500 cells per plate. After a 14-day incubation period, the plates were visually examined to assess the presence of CFU multilineage colonies.

[0257] Mice

[0258] NSG (Jackson Laboratories) and NBSGW (Jackson Laboratories) mice were purchased from the vendor and subsequently bred in the BST3 animal facility at the University of Pittsburgh. They were kept in sterile conditions in sterile cages, provided with free access to sterile water and food suitable for immunocompromised mice. Food and water were changed manually in a biosafety cabinet to maintain the sterile condition. Mice were kept in room temperature with a 12 / 12-hour light / dark cycle.

[0259] Transplantation

[0260] Transplantations were performed either via retro-orbital (RO) injection of adult mice (8-12 weeks) or intrahepatic (IH) injection of neonatal pups. For RO injections mice were anesthetized with isoflurane, at least 4 hours after 250 cGy gamma-irradiation for adult mice or 100 cGy for the pups and administered 100 mL of cell solution using a 1 cc syringe with a 29-gauge 2' to the orbital sinus with the bevel directed away from the eyeball to prevent damage to the eye. To perform the IH injections, pups were sedated by cold until gross movement ceased, which took approximately 5 minutes. Each pup was lightly scuffed on the back in the shoulder area and held in a supine position to visualize the abdomen. The stomach of nursing pups appears as a large milky white spot on the right side of the abdomen. To the left and slightly above the stomach, the liver appears as a large reddish organ just below the rib cage. 30 pl of cell solution was injected using a 0.3 cc syringe with a 31 -gauge1 / 2" needle IH. The needle was inserted bevel up to allow visualization of the inoculum being injected. After the proper volume was dispensed, to prevent backflow the needle was held in place 781 for a few seconds before being withdrawn slowly. Pups were wrapped in nestlet material from their original cage until movement resumed and then were returned to their mother where they remained until weaning at 21 days of age. If a dam was not immediately attentive to the returning pups, the pups were marked with the dam’s scent by encouraging her to urinate on to the pups or on to the investigator’s gloved hands which were then used to rub the pups’ skin.

[0261] Tissue harvest and sampling

[0262] Peripheral blood was collected via cheek puncture with a lancet and collected in EDTA-coated tubes for monitoring of the engraftment after transplantation. No more than 100-200 pl per mouse was collected per month. At the end point of the study, between 20-24 weeks following transplantation, mice were euthanized using a CO2 chamber and peripheral blood and tissues, including spleen, liver, lung, kidney, and femoral and tibial bone marrow were collected. From each tissue, small pieces of each tissue were placed in formalin 10% to be fixed for histopathological studies, small pieces were placed in cryovial and were snap-frozen in liquid nitrogen, and the rest were kept in HBSS on ice to be used subsequently for single-cell solution sample preparation for flow cytometry.

[0263] Limiting Dilution Transplantation (LDA) Assay

[0264] Adult female NSG mice were transplanted via RO injection according to the routine injection protocol following gamma-irradiation with different cell counts of the harvested cells from the co-cultures and monocultures. Results regarding reconstitution of human CD45+ cells in transplanted mice after harvesting the animals were analyzed using the Walter and Eliza Hall Institute of Biomedical Research Extreme Limiting Dilution Analysis website (https: / / bioinf.wehi.edu.au / software / elda / ).

[0265] Flow Cytometry

[0266] Following the preparation of single-cell solutions from peripheral blood, spleen, and bone marrow samples, with red blood cell lysis accomplished using ACK lysis or following harvesting expanded cord blood cells from the co-cultures and monocultures, a series of steps were carried out. The cells were initially placed into flow cytometry tubes and subjected to centrifugation at 300g for 5 minutes. After discarding the supernatant, 50uL of Hanks' balanced salt solution (HBSS) without calcium, magnesium, and phenol red, supplemented with 5% FBS and a 1 :50 dilution of FCG receptor blocker, was added to the pellet in each tube. Gentle flicking was performed to ensure resuspension, followed by a 10-minute incubation at room temperature.

[0267] Subsequently, 100 uL of HBSS with 5% FBS, along with the appropriate flow cytometry antibodies at a 1 :100 dilution, were introduced into each tube and mixed by vertexing. The tubes were then incubated on ice in the dark for a duration of 30 minutes. To complete the preparation, 2 ml of HBSS with 5% FBS was added to each tube, and the cells were pelleted by centrifugation at 300g for 5 minutes. Following this, the supernatant was removed from each tube, and the cells wereresuspended in 100 uL of HBSS with 5% FBS and 7-AAD at a 1 :40 dilution. Flow cytometry analysis (FACS) was subsequently performed while maintaining the cells on ice and in the dark. The list of antibodies: anti-human CD34 (clone, APC, PerCP- Cy5.5, BioLegend), anti-human CD38 (clone, Pacific Blue, PE, BioLegend), antihuman CD45RA (clone, APC-Cy7 or BV785, BioLegend), anti-human lineage cocktail (clone, FITC, BioLegend), anti-human CD90 (clone, PE, BioLegend), anti-human and mouse CD49f (clone, PE-Cy7, BioLegend), anti-human CD71 (clone, PE-dazzle, BioLegend), anti-human CD235a (clone, PE-Cy7, BioLegend), anti-human CD45 (clone, APC or PE or APC-Cy7, BioLegend and BD Biosciences), anti-mouse CD45 (clone, Pacific Blue or BUV 395, BioLegend and BD Biosciences), anti-human CD19 (clone, BV605 or BV510, BioLegend), anti-human CD33 (clone, APC-Cy7 or BV605, BioLegend), anti-human CD3 (clone, Alexa Fluor 700 or PE-Cy7, BioLegend), antihuman CD56 (clone, BV421 or PE-Cy7, BioLegend), Hoechst 33258 (), EdU (Eterneon™ Red 645 Azide, Alexa Fluor 647, BD Biosciences), Zombie NIR fixable viability kit (APC-Cy7, BioLegend).

[0268] Cell Cycle Assay

[0269] To investigate the cell cycle stages in both the heX-embryoid and cytokine-only, we employed a flow cytometric-based EdU assay using the BD Pharmingen™ 647 EdU Click Proliferation Kit (BD Biosciences). Cells in heX- embryoid and cytokine-only culture plates were incubated with EdU for 3 hours, harvested as described earlier, and proceeded to the staining process. Following staining with Zombie NIR fixable live / dead dye in a PFA 4%-based solution, cells were fixed. The click reaction was then performed according to the manufacturer's protocol for the EdU assay, and staining for other surface markers was carried out. The cell cycle phases were subsequently identified by using DNA content staining with Hoechst 33258 in the Lin-CD34+CD38- population of heX-embryoid - and cytokine-only- expanded cells.

[0270] Immunohistochemistry

[0271] Tissue samples collected from mice that underwent cord blood transplantation underwent a series of preparation steps. Initially, these samples were rinsed in PBS and immersed in a 10% formalin solution and were kept overnight. After 18-24 hours, the samples were washed 3 times with PBS, transferred to a 70% ethanol solution, and delivered to University of Pittsburgh Histology Core Laboratory for paraffin embedding and sectioning and 4-pm slices were obtained from each sample.Upon the return of the slides, they underwent further processing. The slides were immersed three times in fresh xylene for 5 minutes each, followed by two washes of 10 minutes each in 100% ethanol, two washes of 10 minutes each in 95% ethanol, and two additional rinses in deionized (DI) water. To facilitate antigen retrieval, the slides were submerged in 1 x citrate buffer (Abeam) and subjected to near-boiling conditions in a microwave oven for 15 minutes. Afterward, the slides underwent three rinses in DI water for 5 minutes each, followed by one rinse in PBS.

[0272] To prepare the samples for immunostaining, they were blocked and permeabilized in a solution of PBS with 8% donkey serum and 0.2% TritonX-100 for a duration of 2 hours at room temperature. Subsequently, primary antibodies were applied to the samples and left to incubate overnight at 4°C. Following this incubation, the slides were rinsed three times in DI water and then washed for 15 minutes in PBS.

[0273] Secondary antibodies were introduced to the samples in a solution of PBS with 2% donkey serum, allowing them to incubate for 2 hours at room temperature. Following this step, the tissues were counterstained with either DAPI or Hoechst, followed by three rinses in DI water, a 15-minute wash in PBS, and sealing with a glass coverslip. These prepared slides were then ready for imaging, and two types of mounting media, Diamond Antifade (Thermo Scientific) or Vectorshield (Vector Labs), were utilized depending on the imaging process.

[0274] Immunofluorescence Staining on Glass Coverslips

[0275] Cells were cultured on circular glass coverslips, either 8mm or 12mm in diameter, that had been pre-coated with Matrigel. The cultures were then fixed at room temperature for a duration of 20 minutes using a 4% paraformaldehyde solution from Electron Microscopy Sciences. Following fixation, the coverslips underwent a thorough washing process, including three rinses with PBS, and were subsequently subjected to a 15-minute permeabilization step with 0.2% Triton X-100 in PBS.

[0276] After permeabilization, the coverslips were washed three times for 5 minutes each with a washing buffer containing 0.05% Tween-20 in PBS and were then blocked for 20 minutes using a solution consisting of 200 pl of wash buffer combined with 5% normal donkey serum from Jackson ImmunoResearch Laboratories.

[0277] The primary antibodies were diluted in PBS containing 5% normal donkey serum and were applied to the tissues for a duration of 1 hour at room temperature. Following this incubation, the coverslips were subjected to three additional washes, each lasting 5 minutes, using the wash buffer.

[0278] Similarly, the secondary antibodies were diluted in PBS with 5% normal donkey serum and were applied to the tissues for 1 hour at room temperature. Subsequently, the coverslips underwent three more washes, each lasting 5 minutes, using the wash buffer.

[0279] To complete the preparation of the coverslips for microscopy, they were mounted onto microscopy glass slides using Prolong Diamond Antifade from Life Technologies. Afterward, they were allowed to cure overnight at room temperature and then sealed with nail polish.

[0280] Wright-Giemsa Staining

[0281] The Wright-Giemsa staining process, in accordance with the manufacturer's instructions (Abeam Cat# 245888), involved several key steps to achieve optimal staining results. Initially, cells were transferred onto a clean microscope slide and left to air dry. Following this, the cells were fixed by immersing the slide in absolute Methanol for a duration of 5 minutes. Subsequently, the slide was placed in a staining tray and generously flooded with Working Wright-Giemsa Solution for another 5 minutes, with occasional agitation to ensure thorough staining.

[0282] After staining, the slide underwent a series of rinsing steps, starting with deionized or distilled water to remove excess stain. The slide was then flooded with Phosphate Buffer Solution, pH 6.8, until no further stain runoff was observed. An additional minute was allowed for the slide to remain in PBS at pH 6.8.

[0283] To complete the staining process, the slide was dipped in distilled water and allowed to air dry at room temperature. In the final step, the slide was immersed several times in Xylene or a Xylene Substitute, effectively clearing and preparing the slide for mounting in synthetic resin. This meticulous staining procedure ensured high- quality results for microscopic examination and analysis.

[0284] Image Acquisition, Processing, and Analysis

[0285] Images were acquired using the Leica DMi8 automated scanning microscope or Leica TCS SP5 confocal microscope and processed using Imaged software (NIH). Any contrast adjustments were made in individual channels and applied evenly across the whole image in that channel. Contrast and color balance for color images was applied evenly across the whole image.

[0286] Bulk RNA Sequencing of heX-embryoid Tissue

[0287] RNA extraction, as previously outlined, was conducted on the samples, and the extracted RNA was subsequently forwarded to the UCLA Technology Centerfor Genomics and Bioinformatics for library preparation and sequencing. The libraries for RNA-Seq were meticulously prepared employing the KAPA Hyper Stranded RNA- Seq Kit. 941 This library preparation workflow encompassed mRNA enrichment, cDNA generation, end repair to create blunt ends, A-tailing, adaptor ligation, and PCR amplification. Distinct adaptors were employed for multiplexing samples within a single sequencing lane. Sequencing was accomplished on the Illumina NextSeq500 platform, using a single-read 75bp run.

[0288] Quality assessment of the data was conducted via Illumina SAV, and the demultiplexing step was executed utilizing the Illumina Bcl2fastq2 version 2.17 program. Further evaluation of raw FASTQ quality was carried out using FASTQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc).

[0289] Reads were subsequently aligned to the latest UCSC transcript set using Bowtie2 version 2.1.0, with gene expression levels being estimated through RSEM vl .2.15. The EdgeR TMM (trimmed mean of M-values) algorithm was applied for the normalization of gene expression data. Additionally, reads were mapped to the latest UCSC genome set via Bowtie2 and Tophat, resulting in a BAM file from which alignment information was gathered using the PicardTools CollectRNASeqMetrics program. A Genebody analysis was carried out utilizing the ngsplot toolkit.

[0290] To visualize the resulting data, heatmaps were generated using the heatmap package in R, following documentation available at (https: / / cran.r962project.org / web / packages / pheatmap / pheatmap.pdf).

[0291] 10x Genomics Sample Preparation for Next-Generation Sequencing

[0292] The samples were prepared following the 10x Genomics Cell Multiplexing Oligo Labeling protocols, ensuring cells with over 80% viability were selected. Single-cell suspensions were obtained from the samples by treating them with Accutase for 20 minutes at 37 °C. To eliminate any aggregates, the cell suspension was passed through a 40pm strainer. Subsequently, each sample suspension underwent centrifugation at 300g for 5 minutes at room temperature, and the supernatant was gently removed using a P1000 pipette. The cell pellets were resuspended in 1 ml PBS+0.04% BSA and underwent another round of centrifugation at 300g for 5 minutes at room temperature. These samples were then resuspended to achieve a target concentration of 1 x106 cells / ml, adjusted based on expected cell densities for each day. After counting the cells using a hemocytometer, volumes weremodified as necessary to reach a final cell count of 1 x106 cells, with additional PBS + 0.04% BSA added to replace removed volumes.

[0293] For Multiplexing Oligo Labeling, samples were once again centrifuged at 300g for 5 minutes at room temperature, and the supernatant was carefully aspirated. Each sample was resuspended in 50 pl of CellPlex Multiplexing Solution from 10x Genomics, with unique multiplexing oligo solutions designated for each sample. Up to 12 samples were labeled simultaneously, incubating for 5 minutes following the addition of the oligo solution to the last sample.

[0294] Post-labeling, 1 .95 ml of 1 x PBS + 1 % BSA was added to each sample, thoroughly mixed, and centrifuged at 300g for 5 minutes at 4°C. The supernatant was carefully aspirated, leaving less than 10pl of supernatant when feasible. Samples were then resuspended in 2 ml of 1 x PBS + 1 % BSA and thoroughly mixed for washing. This washing and centrifugation process was repeated twice more, with the final resuspension adjusted to achieve a cell count of 1 x106 cells / ml, accounting for a 50% cell loss from the initial count after the first protocol. The labeled cell suspensions were placed on ice for transfer to the Pitt Single Cell Core for library creation.

[0295] Following a final count and viability assessment, cells and 10x Genomics reagents were loaded into the single-cell cassette, targeting 25,000 single cells for analysis. This accounted for expected cell loss and doublets resulting from multiplexing, as outlined in the Chromium Single-Cell 3’ Reagent Kit user guide from 10x Genomics. After generating GEMs (Gel Bead-In Emulsions), the cDNA library was prepared by the Pitt Single Cell Core staff, adhering to the relevant steps outlined in the 10x Genomics user guide. Subsequently, the libraries were sent to the UP cc Genome Center for sequencing on a NovaSeq S4-200 platform, aiming for an intended read depth of 100,000 reads per cell with 150 bp paired-end reads. Downstream analysis of the sequencing data yielded varying mean reads per cell, ranging from 40,000 to 150,000 in different samples.

[0296] Single Cell RNA Sequencing 1005 Sample Processing and Quality Control

[0297] The analysis pipeline for the single-cell data followed several steps using the 10x Genomics CellRanger pipeline. Initially, reads were aligned to the reference genome (GRCh38.84) supplemented with transgene sequences. This alignment process assigned reads to individual cells and estimated gene expression based on unique molecular identifier (UMI) counts.

[0298] To ensure data quality, single cells were excluded based on a high ratio of mitochondrial genome transcripts and either unusually high or low feature or UMI counts. Genes with UMI counts in fewer than 5 cells were also filtered out. For subsequent scRNA-Seq data processing and cluster analysis using Seurat, a standardized pipeline was applied. This included SCTransform to regress percent mitochondrial genes, principal component analysis (PCA), and clustering. To determine the optimal number of principal components (PCs) that retained the most variation, jackstraw plots and permuted p-values were employed.

[0299] The quality of clustering was assessed using enrichment analysis of cluster marker genes, which are genes differentially upregulated in a specific cluster compared to all others, with embryo cell type-specific genes. As a quality check, adjustments were made to PC and resolution parameters to confirm the selection of the most biologically relevant clustering. Visualization was achieved using UMAP plots to identify cells, clusters, and selected gene expression in each cell, as well as heatmaps and violin plots illustrating gene expression levels by cluster.

[0300] Subclustering was performed by isolating the cluster with the highest transcript levels of CD34, calculating distances between rows in the data matrix using Euclidean measurements, and applying hierarchical clustering to create a dendrogram representing the distance explained by various numbers of subclusters. The number of subclusters was determined based on the highest observed distance level and was applied to the data. This cluster was then reintegrated into the overall dataset, and markers were identified.

[0301] Cell-cell communication analysis

[0302] Cell-cell communication inference was performed between 1036 cell clusters using the CellChat v.1.5.0 R package (ref: 33597522). The CD34+CD38- population was defined as cells having CD34 expression larger than 0 and CD38 expression equal to 0. All three CellChatDB interaction types (paracrine / autocrine signaling interactions, ECM-receptor interactions and cell-cell contact interactions) were used. All other parameters were set to default.

[0303] SingleCellNet Processing and Statistical Analysis

[0304] h5ad files obtained from https: / / www.biorxiv.org / content / 10-1 101 / 2022.08.03.502475v1 .full.pdf were transformed into Seurat objects using the zellkonverter before being subjected to SingleCellNet processing. The SingleCellNet processing procedure adhered to theguidelines outlined in https: / / pcahan1.github.io / singleCellNet / . Within the pipeline, 50 random cells were generated for the computed comparisons, with a consistent set.seed value of 100 being applied across all comparisons. The assessment of similarity between lists of marker genes was carried out through the utilization of the hypergeometric test for overrepresentation, which is equivalent to a one-tailed Fisher’s Exact Test.

[0305] Gene Enrichment Analysis

[0306] Differential expression data was derived from the TMM normalized reads (counts per million, CPM) by calculating the ratio of the average counts of each sample relative to the average counts of the reference condition for each gene. The resulting fold change list was employed to compile a gene list for each condition, comprising genes with a minimum of two reads and a > 2-fold change compared to the reference condition.

[0307] These gene lists from each condition were subsequently imported into the EnrichR web browser application. The outcomes were employed to establish alignment scoring and significance data for analyses related to cells, pathways, functions, and ontologies. In the case of scRNA-Seq data, gene lists for each cluster were generated using Seurat, which included genes with adjusted p-values <0.05 and an expression fold change of at least 1 .6-fold when compared to the average gene expression in other clusters.

[0308] These lists were then submitted to EnrichR for enrichment analysis, and the results were presented using GraphPad Prism 8.

[0309] Quantification and Statistical Analysis

[0310] In studies involving statistical analyses, unless specified differently, a minimum of three biologically independent replicates was employed. Statistical assessments encompassing three or more conditions were conducted through oneway ANOVA, followed by multiple comparisons testing using Tukey's method, with a significance threshold set at p < 0.05. For comparisons involving only two conditions, either one-tailed or two-tailed t-tests were applied, as indicated, with a significance threshold of p < 0.05.Results

[0311] It is well-known that the fetal liver is the hub for embryonic hematopoiesis and supporting hematopoietic cells differentiation, maturation, and expansion. Recently, it has been shown that the yolk sac performs diverse roles during earlyhuman development, and among these has many functions in common with the fetal liver. YS tissue lineages display distinctive molecular signatures reflective of both soluble and insoluble signaling cues crucial for expanding, maintenance, and quiescence of hematopoietic cells such as THPO and VTN from endodermal cells, WNT5A and FN1 from fibroblasts and smooth muscles, and KITLG and JAG1 from endothelial cells. Moreover, transplantable HSCs with long-term multilineage reconstitution capabilities have been identified in the human yolk sac prior to colonization of the fetal liver. This observation raises questions regarding the yolk sac's potential role in supporting HSCs or the independent emergence of HSCs within the yolk sac, separate from the aorta-gonad mesonephros (AGM) region. Therefore, it is crucial to explore the hematopoietic niche potential of the yolk sac as an extraembryonic environment for the expansion and maintenance of HSCs. Whether the yolk sac and fetal liver comprise similar and redundant niche factors in HSCs to ensure the developmental robustness of the HSC programming, or whether they confer distinct signals within the heterogenous developing HSC population, is unknown. Our analysis of a recently published human yolk sac and fetal liver dataset at CS10 to CS23 shows that human YS and fetal liver have many similar characteristics related to the hematopoietic niche. Our computational analysis depicts the pattern of expression of hematopoietic factors in different cell types across the developmental stages. YS endoderm and FL hepatocytes have the same pattern of expression for niche factors such as IGF2, MDK, ANGPTL3 and DLK1 . YS fibroblasts and mesothelium and fetal liver fibroblast clusters also share the same pattern of expression of factors for expansion like IGFBP3 and PTN, quiescence factor CXCL12, and HSC maintenance factor BMP4. Of note, only YS mesothelium and not FL at CS22 express FLT3LG, a crucial growth factor for hematopoietic expansion, and expresses higher levels of JAG1 , a notch ligand important for functional maturity of HSCs YS and FL endothelium both express expansion growth factors such as ANGPTL4 and DLL4, and the maintenance factor. Hence, the in vivo yolk sac with a similar expression pattern of hematopoietic factors to fetal liver is a potential niche for HSC expansion and it can potentially serve as a niche for supporting HSC expansion and maintenance before the fetal liver emergence.

[0312] We have made an iPSC-derived in vitro model of human embryo postimplantation via combining the engineered iPSC to overexpress GATA transcription factor and WT iPSC cells. This model leads to the morphogenesis of yolk sac-liketissue and the emergence of yolk sac cell types, along with the development of amniotic ectodermal cells derived from WT iPSC cells (FIG. 6). We conducted a comprehensive analysis of the niche signature of heX-embryoid’s cells, aiming to elucidate their composition and their resemblance to the developmental programs observed in the yolk sac and fetal liver. We investigated the development of heX- embryoid yolk sac-like cell types by analyzing gene sets associated with yolk sac and fetal liver endoderm cells, fibroblasts, and endothelial cells at days 5, 10, and 17. Our analysis revealed an increase in the expression of endodermal genes such as AFP, SERPINA1 , FOXA2, and HNF4A, fibroblast-related genes like LUM (a yolk sac- specific fibroblast marker), DES, and COL2A1 (an extracellular matrix product), and endothelial genes including CDH5, PECAM1 , and the yolk sac-specific endothelial gene PLVAP from day 5 to day 17. Additionally, we observed the expression of genes responsible for the proliferation of endodermal cells, fibroblasts, and endothelial cells. The expression of cell migration genes also highlights the dynamic nature of the heX- embryoid self-organization. Our analysis through immunofluorescent (IF) staining corroborated a highly vascularized (CD31 + cells) network tissue with the presence of HNF4A+ endoderm cells, Desmin+ fibroblasts, Nestin+ pericytes, and DLK1 expressing cells (FIG. 7).

[0313] To further investigate the yolk sac and fetal liver niche characteristics of heX-embryoid at the transcriptomic level we performed single-cell RNA sequencing (scRNA-seq) of our tissue on day 21 . We used SingleCellNet

[0046] to classify the heX- embryoid clusters to cell types of the in vivo yolk sac and embryonic liver datasets. The endoderm clusters were classified as yolk sac endoderm rather than embryonic liver hepatocytes. We also adapted the scRNAseq data from day 21 of heX-embryoid and 220 compared it against the in vivo developmental identity of the yolk sac and fetal liver. Our analysis reveals that transcriptomes of cells comprising the endoderm- , fibroblast-, endothelial-, and macrophage-like lineages have highly significant similarity to corresponding YS and fetal liver tissue transcriptomes between CS1 1 (4 weeks) and CS23 (17 weeks) of human development. The endoderm populations within heX-embryoid aligned most strongly to the human yolk sac between CS1 1 and CS18, but had low similarity to yolk sac samples beyond this age; notably, these endoderm clusters maintained strong significant similarity to fetal liver stages between CS18 and CS23 (17 weeks) of development, suggesting that the tissue state can cover a large range of functional states equivalent to different functional stages of each ofthese tissues. Unlike the endoderm population, the transcriptome of the fibroblast and pericyte populations of heX-embryoid were more resembling yolk sac counterparts from CS1 1 to CS23 with the exception of CS15. The endothelial cluster was equally similar to both yolk sac and fetal liver. Hence, our analysis demonstrated the resemblance of heX-embryoid's yolk sac-like cell types to both yolk sac and fetal liver cells, which originates from the shared molecular signatures of these two tissues in vivo suggested by others.

[0314] Having interrogated the similarity of yolk sac and fetal liver and the potential of yolk sac as a hematopoietic niche, and the similarity of heX-embryoid cells to in vivo yolk sac and fetal liver, we sought to investigate the hematopoietic niche signature of heX-embryoid. Bulk RNA-seq of heX-embryoid on days 5, 10, and 17 depicts the upregulation of many soluble and insoluble hematopoietic signaling effectors. Our single-cell RNA-seq analysis revealed the expression of some of the hematopoietic factors in a cell-type-specific manner. The cell-type specificity in the expression of these factors is aligned with in vivo tissue. Remarkably, the amniotic ectoderm cells of the heX-embryoid which is lacking in the yolk sac in vivo express important expansion growth factors such as DLK1 , IGF2, IGFBP2, IGFBP3, IGFBP5, PTN, MDK and WNT4 suggesting that these non-yolk sac cells are contributing to the heX-embryoid’s hematopoietic niche. Taken together, these data indicate that heX- embryoid tissue signature aligns with both yolk sac and embryonic liver, with the presence of cell populations expressing hematopoietic factors in a cell-specific and physiologically relevant manner, making it a highly promising potential niche for HSCs expansion and differentiation. Additionally, this platform can be used to test whether YS can be a tissue for the expansion and maintenance of HSC as an in vitro surrogate.

[0315] heX-embryoid autonomously produces a plethora of signaling cues in a soluble and insoluble format, providing an in vivo-like yolk sac-like hematopoietic niche. This characteristic reflects a physiological milieu where many signaling cues act in tandem to promote HSC homeostatic expansion, maintenance, and differentiation. As such we interrogated whether heX-embryoid as an in vitro model of yolk sac can function as a unique multicellular niche for expansion of hematopoietic stem cells. The expansion of HSCs during fetal liver development coincides with the expansion of portal vessels and associated pericytes (PMID: 26634440). Drawing inspiration from this observation, we proceeded to explore the dynamics of heX- embryoid cellular niche development in order to identify an appropriate timeframe toalign HSC expansion and cellular niche expansion in heX-embryoid. The principal hematopoietic cellular niches within heX-embryoid, expressing soluble and insoluble hematopoietic factors, comprise endodermal, fibroblast / pericytes, and endothelial cells. We evaluated the progression of these cellular niches and noted a dynamic increase occurring between day 10 and day 20 of the culture (FIG. 8). Hence, we designated this timeframe as optimal for culturing HSCs with heX-embryoid. We used cord blood CD34+ cells (CB CD34+) as an enriched source of HSCs. Initially, we cultured CB CD34+ on day 10 heX-embryoid adding the regular concentration of conventionally used cytokines: SCF (100 ng / mL), TPO (50 ng / mL), and FLT3L (100 ng / mL) into the media (heX-embryoid + Regular cytokine level). The magnified sections of the phase image microscopy illustrate the dynamic expansion of morphologically distinguishable spherical CB CD34+ cells throughout the entire coculture period. To investigate whether the observed expanding cells are derived from CB CD34+ cells and are not the heX-embryoid hematopoietic progenitors, we lentivirally labeled the CB CD34+ cells with a constitutive fluorescent marker (mKate) and observed the expansion of mKate+ cells. This strategy also allowed us to track the localization of CB CD34+ cells. Fluorescent imaging revealed that the CB CD34+ cells were spatially positioned near the NES+ stromal cells and CD31 + vasculature. Next, we evaluated the outcome of CB CD34+ expansion on heX-embryoid under the regular cytokine level. The total cells expansion fold change of the initial population was similar between the heX-embryoid with regular cytokine level 283 condition and the cytokine-only control group (p-value >0.1 ) (FIG. 9). However, the heX-embryoid supported a 170-fold expansion of the phenotypically defined HSC population (Lin- CD34+CD38-CD45RA-CD49f+) compared to the ~20 fold expansion in the cytokine- only control (p-value <0.0001 ). In addition to expanding the HSC population, it is also crucial to expand the immediate progenies of HSCs, such as multipotent progenitors (MPPs) or multilymphoid progenitors (MLPs), which are enriched in the Lin- CD34+CD38- population, to enable host recovery from lethal conditioning in HSC transplantation before HSC activation. Culturing CB CD34+ cells with the heX- embryoid niche led to a 15-fold increase in the Lin-CD34+CD38- population, compared to the ~2.5-fold increase observed in the cytokine-only control (FIG. 9). Mock harvest of media of heX-embryoid on day 20 cultured in regular cytokine condition without the CB CD34+ cells did not show any cells with phenotypic HSC signature.

[0316] Using single-RNA sequencing, we assessed the expression of potential ligands (on niche) and receptors (on expanded CB CD34+ in heX-embryoid with regular cytokine level), such as IGF2-IGFR2, KITLG-KIT, IGFBP2-PTPRB, WNT5A- FZD6, TGFB2-TGFBR2, PTN-LRP1 , and MDK-LILRB1 (FIG. 9). We then used CellChat to investigate the cell-cell communication between the niche and CB CD34+ cells. Cellchat could identify the expected signaling such as MDK-NCL, PTN-NCL, IGF2-IGF1 R, KITLG-KIT, and DLK1 -NOTCH1. Of note, the DLK1 -NOTCH1 interaction score was computed more for the ectodermal cluster compared to other heX-embryoid cell types. This analysis also identified previously-unknown novel pathways involved in hematopoietic expansion and maintenance, including endothelial cell-derived Resistin-CAP1 signaling, pericyte-derived SEMA3C-PLXND1 , and endoderm-derived GDF15-TGFBR2, EFNB3-EPHB6, and CDH1 -KLRG1.

[0317] The clonogenic potential of progenitor cells derived from expanded CB CD34+ cells in both heX-embryoid and cytokine-only control cultures was investigated through a colony-forming unit (CFU) assay. In control cultures, HSCs exclusively generated CFU-GM colonies, indicative of a restricted lineage commitment towards granulocyte-macrophage progenitors. Conversely, heX-embryoid expanded cells generated all colony types and displayed higher overall colony numbers, highlighting the ability of the heX-embryoid niche to support the generation of multipotent hematopoietic progenitor cells. To evaluate the maintenance of the HSC sternness when expanded on heX-embryoid, we next performed subsequent passages on the expanded CD34+ cells by harvesting the cells and re-seeding them onto another day 10 heX-embryoid niche or cytokine-only control cultures. heX embryoid maintained HSCs in culture for over a month resulting in overall 1000-fold expansion of HSCs over time. In contrast, cells expanded in the cytokine-only cultures exhausted quickly, with no HSCs detected at the end of the culture period. Moreover, we investigated the expression of bone marrow homing markers in the expanded CD34+ cells cultured on both the heX-embryoid niche and cytokine-only control conditions. The higher rate of of VLA-4, CD44, and PSGL-1 positive cells among the CB CD34+ cells expanded on the heX-embryoid niche compared to the control indicates that possibly the niche offers an optimal milieu for HSC expansion while better preserving their capacity for homing to the bone marrow.

[0318] We then performed a comprehensive analysis of the single cell RNA-seq data to better dissect the biological difference between the expanded CB CD34+cultured with heX-embryoid compared to cytokine-only control in regular cytokine levels. To examine HSCs-enriched populations, we identified the CD34+ cluster for each condition and performed sub-clustering. Both heX-embryoid-cultured and control-cultured cells contained a “CD34+ Cycling HSC” subcluster, which expressed CD34, canonical HSC genes, and the proliferative marker MKI67, and a “Committed Cycling HSC” subcluster with low expression of CD34 and canonical HSC and high MKI67 expression. heX-embryoid also included two non-proliferative clusters expressing canonical HSC genes and either high CD34 expression (CD34+ Quiescent HSC) or low CD34 expression (CD34low Quiescent HSC) which were not present in cytokine-only control. This suggests that in the control group HSCs are induced to enter the cell cycle, while a higher degree of regulation is exercised in , potentially maintaining more potent populations in culture for longer including the highly potent CD34- HSC. The cells expanded in the cytokine-only condition notably have upregulated HOXA genes, known to be expressed in myeloid cells and myeloid- committed progenitors, which provides further support for the notion that these cells are myeloid-biased. Excluding committed subclusters, the frequency of HSCs is over six fold higher in heX-embryoid.

[0319] Looking at the UMAP projection of single cell RNA-seq data, we noticed the presence of erythroid cells in heX-embryoid expanded cells absent in cytokine- only. This cluster expresses the erythroid markers GYPB and HBG1. We also found the high expression of the transferrin receptor gene (TFRC) in this cluster. We could identify the CD71 hi population in flow cytometry, and Wright-Giemsa staining of MACS-sorted CD71 cells showed the erythroid-like morphology of this population. The yolk sac is a primary tissue for erythropoiesis in primitive and definitive waves of yolk sac hematopoiesis. Using the difference in sex-specific genes between the cord blood (female) and heX-embryoid (male), we confirmed that the erythroid cells are not derived from heX-embryoid and are a result of CB CD34+ cells differentiation. This result suggests that like the fetal liver, the yolk sac microenvironment also contains growth factors leading to HSC-derived erythropoiesis.

[0320] Gene set enrichment analysis of differentially expressed genes in the HSC-enriched Lin-CD34+CD38- fraction of the expanded cells revealed a number of differences in signaling and metabolism between the two conditions. Response to interferons was highly enriched in heX-embryoid-cultured cells, which are known to be active in human fetal liver HSCs to help promote their functional maturation andexpansion as well as influence HSC niche interactions. Importantly, interferons are known to have contradictory effects on HSCs based on context and their role during early fetal liver colonization is incompletely understood. An inflammatory signature was enriched in heX-embryoid-cultured cells which is known to promote HSC selfrenewal and expansion. mTOR signaling was enriched in cytokine-only-cultured cells which is known to be inversely correlated with HSC maintenance. Signaling via DLK1 , which is abundantly expressed in heX-embryoid and the human fetal liver, has been shown to inhibit mTOR signaling and mitochondrial metabolism which may explain this phenotype in monoculture. Glycolysis was enriched in heX-embryoid cells which is known to occur in HSCs with higher engraftment potential. Conversely, oxidative phosphorylation was enriched in cytokine-only-cultured cells. Conflicting evidence exists for the relationship between HSC metabolic activity, proliferation, and potency. Some studies suggest that active HSCs utilize glycolysis while others indicate oxidative phosphorylation; some evidence suggests the most potent HSCs utilize glycolysis, while other evidence points to oxidative phosphorylation. It was previously thought that fetal liver HSCs prefer oxidative pathways based on a comparison with adult HSCs but these data suggest that our understanding may be incomplete. Our data indicate that cytokine-only-cultured cells undergo oxidative phosphorylation driven by mTOR signaling with high protein synthesis and DNA damage, an established phenotype that leads to HSC exhaustion. heX-embryoid-cultured cells conversely exhibit a hypoxic, inflammatory response fueled by glycolysis. Bile acids in the fetal liver have been reported to protect expanding HSCs from ER stress and DNA damage and heX-embryoid was found to pervasively express components of the bile acid and salt synthesis pathway. This finding suggests that relative to cytokine-only, heX-embryoid-cultured cells are protected from insult potentially via a more physiologically relevant mechanism. Altogether, we showed superior expansion of HSCs on heX-embryoid in regular cytokine conditions with an augmented molecular profile compared to the cytokine-only control.

[0321] The hematopoietic niche within the hex-embryoid encompasses both soluble and insoluble factors essential for expanding HSCs. Hence, we delved into whether this microenvironment could obviate the necessity for regular cytokine levels employed in expansion. We significantly decreased the levels of growth factors by 99%: SCF (1 ng / mL), TPO (0.5 g / mL), and FLT3L (1 ng / mL). The heX-embryoid with low-cytokine levels resulted in a 50-fold expansion of Lin-CD34+CD38-CD45RA-CD49f+ phenotypic HSCs, with no expansion in the low-cytokine control group. Additionally, the Lin-CD34+CD38- cells expanded 2-fold in the heX-embryoid with low- cytokine levels (FIG. 10). To investigate the functionality of the HSCs expanded on heX-embryoid in low-cytokine condition, we transplanted the expanded cells into immunocompromised NSG / NBSGW recipient mice. After transplanting the total expanded cells from the same number of input CB-CD34+ cells, we discovered that the heX-embryoid-expanded cells could reconstitute mice with human hematopoietic cells significantly higher than the controls across peripheral blood, bone marrow, and spleen. Around 80% of the recipient mice (16 / 20) transplanted with heX-embryoid expanded cells showed over 2% of human CD45+ cells (out of total human plus mice CD45+ cells), as a marker of human hematopoietic cells, and 70% of the recipient mice had over 10% human CD45+ cells reconstitution in bone marrow. The reconstitution was significantly lower in mice being transplanted with low-cytokine control cells, with only 1 1 % of recipient mice showing over 2% human CD45+ cells / mice CD45+ cells (1 / 9) and no mice showing over 10% reconstitution of human CD45+ cells in their bone marrow (FIG. 11). Mice transplanted with heX-embryoid expanded cells exhibited multilineage reconstitution of human hematopoietic cells developing various downstream lineage cell types, including human B cell, T cell, NK cell, and myeloid cells, while in the control transplantations, this multilineage reconstitution was not observed, and the downstream lineages were mostly limited to B cells. We also found human erythroid cells (hCD235a+ cells) in RBC-unlysed bone marrow samples of heX-embryoid recipient mice but not in control transplants (FIG. 12).

[0322] Immunodeficient mice exhibit abnormalities in their immune system function due to the improper development of primary and secondary lymphoid organs. As a result, NSG mice have underdeveloped spleens. We observed substantially larger spleen sizes in recipients of heX-embryoid-expanded cells compared to those in cytokine-only control, potentially indicative of repopulation with human cells, as the mice immune cells in this strain cannot repopulate the spleen effectively. Immunohistochemistry (IHC) of spleen sections also revealed a higher frequency of human B and T cells in recipients of heX-embryoid-expanded cells, organizing in germinal center-like structures, while a substantially smaller number of cells were captured in cytokine-only recipients. This may indicate the presence of auxiliary cells required for the germinal center response. Higher frequencies of B and T cells werealso found in the liver, lung, and kidney of heX-embryoid transplant recipient mice in IHC studies compared to cytokine-only controls.

[0323] Repopulating functional T cells following bone marrow transplantation (BMT) is a challenging and slow process, as it is primarily thymus-dependent. In the initial wave of hematopoiesis post-BMT, mainly LMPs and LMPPs are capable of repopulating blood cells, yet they often fail to engraft in the thymus and thus do not lead to the generation of T cells, which mainly rely on donor-derived HSCs capable of reconstituting the thymus. Of note, heX-embryoid transplants showed significantly higher rates of T cell reconstitution over the long term compared to cytokine-only controls. This fact also underscores that heX-embryoid-expanded cells likely had a higher rate of specification of functional LT-HSCs, which could repopulate T cells within the hematopoietic organs of the recipient mice significantly more than cytokine- only control. LT-HSCs are capable of long-term survival and functionality in the recipients following bone marrow transplantation and can be transplanted multiple times and reconstitute blood lineages. To investigate the presence of functional LT- HSCs expanded ex vivo on heX-embryoid or in cytokine-only control conditions, we extracted bone marrow cells of the primary recipient mice being reconstituted with human CD45+ cells and transplanted them into secondary recipient mice at 1 .5-2 x 106 cells per secondary mouse. Bone marrow cells of primary mice transplanted with heX-embryoid-expanded CBC-CD34+ were capable of reconstituting secondary transplant recipients with three out of ten total secondary recipients found to be reconstituted with human CD45+ cells, while no secondary recipient of primary cytokine-only control cells could reconstitute human CD45+ cells. This result depicts the preserved HSC sternness and functional quality of CB-CD34+ expanded on heX- embryoid and its loss in the HSCs cultured in cytokine-only condition.

[0324] To probe the gene expression pattern of the expanded CB CD34+ cells in low-cytokine conditions, we subsequently performed single-cell RNA-seq analysis on them. Examination of gene expression across the entire expanded cell population revealed elevated levels of CD34, HOPX, and SPINK2, three canonical hematopoietic stem cell (HSC) markers, in cord blood cells cultured with hex-embryoid compared to the control group. This finding aligns with the flow cytometry data indicating enhanced expansion of HSCs in cocultured samples. Furthermore, we observed increased expression of GYPA, an erythroid lineage marker as observed in regular-cytokine condition, supporting the resemblance of the yolk sac and fetal liver-likemicroenvironment and indicating noticeable erythropoiesis compared to the control group, which exhibited increased myeloid lineages (CD33).

[0325] Next, we narrowed down our analysis to the HSC-enriched population defined as Lin-CD34+CD38-. Analyzing the HSC-specific markers (HLF, AVP, PRDM16, MECOM, PROM1 , PBX1 ) shows the higher number of cells expressing these markers in heX-embryoid cultured cells suggesting the expansion of a higher expansion of HSCs. heX-embryoid-cultured cells showed less activation of the AHR signaling pathway which inhibits HSC self-renewal capability, as shown by less expression of AHR and CYP1 B1 ; a downstream product of AHR pathway activation. The expression of MSI2, a gene contributing to HSC self-renewal by inhibition of the AHR pathway, did not differ between low cytokine-only and heX-embryoid-cultured conditions, suggesting another mechanism heX-embryoid used to inhibit AHR which warrants further investigation. Importantly, the expression level of MLLT3, a protein crucial for HSC self-renewal was higher in heX-embryoid-cultured HSCs. Our analysis also revealed that a remarkably higher number of the cells in with MYCT1 expression as an important factor for HSC self-renewal and engraftability in heX-embryoid cultured cells. Additionally, we investigated the CDK6 expression, a marker for HSC activation and short-term HSCs. Most Lin-CD34+CD38- cells in both conditions express this gene. However, the number of inactivated LT-HSCs (without CDK6 expression) is higher in heX-embryoid cultured condition compared to low-cytokine- only. The heX-embryoid microenvironment reconstitutes the environment of the earliest embryonic niche which proceeds the emergence of CB CD34+ cells. | In other words, in our approach, we treat the CB CD34+ cells with a micro-environment that they have been in touch with 8 months earlier. Thus, we hypothesized whether heX- embryoid can reprogram the HSC state from CB to a younger state. We compared the HSC-enriched Lin-CD34+CD38- populations of low cytokine heX-embryoid and cytokine-only conditions to a recently published atlas of HLF+ HSCs at different developmental stages. Remarkably, we observed the high similarity of heX-embryoid- expanded Lin-CD34+CD38- cells to 6 weeks fetal liver HSCs (CS17) due to the similarity of the yolk sac and fetal liver niche, while the cytokine-only expanded cells preserve their similarity to 40 weeks CB HSCs. A similarity to 15 weeks of liver HSCs also is observed in cytokine-only cells which are due to the expression of ribosomal genes. Fetal liver HSCs have been shown to have the highest competitiveness in engraftment of mice compared to other embryonic and adult HSCs. Hence, the highreconstitution ability of low cytokine heX-embryoid expanded cells might be due to this reprogramming. Further investigation of the expanded HSCs on heX-embryoid can shed light on the factors leading to such competitiveness which is hardly accessible in studying humans in vivo.

[0326] Additionally, pathway analysis using GSEA revealed a significant enrichment of genes associated with glycolysis, translation, and DNA repair and negative regulation of apoptosis in heX-embryoid-cultured low-cytokine conditions compared to monoculture. However, the enriched pathways in low-cytokine monoculture cells compared to low-cytokine heX-embryoid-cultured cells were increased ROS formation, antigen presentation, higher inflammation, and lysosomal protein degradation.

[0327] Next, we compared the engraftment efficiency of expanded CB CD34+ cells on heX-embryoid with regular- and low-cytokine conditions transplanting the same number of expanded cells. We found significantly higher levels of hCD45 reconstitution in recipient mice transplanted with the low-cytokine heX-embryoid group in all three assessed organs — peripheral blood, bone marrow, and spleen. Almost 70% of replicates in the low-cytokine heX-embryoid condition showed hCD45 reconstitution levels of over 10% compared to around 35% of recipient mice in the regular-cytokine heX-embryoid condition. Moreover, quiescence is considered as a critical functional capability in LT-HSCs, where the cells will be preserved at a quiescent stage in the niche and can get activated and give rise to downstream progenitors and blood cell lineages whenever demanded. To evaluate the level of quiescence among the ex vivo expanded HSCs in our cultures, we further investigated the frequency of quiescent cells among the HSC-enriched population (CD34+CD38-) in heX-embryoid-expanded cells compared to cytokine-only controls, in both regular and low cytokine concentrations, to assess the level of quiescent cells preserved in the culture under each condition. In this investigation, a significantly higher percentage of CD34+CD38- cells were found to be in the G0-G1 phase in heX-embryoid-low-cytokine compared to the regular-cytokine concentration. Cytokine-only at regular concentration had the highest S phase level among the conditions, along with showing almost the highest total number of cells after expansion among conditions with a trivial percentage of CD34+CD38- and HSCs (CD34+CD38-CD45RA-CD49f+). Regular cytokine- supplemented heX-embryoid showed a higher percentage of desired CD34+CD38- cells and HSCs than the GF controls, among which a higher proportion of cells werein G0-G1 phase. This suggests that there are potential inhibitory mechanisms in heX- embryoid working to balance the level of quiescence, self-renewal capabilities, and differentiation of the HSCs. Moreover, the cells expanded on heX-embryoid at low- cyokine concentration showed a significantly higher percentage of cells in G0-G1 and a lower percentage of cells in S compared to regular-cytokine-supplemented heX- embryoid and cytokine-only controls. Despite showing less overall expansion in terms of total cell number at the endpoint, low-cytokine supplemented heX-embryoid possessed a significantly higher percentage of CD34+CD38- and HSCs out of total cells compared to the regular cytokine supplemented heX-embryoid and cytokine-only controls. The low-cytokine supplemented heX-embryoid, therefore, seemed to provide the most optimal environment among the conditions, in which the cells could expand, at the same time, self-renew and preserve their functional sternness qualities at a more balanced rate, in line with the other in vitro, single-cell RNAseq, and in vivo findings presented earlier.

[0328] In order to investigate the mitochondrial metabolic activity of cells expanded on heX-embryoid or in cytokine-only control conditions, we incubated the cells with MitoT racker Red CMXRos dye, which indicates active mitochondrial content, and MitoSOX Red dye, which detects mitochondrial superoxide production. We observed the highest active mitochondrial content and superoxide production on flow cytometry among both the total cell population and the Lin-CD34+CD38- cells in the refular cytokine heX-embryoid. The Low-cytokine heX-embryoid cells exhibited the highest ratio of MitoT racker negativity among CD34+CD38- cells. This finding aligns with a higher rate of quiescence among the CD34+CD38- cells in the low-cytokine heX-embryoid. This subset of cells seems to have a lower rate of proliferation and expansion, suggesting that heX-embryoid may preserve a proportion of CD34+CD38 cells in a steady state compared to other culture conditions. Mitochondrial superoxide production appeared to correlate with the rate of expansion in the culture; the greater the number of total cells at the end of the culture, the higher the mitochondrial superoxide production seemed to be.Example 2Materials and Methods

[0329] Cell culture

[0330] All cells and tissues were placed in a humidified incubator set at 37°C with 5% CO2 for the duration of culture. Our hiPSC lines were grown in a sterileenvironment using mTeSR-1 medium (Stem Cell Technologies) with daily medium changes. To prepare tissue culture plates, hESC qualified Matrigel (Corning), diluted to a working concentration in ice cold DMEM / F-12 (Corning) following the manufacturer’s recommended dilution factor per-lot, was applied and allowed to coat the plates at room temperature for 1 hour. For routine passaging, hiPSC colonies were incubated with Accutase (Sigma) for up to 10 minutes at 37°C. Subsequently, the suspension was collected, mixed with 2 mL of DMEM / F-12 medium containing 10 pM Y-27632 (Stem Cell Technologies), and centrifuged at 300g for 5 minutes. After centrifugation, the cells were resuspended in 3ml DMEM / F-12 supplemented with 10 pM Y-27632 for cell counting purposes. The cells were then seeded at a density of 25,000 cells per cm2 for regular maintenance.

[0331] Human cord blood (CB) processing and CD34+ cells isolation

[0332] Human CB samples were sourced either from the University of Arizona Biorepository (Tuscon, AZ) or obtained from Vitalant-Cord Blood Services (Pittsburgh, PA). CB was diluted 1 :1 with PBS containing 2% FBS and 1 mM EDTA, layered over Lymphoprep (Stem Cell Technologies Cat# 07801 ) and centrifuged at 1200x g for 20 minutes (brake off). The buffy coat was carefully separated and washed and subjected to CD34+ cells isolation using EasySep Human Cord Blood CD34 Positive Selection Kit II (Stem Cell Technologies Cat# 17896), according to the manufacturer's provided instructions. Isolated CB CD34+ cells were either used immediately or preserved through cryopreservation.

[0333] Generation of heX-embryoids and heX-embryoids-CB CD34+ cells coculture

[0334] To generate heX-embryoids parental cell population, we electroporated the PGP1 iPSC line (Coriell Institute), using the dox-inducible GATA6-encoding and reverse tetracycline-controlled transactivator (rtTA) plasmids described before74. Electroporation was performed using Lonza® 4D-Nucleofector™ X Unit to program code CA-137 with total 800 ng of DNA. After rounds of antibiotic selection (Puromycin at 0.5 ug / mL), we confirmed that the engineered population contained both cells with plasmid integration (iGATA6) and cells without the integration (WT), at a ratio conformed to the expected morphology and WT area by day 5, matching the 81 / 5 iGATA6 / WT combination described previously9. To make heX-embryoids cultures, the generated parental line was seeded at the density of 30,000 cells per cm2. After 24 hours, the medium was replaced with an induction medium (mTeSR-1 supplementedwith 1 ng / mL doxycycline) and refreshed daily. On day 5, the induction culture medium was changed to IMDM, supplemented with 10% FBS, as well as 1 x concentrations of GlutaMax, MEM Non-essential Amino Acids Solution, and Penicillin-Streptomycin (Gibco).

[0335] Upon reaching the tenth day of culture, CB CD34+ cells were introduced to the culture at a cell density of 10,000 per cm2. For regular cytokine conditions, the medium was enriched with 100 ng / mL of hSCF, 100 ng / mL of hFLT3L, and 50 ng / mL of hTPO. For low cytokine conditions, the medium was enriched with 1 ng / mL of hSCF, 1 ng / mL of hFLT3L, and 0.5 ng / mL of hTPO. Throughout the CB culture period, half of the medium volume in each well was manually replaced daily. To harvest the CB CD34+ cells, the wells were gently pipetted by manually aspirating and expelling the contents, followed by two washes with HBSS (without calcium, magnesium, or phenol red; Gibco).

[0336] For FIGS. 26-27, to investigate the enduring maintenance of multipotent progenitors within the culture, a passaging procedure was employed. Following ten days of co-culture, the cells were counted and splitted into five wells of day 10 heX- embryoid cultures. These co-cultures were sustained for an additional ten days before being harvested using the same protocol. The cumulative cell counts for each well in each passage were recorded. This process was repeated until the cell population from the cytokine-only control was exhausted and showed no further expansion compared to the previous passage. At the end of this period, the cells were collected for flow cytometric analysis.

[0337] Flow cytometry and FACS for HSC assays

[0338] At the end of the culture period, the cells were harvested by gently pipetting and manually aspirating the culture medium. The cells were then washed twice with HBSS (without calcium, magnesium, or phenol red; Gibco) to prepare single-cell suspensions. The suspensions were first treated with Fc block solution (Thermo Fisher) and incubated on ice for 10 minutes. Next, the antibody mix containing anti-human lineage cocktail (FITC, BioLegend), anti-human CD34 (clone 581 , APC, BioLegend), anti-human CD38 (clone HB-7, Pacific Blue, BioLegend), antihuman CD45RA (clone HI100, APC-Cy7, BioLegend), anti-human CD90 (clone 5E10, PE, BioLegend), anti-human CD49f (clone GoH3, PE-Cy7, BioLegend), was added at a final dilution of 1 :400 and incubated for 30 minutes on ice. Following a washing step,the 7-AAD (BD Biosiences) was added for dead cell exclusion. Flow cytometric analysis was performed on BD Fortessa, BD LSR II, or Cytek Aurora flow cytometers.

[0339] Calculation of fold change of expanded HSCs and HPSCs

[0340] For the quantification of the expansion fold change of HSPC and HSCs in FIGS. 23, 25, 30, the initial uncultured CB CD34+ cells were analyzed by flow cytometry to find the frequency of the immunophenotypic HSPCs and HSCs in the input uncultured cells and post-expansion. Fold change of expansion was calculated as the ratio of the number of immunophenotypic HSCs / HSPCs post-expansion to the number in the initial population.

[0341] Production of lentiviral Particles

[0342] To generate lentivirus for labeling the CB cells with mKate fluorescent protein, HEK293FT cells (Life Technologies) were cultured following the manufacturer's recommendations. One day before transfection, 8 million HEK293FT cells were seeded onto a collagen l-coated (Gibco A10483-01 ) 15 cm2 tissue culture dish. On the day of transfection, cells were co-transfected with 15 pg psPAX2 (Addgene Plasmid 12260), 3.75 pg pCMV-VSV-G (Addgene Plasmid 8454), and 11 .25 pg of the plasmid to be packaged, using 90 pg of linear polyethylenimine (Polysciences, Inc 23966-1 ). The following morning, the medium was changed, and the supernatant was collected after 48 and 72 hours. Pooled supernatant was passed through a 0.45 pm low protein binding filter (Corning) and concentrated in Amicon Ultra 15 filter columns (100 kDa cutoff, Millipore) at 4,000 g for 23 minutes. The concentrated virus was then divided into aliquots, snap frozen, and stored at -80 °C. Lentiviral concentrate was diluted 2000-fold in phosphate-buffered saline (PBS) and its titer was determined via qRT-PCR using a commercially available kit (ABM LV900). Titers were calculated following the manufacturer's instructions.

[0343] Lentiviral Transduction

[0344] For mKate2 labeling of the CB CD34+ cells, 100,000 CB CD34+ cells were thawed and cultured at regular cytokine conditions containing 8 pg / mL polybrene, and the hEF1 a-mKate2 virus was added to the medium to reach 100 MOI. The cells were kept in the incubator overnight and washed with IMDM with 10%FBS for three times before starting the coculture with heX-embryoids.

[0345] Colony Forming Unit Assays

[0346] Colony forming unit assays were conducted using methylcellulose- based media (specifically, MethoCult H4434 Enriched from StemCell Technologies)following the manufacturer's guidelines. Uncultured and cultured CB CD34+ cells were FACS-sorted for Lin-CD34+CD38- population and were plated at 500 cells per plate in duplicate cultures. After a 14-day incubation period, the plates were visually examined to count the number of CFU-multilineage colonies.

[0347] Transplantation

[0348] NOD.Cg-Prkdcscidll2rgtm1 Wjl / SzJ (NSG) and NOD.Cg-KitW-41 J Tyr+Prkdcscid H2rgtm1 Wjl / ThomJ (NBSGW) mice were purchased from Jackson Laboratories or bred in house. Transplantations were performed via retro-orbital (RO) injection of female adult mice (8-12 weeks). NSG mice were anesthetized with isoflurane, at least 4 hours after 250 cGy gamma-irradiation and administered with 100 mL of cell solution to the orbital sinus. NBSGW mice are highly immunodeficient due to a mutation in the c-kit gene, which eliminates the need for preconditioning. For comparison of the reconstitution of heX-embryoids-expanded CB cells versus low- cytokine control, the total expanded cells from the same number of CB CD34+ cells were injected into the mice. For the comparison in Extended Data Fig. 8b, the same number of expansion outputs were transplanted into the mice.

[0349] For secondary transplantation, primary recipients of different conditions were harvested, and femoral and tibial bone marrow cells were collected in a cell culture hood. 1.5x106cells from each sample of mouse bone marrow were counted and injected to secondary 8-12 weeks mice via RO injection.

[0350] The animal study was reviewed and approved by the Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. Mice were housed in sterile conditions at 20-26 °C ambient temperature with a 12 / 12-hour light / dark cycle.

[0351] Tissue harvest and sampling

[0352] Peripheral blood was collected via cheek puncture for monitoring of engraftment after transplantation. At the endpoint of the study, mice were euthanized and peripheral blood and tissues, including spleen, liver, and femoral and tibial bone marrow were collected. Small pieces of the tissues were placed in formalin 10% to be fixed for histopathological studies, and the rest were kept in HBSS on ice to be used subsequently for single-cell solution sample preparation for flow cytometry.

[0353] Flow Cytometry on harvested mouse tissues and blood

[0354] Spleen tissue was passed through a cell strainer, rinsed with HBSS (without calcium, magnesium, or phenol red; Gibco). For bone marrow, the femur andtibia were carefully dissected from the mouse legs, and the epiphyseal regions were removed. Bone marrow was flushed with cold HBSS. Both spleen and bone marrow samples were centrifuged at 300 g for 5 minutes and the pellet were resuspended in 200 pL of HBSS. A 50 pL aliquot was aside for unlysed bone marrow. ACK lysis was performed on the collected peripheral blood, spleen and the remaining bone marrow samples. After 5 minutes incubation, the samples were centrifuged, and the pellets were washed with FASC buffer before proceeding to staining. The single-cell suspensions were initially incubated with Fc block solution (Thermo Fisher) on ice for 10 minutes. Human cell engraftment was assessed after staining and performing flow cytometry using the following antibodies with 7-AAD added for dead cell exclusion: anti-human CD45 (clone HI30, or PE or APC-Cy7, BioLegend), anti-mouse CD45 (clone 30-F11 , Pacific Blue or BUV 395, BioLegend, anti-human CD19 (clone HIB19, BV605 or BV510, BioLegend), anti-human CD33 (clone P67.6, APC-Cy7 or BV605, BioLegend), anti-human CD3 (clone UCHT 1 , Alexa Fluor 700 or PE-Cy7, BioLegend), anti-human CD56 (clone MEM-188, BV421 or PE-Cy7, BioLegend), and anti-human CD235a (clone HIR2, PE-Cy7, BioLegend).

[0355] Cell Cycle Assay

[0356] To investigate the cell cycle stages in both the heX-embryoids CB coculture and cytokine-only control, we employed a flow cytometric-based EdU assay using the BD Pharmingen™ 647 EdU Click Proliferation Kit (BD Biosciences). Before harvesting for flow cytometry, cells were incubated with EdU (Eterneon™ Red 645 Azide, Alexa Fluor 647, BD Biosciences) for 3 hours. Following incubation, cells were harvested as described earlier and subjected to the staining process. Following staining with Zombie NIR fixable live / dead dye (APC-Cy7, Biolegend), cells were fixed in a PFA 4%-based solution. The click reaction was then performed according to the manufacturer's protocol for the EdU assay, and staining for other surface markers was carried out.

[0357] Mitochondrial Assay

[0358] To assess mitochondrial activity, cells were stained with MitoTracker™ probes (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, cells were incubated with MitoTracker™ Red CMXRos (ThermoFisher Scientific). After incubation, cells were washed and stained against CD34, CD38 and lineage cocktail.

[0359] Immunohistochemistry

[0360] Spleen sections from mice were fixed in 10% formalin for 24 hours embedded in paraffin, and sectioned. Deparaffinization was achieved in xylene (5 minutes, X3), 100% ethanol (10 minutes, X2), 95% ethanol (10 minutes, X2), and deionized water (X2). To facilitate antigen retrieval, the slides were submerged in 1 x citrate buffer (Abeam) and subjected to near-boiling conditions in a microwave oven for 15 minutes. Afterward, the slides underwent three rinses in deionized water for 5 minutes each, followed by one rinse in PBS.

[0361] For immunostaining, deparaffinized sections were blocked and permeabilized in a solution of PBS with 8% donkey serum and 0.2% TritonX-100 for a duration of 2 hours at room temperature. Subsequently, primary antibodies were applied to the samples and left to incubate overnight at 4°C. Following this incubation, the slides were rinsed three times in deionized water and then washed for 15 minutes in PBS.

[0362] Secondary antibodies were introduced to the samples in a solution of PBS with 2% donkey serum, allowing them to incubate for 2 hours at room temperature. Followed by this, the tissues were washed with three rinses in DI water, a 15-minute wash in PBS, and sealing with a glass coverslip. These prepared slides were then ready for imaging, and two types of mounting media, Diamond Antifade (Thermo Scientific) or Vectorshield (Vector Labs), were utilized depending on the imaging process.

[0363] Immunofluorescence Staining

[0364] Cells were cultured on one of the following substrates: circular glass coverslips, either 8 mm or 12 mm in diameter, or ibidi 96 well optical graded plastic bottom plates. Each of these had been pre-coated with Matrigel, diluted according to manufacturer’s specifications in DMEM / F-12 as described above. At experiment termination, cultures were fixed at room temperature for a duration of 20 minutes using a 4% paraformaldehyde solution (Electron Microscopy Sciences). Following fixation, the samples underwent three rinses with PBS and were subsequently subjected to a 15-minute permeabilization step with 0.2% Triton X-100 in PBS. After permeabilization, the samples were washed three times for 5 minutes each with a washing buffer containing 0.05% Tween-20 in PBS and were then blocked for 20 minutes using a solution consisting of 200 pl of wash buffer combined with 10% normal donkey serum from Jackson Immuno Research Laboratories. The primary antibodieswere diluted in PBS containing 5% normal donkey serum and were applied to the tissues for a duration of 1 hour at room temperature. Following this incubation, the coverslips were subjected to three additional washes, each lasting 5 minutes, using the wash buffer. Similarly, the secondary antibodies were diluted in PBS with 5% normal donkey serum and were applied to the tissues for 1 hour at room temperature. Subsequently, the samples underwent three more washes, each lasting 5 minutes, using the wash buffer. To complete the preparation of the coverslips for imaging, they were mounted onto microscopy glass slides using Prolong Diamond Antifade from Life Technologies. Afterward, they were allowed to cure overnight at room temperature.

[0365] Image Acquisition, Processing, and Analysis

[0366] Phase images were acquired by EVOS M700 automated scanning microscope (software version 2.0.2094.0). Images of IHC slides were acquired using Fritz slide scanner (Precipoint, MicroPoint software version 2021 -01 ). Immunofluorescence images were acquired by EVOS M700 automated scanning microscope (M7000 Software Revision v.2.0.2094.0), and Nikon A1 confocal microscope. Fiji / lmageJ (NIH) was utilized for image processing and quantification. Contrast adjustments were made for each channel was evenly applied for the whole image in that channel.

[0367] For FIG. 21 , two coverslips for each timepoint were immuno-stained for respective markers. Replicate images for each marker were binarized, and four random areas of each image were cropped. The total surface area percentage covered by cells positive for the marker in each cropped binarized image was measured using Imaged. For Extended Data Fig. 7b, the histogram was created using the “Plot Profile” function of Imaged for a horizontal line on the shown image. For the plot (right), the channel for the CD34 marker was binarized. 12 areas around the WT- derived structure and 12 areas in between the WT-derived structure were cropped, and the total surface area percentage covered by cells positive for CD34 was quantified. For this quantification, two samples of day 12 heX-embryoids were used.

[0368] For FIG. 44, three D20 heX-embryoid-CB co-cultured samples were gently fixed with 4% PFA for one hour to retain the CB-niche interaction. The images were taken in 4 channels including CD34, LYVE1 , CXCR4, and phase. To quantify the proximity of CD34+ CB cells to each vascular niche, more than 15 random areas (in three biological replicates) containing expanded CB CD34+ clusters (cluster wasdefined as an identifiable population of CB cells in one area) were cropped based on CD34 and Phase channels without looking at the type of vascular niche. Then, the type of vasculature niche and proximity were defined by quantifying the border of the CB cluster distance to each vascular niche to be up to 50 pm.

[0369] Bulk RNA Sequencing of heX-embryoid

[0370] RNA extraction, as previously outlined, was conducted on the samples, and the extracted RNA was subsequently forwarded to the UCLA Technology Center for Genomics and Bioinformatics for library preparation and sequencing. The libraries for RNA-Seq were meticulously prepared employing the KAPA Hyper Stranded RNA- Seq Kit. This library preparation workflow encompassed mRNA enrichment, cDNA generation, end repair to create blunt ends, A-tailing, adaptor ligation, and PCR amplification. Distinct adaptors were employed for multiplexing samples within a single sequencing lane. Sequencing was accomplished on the Illumina NextSeq500 platform, using a single-read 75bp run.

[0371] Quality assessment of the data was conducted via Illumina SAV, and the demultiplexing step was executed utilizing the Illumina Bcl2fastq2 version 2.17 program. Further evaluation of raw FASTQ quality was carried out using FASTQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc).

[0372] Reads were subsequently aligned to the latest UCSC transcript set using Bowtie2 version 2.1.0, with gene expression levels being estimated through RSEM vl .2.15. The EdgeR TMM (trimmed mean of M-values) algorithm was applied for the normalization of gene expression data. Additionally, reads were mapped to the latest UCSC genome set via Bowtie2 and Tophat, resulting in a BAM file from which alignment information was gathered using the PicardTools CollectRNASeqMetrics program. A Genebody analysis was carried out utilizing the ngsplot toolkit75.

[0373] To visualize the resulting data, heatmaps were generated using the heatmap package in R.

[0374] 10x Genomics Sample Preparation for Next-Generation Sequencing

[0375] The samples were prepared following the 10x Genomics Cell Multiplexing Oligo Labeling or Hashing protocols, ensuring cells with over 80% viability were selected. Single-cell suspensions were obtained from the samples by treating them with Accutase for 20 minutes at 37°C. To eliminate any aggregates, the cell suspension was passed through a 40 pm strainer. Subsequently, samples underwent centrifugation at 300 g for 5 minutes at room temperature, and the supernatant wasgently removed. The cell pellets were washed with 1 ml PBS+0.04% BSA. These samples were then resuspended to achieve a target concentration of 1 X 1 06cells / ml, adjusted based on expected cell densities for each day. After counting the cells using a hemocytometer, volumes were modified as necessary to reach a final cell count of 1 x106cells, with additional PBS + 0.04% BSA added to replace removed volumes.

[0376] During the Multiplexing Oligo Labeling procedure, samples were centrifuged once more at 300 g for 5 minutes at room temperature, and the supernatant was carefully discarded. Following this, the samples were resuspended in 50 pl of CellPlex Multiplexing Solution from 10x Genomics, using specific multiplexing oligo solutions assigned to each individual sample. Up to 12 samples could be labeled concurrently, with a 5-minute incubation period observed after the oligo solution was added to the last sample.

[0377] For multiplexing using Cell-Hashing, the samples were once again centrifuged at 300g for 5 minutes at 4eC degree, and the supernatant was carefully aspirated. The pellet was resuspended in cold PBS+2%FBS and 1 pl of the Hashing antibody (TotalSeq-B antibodies against CD298 / [32-microglobulin, Biolegend, Clone LNH-94; 2M2) was added to the cell suspension and incubated at 4 degrees for 30 minutes.

[0378] Post-labeling, 1 .95 ml of 1 x PBS + 1 % BSA was added to each sample, thoroughly mixed, and centrifuged at 300 g for 5 minutes at 4°C. The supernatant was carefully aspirated, leaving less than 10 pl of supernatant when feasible. Samples were then resuspended in 2 ml of 1 x PBS + 1 % BSA and thoroughly mixed for washing. This washing and centrifugation process was repeated twice more, with the final resuspension adjusted to achieve a cell count of 1 X 1 06cells / ml, accounting for a 50% cell loss from the initial count after the first protocol. The labeled cell suspensions were placed on ice for transfer to the Pitt Single Cell Core for library creation.

[0379] Following a final count and viability assessment, cells and 10x Genomics reagents were loaded into the single-cell cassette, targeting 25,000 single cells for analysis. This accounted for expected cell loss and doublets resulting from multiplexing, as outlined in the Chromium Single-Cell 3’ Reagent Kit user guide from 10x Genomics. After generating GEMs (Gel Bead-In Emulsions), the cDNA library was prepared by the Pitt Single Cell Core staff, adhering to the relevant steps outlined in the 10x Genomics user guide. Subsequently, the libraries were sent to the UPMC Genome Center for a sequencing on a NovaSeq S4-200 platform, aiming for anintended read depth of 100,000 or 125,000 reads per cell with 150 bp paired-end reads. Downstream analysis of the sequencing data yielded varying mean reads per cell, ranging from 40,000 to 150,000 in different samples.

[0380] Single Cell RNA Sequencing Sample Processing and Quality Control, and analysis

[0381] The analysis pipeline for the single-cell data followed several steps using the 10x Genomics CellRanger pipeline. Initially, reads were aligned to the reference genome (GRCh38.84) supplemented with transgene sequences. This alignment process assigned reads to individual cells and estimated gene expression based on unique molecular identifier (UMI) counts.

[0382] To ensure data quality, single cells were excluded based on a high ratio of mitochondrial genome transcripts and either unusually high or low feature or UMI counts. Genes with UMI counts in fewer than 5 cells were also filtered out. For subsequent scRNA-Seq data processing and cluster analysis using Seurat V4, a standardized pipeline was applied. This included SCTransform to regress percent mitochondrial genes, principal component analysis (PCA), and clustering. To determine the optimal number of principal components (PCs) that retained the most variation, jackstraw plots and permuted p-values were employed.

[0383] The quality of clustering was evaluated through enrichment analysis of marker genes for each cluster, which are genes that are significantly upregulated in a particular cluster when compared to all other clusters, alongside embryo cell typespecific genes. For quality assurance, adjustments were made to the principal component (PC) and resolution parameters to ensure the selection of the most biologically pertinent clustering. Seurat V5 was used for the subsetting of the population based on gene expressions, and data visualization such as UMAP plots, violin plots, dot plots, feature plots, and feature scatter plots.

[0384] For dot plot in the Extended Data Fig. 7f, the “Stalk mesoderm” population was made by subsetting the mesodermal population in day 16 hexembryoid with HQXA10>0 expression. For Allantois, the endoderm cluster was subclustered and the subcluster containing the cells with expression of SOX17, CDX2, SHH, FGF9, WNT5B, and CST1 was named “Allantois”. For different endothelial cell subtypes, the endothelium cluster was subclustered and they were named based on the expression of genes shown in FIG. 42.

[0385] Cell-cell communication analysis

[0386] Cell-cell communication inference was performed between cell clusters using the CellPhoneDB V5.0033. All three CellPhoneDB v5 interaction types (paracrine, cell-adhesion, and juxtracrine signaling) were used. All other parameters were set to default. The receiver population was the cluster in the UMAP of expanded cord blood with CD34 expression. The sender sample was the heX-embryoids niche that was used in the same experiment for the expansion of the receiver cells on day 20 in regular cytokine conditions. All three CellPhoneDB v5 interaction types (paracrine, cell-adhesion, and juxtracrine signaling) were used. All other parameters were set to default. The receiver population was the cluster in the UMAP of expanded cord blood with CD34 expression. The sender sample was the heX-embryoids niche that was used in the same experiment for the expansion of the receiver cells on day 20 in regular cytokine condition.

[0387] Gene Set Enrichment Analysis

[0388] For gene set enrichment analysis, the comparing datasets were first merged using “merge” function of Seurat. After subsetting, the Lin-CD34+CD38- population (CD34 > 0 & CD38 == 0 & CD14 ==0 & GYPA==0 & GP1 BA==0 & CD3D==0 & CD3E==0 & FCGR3A==0 & CD19==0 & MS4A1 ==0 & NCAM1 ==0), the DEseq2 R package was used to find the differentially expressed genes between the two datasets. The fgsea R package was used to assess the MsigDB c2 curated pathways and Hallmarks pathways. The results were visualized using the Graph Pad Prism 9.

[0389] Quantification and Statistical Analysis

[0390] In studies involving statistical analyses, unless specified differently, a minimum of three biological replicates were employed. Statistical tests were noted in the legend of each corresponding figure panel. For comparisons involving only two conditions, unpaired t-tests were applied, as indicated, with a significance threshold of p < 0.05. Statistical assessments encompassing three or more conditions were conducted through one-way ANOVA and post hoc Tukey’s test.

[0391] For comparison of heX-embryoids cell type similarity to in vivo YS in FIG. 19, we assessed the resemblance between two sets of marker genes, A and B, by employing the hypergeometric test to determine overrepresentation. This approach is similar to conducting a one-tailed Fisher’s Exact Test.Results

[0392] Development of YS and pre-umbilical cord like niches within heX- embryoids

[0393] We previously reported heX-embryoids, a post-implantation in vitro model of the human embryo derived from human induced pluripotent stem cells (hiPSCs). These embryoids are generated by combining iPSCs engineered to overexpress the GATA6 transcription factor (iGATA6-iPSCs) with wild-type (WT) iPSCs (WT-iPSCs) (FIG. 13). heX-embryoids recapitulate aspects of yolk sac (YS)- like hematopoiesis characteristic of early post-implantation human embryos. The YS- like niches formed in this system hold potential for regenerative medicine applications and for investigating YS functions. However, the full spectrum of cell types and the extent to which heX-embryoids can model post-implantation developmental stages in vitro remained unexamined.

[0394] Analysis of single-cell RNA sequencing (scRNA-seq) data from day 16 heX-embryoids confirmed the expression of genes specific to in vivo YS lineages, including YS-like endoderm (e.g., APOM, ASGR1 , HNF4A), YS-like fibroblasts (LUM, PDGFRA), pericytes (ACTA2, ITGA1 ), and mesothelium (KRT19, PDPN) (FIGS. 14- 15). Furthermore, neural ectoderm (SIX3, SOX2) and amniotic tissue (TFAP2C, GAT A3, WNT6), derived from the WT-iPSC compartment, were also observed (FIGS. 14-15). By day 14 in vitro, heX-embryoids develop an interconnected network of CD31 + endothelial cells. This structure is supported by HNF4a+ endodermal cells, Desmin+ fibroblasts, Nestin+ pericytes, and Vimentin+ mesoderm derivatives, collectively forming a YS-like three-dimensional tissue organization (FIG. 16).

[0395] Deeper characterization of the endodermal, mesodermal, and endothelial populations within heX-embryoids identified cell types not characteristic of the bona fide YS. These cells exhibited high enrichment for HOXA10 and HOXA1 1 expression, genes known in mice to mark the connecting stalk, the precursor to the umbilical cord. Consistent with this, analysis of a human Carnegie Stage 8 (CS8) spatial scRNA-seq dataset revealed HOXA10 and HOXA1 1 expression in the human connecting stalk. Phase-contrast imaging of heX-embryoids showed cord-like structures extending from the WT-iPSC compartment towards the YS-like interface (FIG. 17). Immunofluorescence (IF) staining for HOXA1 1 , HAND1 , and CD31 in third- week heX-embryoids confirmed the presence of HOXA1 1 + endothelial and mesodermal cells (FIG. 18). These cells displayed a spiral arrangement and wereconnected by endothelial cells to the interface, a morphology potentially reflecting the developmental transition of the connecting stalk into the umbilical cord (FIG. 18). In some instances, these HOXA1 1 + mesodermal and endothelial cells were observed in close proximity to this interface (FIG. 17). The endothelial cells were SOX17+ showing their arterial identity.

[0396] Previous work indicated that the human allantois arises from an invagination of YS visceral endoderm and hindgut. While the CS8 spatial dataset did not identify a distinct endodermal cell type within the connecting stalk, it reported high expression of signaling factors such as WNT5B, FGF9, and SHH. Notably, we detected expression of these same genes in HOXA10+ endodermal cells within heX- embryoids (FIG. 19). These cells also highly expressed SOX17 and CST1 , markers for visceral endoderm and hindgut, respectively, which are also present in the CS8 embryo's connecting stalk (FIG. 19). IF staining for SOX17 and CST1 revealed coexpressing cells in two locations: at the WT-iPSC / YS-like interface, consistent with visceral endodermal cells, and as tube-like structures developing within the spirally oriented, SOX17+ arterial endothelial cells reflecting the morphogenesis of allantois diverticulum (FIG. 19).

[0397] In conclusion, heX-embryoids recapitulates the formation of yolk sac cell types and structures, amnion, and cell types of human pre-umbilical cord.

[0398] These findings demonstrate the in vitro reconstitution of YS, connecting stalk, and allantois-like structures within heX-embryoids, with the latter two being reported for the first time in an embryoid model. Importantly, the integrated analysis of scRNA-seq from heX-embryoids and IF staining, corroborated by the human CS8 spatial dataset, provides detailed molecular signatures and structural insights into a pre-umbilical cord-like structure. This addresses a significant knowledge gap in early human development, previously limited by ethical considerations, technical challenges, and sample scarcity.

[0399] heX-embryoids support a homeostatic balance between HSC expansion and maintenance under low cytokine conditions

[0400] Having successfully reconstructed YS and pre-umbilical cord like niches, we next evaluated their ability to support the expansion and maintenance of cord blood (CB) CD34+ cells while preserving their functionality (FIG. 20). CB CD34+ cells were introduced to day 10 heX-embryoids and co-cultured for 10 days, which was determined to be the optimal time point for cell addition and culture duration based onprior assessments. As cytokines are conventionally used for HSC expansion, we supplemented heX-embryoids-CB cocultures with SCF, TPO, and FLT3L at full concentrations (100, 50 and 100 ng / mL, respectively), and reduced these levels to 10%, 1 %, and 0% (FIG. 21). Morphologically distinguishable spherical CB cells on the heX-embryoids cultures, and fluorescent labeling of CB CD34+ cells prior to introduction to the heX-embryoids niche confirmed that the expanding cells were derived from CB rather than de novo generation from heX-embryoids.

[0401] While increasing cytokine concentrations resulted in an overall increase in total cell numbers, the proportion of immunophenotypic HSCs (Lin-CD34+CD38- CD45RA-CD49f+CD90+) relative to the total cell population revealed a different trend (FIG. 21). The highest percentage of HSCs was observed at 1 % cytokine level, whereas higher concentrations resulted in reduced HSC frequency, suggesting more differentiation of the cells (FIG. 21).

[0402] At the 1 % cytokine level, heX-embryoids supported both expansion and maintenance of the HSCs in culture, while no expansion or maintenance was detected in the low-cytokine control group. Additionally, even in the absence of exogenous cytokines, heX-embryoids maintained and expanded HSCs, while no HSCs were maintained in the 0% cytokine control group. These findings suggest that heX- embryoids, and potentially the human YS and pre-umbilical cord niche, provide essential hematopoietic growth factors that support HSC expansion and maintenance.

[0403] We next compared the total cell number, immunophenotypic HSPCs (Lin-CD34+CD38-) and immunophenotypic HSCs (Lin-CD34+CD38-CD45RA- CD49f+CD90+) in CB-heX-embryoids cocultures supplemented with 1 % cytokine and 100% cytokine-only conditions. While the 100% cytokine-only condition yielded a higher total cell number, coculture with heX-embryoids, resulted in an 18-fold expansion of immunophenotypic HSCs (Lin-CD34+CD38-CD45RA-CD49f+CD90+). This increase was twice the fold expansion observed in the 100% cytokine-only condition (p-value= 0.0003) (FIG. 22).

[0404] Moreover, the frequency of phenotypic HSCs increased following expansion on heX-embryoids compared to the input CB CD34+ population, whereas it declined in the 100% cytokine control. This indicates that supra-physiological cytokine levels promote more differentiation and favor progenitor cell generation over HSC self-renewal. Notably, heX-embryoid co-cultures retained a significantly higher proportion of Lin-CD34+ (50%) and Lin-CD34+CD38- (33%) populations, indicative ofa preserved HSPC pool (FIG. 23, e). In contrast, cultures with 100% cytokines showed a sharp reduction in these populations (14% and 2%, respectively), indicating accelerated differentiation and potential HSC exhaustion (FIGS. 23-24). This was reflected in the loss of CD34, associated with multipotency and self-renewal, and the increased expression of CD38, a marker of lineage commitment (FIGS. 23-24). These results demonstrate that heX-embryoids provide a balanced culture environment that promotes HSC self-renewal and limits excessive differentiation.

[0405] To further evaluate the ability of heX-embryoids to prevent HSC exhaustion, we re-cultured the cells onto fresh day-10 heX-embryoids every 10 days. This strategy supported robust HSC expansion achieving up to a 1000-fold increase, while monocultures with reduced cytokine levels mostly collapsed. Moreover, we could maintain cells for over 40 days on the niche and passage multiple times (FIG. 25). In contrast, 100% cytokine-only cultures, which are the conventional method of expansion of HSCs so far, failed to sustain HSC self-renewal, instead promoting differentiation and depleting HSC pool, with no detectable HSCs remaining by the end of the long-term culture period (FIG. 25). These results highlight the ability of heX- embryoids to provide a supportive niche that maintains HSC integrity and function during culture period.

[0406] We also found an increase in the number of immunophenotypic HSPCs (Lin-CD34+CD38-). To assess the differentiation and proliferation abilities of these cells, we performed colony-forming unit (CFU) assay using an equal number of immunophenotypic HSPCs (Lin-CD34+CD38-) across different conditions. While regular-cytokine cultures primarily yielded granulocyte-macrophage progenitor colonies (CFU-GM), indicative of restricted lineage commitment (FIG. 26), HSPCs expanded in heX-embryoids generated all colony types with markedly higher numbers. This suggests a significant augmentation of the functional hematopoietic progenitor pool (FIG. 26). We also observed a notable increase in the frequency of CFU-GEMM colonies compared to the uncultured cells that indicates that heX-embryoids support the expansion or survival of multipotent progenitor cells with the competency to produce granulocyte, erythrocyte, macrophage, and megakaryocyte lineages. The frequency of other committed progenitors including the CFU-E, BFU-E, and CFU-GM colonies remained comparable to uncultured cord blood HSPCs (FIG. 27). Together, these results demonstrate that unlike conventional expansion methods, heX-embryoids maintain HSCs a without drastically depleting or over-expanding the more committed lineages.

[0407] Hematopoietic signatures in heX-embryoids

[0408] Given the marked hematopoietic expansion capability observed in heX- embryoids, we sought to comprehensively investigate and compare the hematopoietic niche of heX-embryoids and in vivo YS, which had not been thoroughly characterized before. We manually generated a curated list of genes pertinent to the hematopoietic niche and regulation of HSCsl 7-20 and analyzed their expression pattern in a publicly- available bona fide in vivo YS dataset (CS10-23)10, as well as in day 16 heX- embryoids just after expansion is observed to begin. We found that the heX-embryoids express hematopoietic factors in a cell type-specific manner strongly mirroring patterns observed in the in vivo YS tissues.

[0409] We observed that expansion factors such as insulin-like growth factor 2 (IGF2)21 and its regulators (IGFBP2 and IGFBP3), midkine (MDK)24, as well as non- canonical notch ligand DLK125, which contributes to HSC maintenance, are expressed by endoderm, mesoderm, and endothelium of both in vivo YS and heX- embryoids. However, these three lineages also express different signaling molecules with differential functions in HSC maintenance and expansion.

[0410] The endoderm in both YS and heX-embryoids exclusively express factors contributing to HSC maintenance and self-renewal, including thrombopoietin (THPO) and angiogenin (ANG), both of which are also expressed in both fetal liver and bone marrow26. Additionally, angiopoietin-like protein 3 (ANGPTL3), which supports HSC expansion in the fetal liver and bone marrow niches, are expressed by both heX-embryoids and YS endoderm.

[0411] We observed that different mesodermal cell types, including fibroblasts, pericytes, and mesothelium, show a similar expression pattern of the curated set of hematopoietic-related factors. The mesoderm in YS and heX-embryoids show exclusive expression of several factors essential for HSC maintenance, including non- canonical WNT ligands (e.g., WNT5A), angiopoietin (ANGPT1 ), BMP4, and CXCL12, all of which support HSC maintenance in the fetal liver and bone marrow niche. The mesoderm also expresses expansion factors including ANGPTL217, IGFPB5 and PTN24, and interleukins (IL6, IL1 1 ).

[0412] The endothelium in both YS and heX-embryoids also plays a role in HSC maintenance, with the expression of stem cell factor (KITLG [SCF]), TGFB1 , and NOTCH ligands such as JAG1 , DLL1 , DLL4.

[0413] Our analysis reveals that heX-embryoids recapitulate YS hematopoietic signatures in a cell type-specific manner.

[0414] heX-embryoid expanded HSCs show high engraftability and multilineage reconstitution

[0415] Next, we assessed the in vivo functionality of these cells via transplantation into immunocompromised mouse models, that support human cell engraftment. At 20 weeks post-transplantation, we assessed human cell chimerism in peripheral blood, bone marrow, and spleen (FIG. 28). We found that heX-embryoids- expanded cells reconstitute mice with human blood cells at significantly higher levels (FIG. 29). This observation was confirmed across peripheral blood, bone marrow, and spleen (p< 0.0001 for each organ). All recipient mice (14 out of 14) of the heX- embryoids-expanded cells demonstrated over 10% engraftment of human CD45+ cells in bone marrow, compared to only 2 out of 8 in the control (FIG. 29). Moreover, we observed enhanced multilineage reconstitution of human hematopoietic cells, evidenced by the generation of CD19+ B cells, CD3+ T cells, CD33+ myeloid cells, CD56+ NK cells, and CD235a+ erythroid cells in mice transplanted with heX- embryoids-expanded cells (FIG. 30). Given the lymphoid-biased differentiation typically observed in these mouse models64, the increased myeloid (p-value=0.0245) and erythroid (p> 0.05) output, further supports the maintenance of the differentiation capacity of functional HSCs. Successful humanization of these mice following transplantation resulted in larger spleen sizes in recipients of heX-embryoids- expanded cells, likely due to improved tissue cellularity and the formation of follicular structures within the organ. In immunohistochemistry (IHC) analysis of spleen sections, we also observed hCD3+ T cells and hCD20+ B cells were organized into tissue foci (FIG. 31).

[0416] As long-term multilineage reconstitution relies on the retention of HSPCs in bone marrow66, we evaluated the CD34+ cell chimerism and detected an average of 20% human CD34+ HSPCs-enriched cells in the bone marrow of mice transplanted with cells expanded on heX-embryoids, compared to 2% in the control condition (FIG. 32). We next performed secondary transplants using primary reconstituted mice bone marrow (FIG. 33) and found human CD45+ chimerism in all (5 out of 5) secondaryrecipients of the bone marrow cells from primary mice from the heX-embryoids group. Each recipient demonstrated chimerism levels above the 1 % threshold (FIG. 34), whereas the secondary recipient of primary control mice showed reconstitution below 0.01 % (FIG. 34). The ability to achieve chimerism in secondary transplants highlights the presence of LT-HSCs and the self-renewal capacity of the HSCs in the co-cultured samples. This was further supported by the sustained human hematopoietic reconstitution beyond 24 weeks post-transplantation. In the heX-embryoids cultured transplanted mice, high hCD45+ reconstitution was maintained from 18 to 35 weeks, while no hCD45 reconstitution was detected in the control group (FIG. 35). We also detected a shift in the balance towards T cell predominance over B cells after 18 weeks, reflecting the presence of LT-HSCs66 (FIG. 36). These results demonstrate that heX-embryoids co-cultured CB CD34+ cells preserved and expanded functional LT-HSCs with self-renewal and differentiation potential.

[0417] We then sought to assess whether heX-embryoids supplemented with low-cytokine levels offer improved in vivo functional outcomes compared to cytokinebased regimens at regular levels. After transplanting an equal number of expanded cells, the heX-embryoid co-culture resulted in significantly higher multilineage hCD45+ reconstitution in the bone marrow and spleen. This finding suggests that culture of CB cells using heX-embryoids, despite yielding fewer total expanded cells, provides a more effective strategy.

[0418] Therefore, heX-embryoids support the expansion of highly engraftable HSCs capable of multilineage humanization of mice immune system.

[0419] Embryonic niches within heX-embryoids maintain the qualities of CB- HSCs

[0420] We next performed scRNA-seq on uncultured CD34+ cells, expanded CD34+ cells with heX-embryoids in low cytokine conditions, and CD34+ cells expanded with regular cytokine levels without heX-embryoids. We observed higher expressions of CD34, PROM1 (CD133), MECOM, and ETV6, established markers crucial for HSC self-renewal and multipotency, in the heX-embryoid cocultured cells compared to uncultured cells (FIG. 37). In contrast, cells expanded in regular cytokine levels without heX-embryoids showed a substantial loss of stem cell characteristics (FIG. 37). This suggests that heX-embryoid coculture under low cytokine levels maintained the transcriptional signature of primitive HSCs.

[0421] heX-embryoids cultured cells also maintained a very similar high expression profile of MLLT3, a gene associated with the maintenance and self-renewal of HSCs (FIG. 38). A similar pattern was observed for MYCT1 , another key factor for HSC self-renewal and engraftability (FIG. 38). This data implies that co-culture with heX-embryoids is superior in maintaining the HSC self-renewal program compared to conventional expansion methods.

[0422] scRNA-seq analysis also showed that heX-embryoid-cultured cells in low cytokine levels exhibit decreased activation of the aryl hydrocarbon receptor (AHR) pathway linked to poor HSC self-renewal, deciphered from higher CYP1 B1 (downstream product of AHR pathway) expression (FIG. 39).

[0423] The addition of SR1 (an AHR inhibitor) did not impact HSPC expansion in heX-embryoids cultures, while significantly boosting it in cytokine-only control, highlighting that AHR inactivation is a mechanism used by heX-embryoids and potentially YS for maintaining HSC self-renewal program which warrants further investigation (FIG. 40).

[0424] Maintaining a quiescent (GO) state is a critical characteristic of engraftable HSCs. To assess the impact of different expansion conditions on the cell cycle status of CD34+ cells, we analyzed the expression of the quiescence marker CDKN1 C (p57) and directly quantified cell cycle phases. Cells expanded in the heX- embryoid low cytokine coculture exhibited the highest average expression of CDKN1 C (FIG. 41 ). In contrast, cells cultured in regular cytokine control conditions showed very low or negligible CDKN1 C expression, predominantly clustered at zero (FIG. 41 ). Uncultured CD34+ cells displayed an intermediate level of CDKN1 C expression, suggesting that the heX-embryoids might even enhance the expression of this quiescence-associated gene compared to the fresh uncultured state (FIG. 41 ).

[0425] Gene set enrichment analysis (GSEA) highlighted significant enrichment of HSF1 activation, proteasome activity, and aggrephagy, pathways essential for HSC function. Autophagy and fatty acid metabolism, crucial pathways for preventing exhaustion and maintenance of the HSC pool, were also enriched in heX-embryoids cultures.

[0426] These transcriptional findings were directly corroborated by our cell cycle analysis of Lin-CD34+CD38- cells (FIG. 42). A large proportion of cells in the heX-embryoid low cytokine coculture (approximately 75%) were found in the G0-G1 quiescent phase. (This data lacks uncultured cells and should be repeated to includeuncultured cells / low cytokine control should be also removed). This indicates that the heX-embryoids maintains a quiescent state in expanded HSCs.

[0427] As low mitochondrial activity is another established hallmark of quiescent, primitive HSCs, we further investigated the metabolic state of immunophenotypic HSPCs (Lin-CD34+CD38-), flow cytometry analysis using MitoTracker dye to label active mitochondria was performed. A distinctive population in immunophenotypic HSPCs (Lin-CD34+CD38-) with inactive mitochondria was observed in the heX-embryoid low cytokine coculture implying a preserved metabolically quiescent state of the cells (FIG. 43). We further investigated the oxidative stress levels within immunophenotypic HSPCs (Lin-CD34+CD38-) by quantifying mitochondrial reactive oxygen species (ROS) using MitoSOX staining. Elevated ROS levels are typically associated with increased metabolic activity, proliferation, differentiation, and cellular stress, which can compromise stem cell function. In contrast, primitive, quiescent HSCs are known to exhibit low intracellular ROS levels, primarily relying on anaerobic glycolysis over oxidative phosphorylation (OXPHOS). The heX-embryoid low cytokine condition, which showed superior maintenance of HSC sternness and quiescence in previous analyses, exhibited a relatively low percentage of MitoSOX+ cells (FIG. 44).

[0428] Beyond stem cell identity markers and metabolic state, the capacity of HSCs to home to and engraft in the bone marrow niche is critically dependent on the expression of specific cell surface adhesion molecules and chemokine receptors. We therefore analyzed the expression profiles of key homing and adhesion markers on Lin-CD34+CD38- cells across different culture conditions. The higher expression of CXCR4 and the sustained expression of CD44, VLA-4, and PSGL-1 in the heX- embryoid low cytokine condition suggests that CD34+ cells expanded in this culture condition are likely to retain their in vivo engraftment capabilities. (FIGS. 45A-45B).

[0429] To further characterize the functional potential of expanded CD34+ cells, we analyzed the expression of PROCR and STAT1 , markers associated with superior in vivo hematopoietic function. The increased expression of these markers in heX- embryoid low cytokine condition, compared to both uncultured cells and cells expanded under regular cytokine condition, suggests that heX-embryoids niche not only maintains the sternness of HSCs but also enhances functional properties for successful engraftment, and long-term hematopoietic reconstitution in a physiological setting (FIG. 46).

[0430] YS, amnion, and pre-umbilical cord niches differentially affect HSCs

[0431] As described earlier, heX-embryoids cultured under low-cytokine conditions enhance the proliferation of CB-HSCs, support phenotypic expansion suggestive of self-renewal, and maintain hallmark stem cell features such as low mitochondrial activity. Given the cellular heterogeneity of heX-embryoids — which include embryonic and extraembryonic lineages, we sought to determine whether these effects could be attributed to specific niche components.

[0432] To dissect spatial interactions between CB-HSCs and their microenvironment, we tracked the distribution of CD34+cells within the heX- embryoids. These cells are consistently localized at the boundary between embryonic and extraembryonic compartments, suggesting that this interface may serve as a regulatory site. The region encompasses signals coming from visceral endoderm and WT-derived lineages such as amnion, neural tissue, and connecting stalk mesoderm, positioning it as a potential signaling hub.

[0433] To test whether expansion of this interface enhances niche function, we incorporated additional WT cells during embryoid formation. This modification resulted in a subtle reduction in total cell output, accompanied by an increased frequency of quiescent cells. Notably, self-renewal capacity was elevated, and mitochondrial activation was further suppressed. Conversely, embryoids depleted of WT cells — thereby lacking amnion, neural tissue, allantois, stalk, and surprisingly endothelial components — displayed increased mitochondrial activity and loss of self-renewal, despite maintaining robust proliferation. These findings underscore a critical role for interface signaling hub, in sustaining self-renewal and regulating metabolic quiescence, independent of proliferative cues.

[0434] To isolate the contribution of specific cell types, we inhibited BMP4 signaling from days 3 to 6 to selectively block extraembryonic mesoderm formation. This intervention abrogated the development of yolk sac mesoderm (YSM) and endothelium. Co-cultures with these BMP4-deficient embryoids exhibited negligible CB cell proliferation, implicating YSM and endothelium as a primary driver of proliferative signaling.

[0435] To further validate lineage-specific roles, we FACS-isolated defined cell populations and co-cultured them with CB cells in low-cytokine conditions. Among these, BST2+YSM cells markedly enhanced proliferation, while other lineages had limited impact. Interestingly, amnion and pericytes promoted self-renewal, whereasendothelial cells, neural tissue, and yolk sac endoderm (YSE) did not exhibit such effects.

[0436] Although surface markers for connecting stalk lineages remain undefined resulting in incapability to isolate them, we tested their contribution by supplementing cultures with their secreted factors. The addition of IGFBP6 (allantois) and HGF (connecting stalk mesoderm) to monoculture and co-culture systems led to a significant reduction in mitochondrial activity, suggesting that connecting stalk- derived signals are instrumental in maintaining metabolic dormancy in HSCs. We also found out that inhibition of WNT signaling decreases the formation of stalk and allantois while not changing the other lineages. Cord blood seeded in WNT-inhibited heX-embryoids showed increased mitochondrial activity. These results are consistent with in vivo data showing that HSCs within the four-week-old embryonic cord exhibit reduced mitochondrial gene expression compared to their AGM-derived counterparts.

[0437] We used our single-cell transcriptomics to identify a diverse array of cell types within our synthetic niche. Immunofluorescence staining confirmed the presence of different populations in culture. Single-cell analysis in tandem with staining enabled us to identify reliable surface markers for various cell types, which we then used to isolate and culture each independently qPCR analyses confirmed high expression of lineage-specific markers in the purified populations.

[0438] To dissect the contributions of individual cell types, we cultured CB- HSCs on isolated populations (day 4, post-isolation), reconstituted combinations, or the full tissue. After 7 to 10 days in supplemented low-cytokine conditions, we assessed expanded cell numbers, self-renewal markers (via flow cytometry), and mitochondrial activity. Through this analysis, we observed that yolk sac endoderm, fibroblasts, mesothelium were the most effective in driving CB cells proliferation (Fig 5j). Pericytes and amnion-like cells were most potent in supporting self-renewal, independent of proliferation-promoting populations. A subset of mesodermal cells (named pre-umbilical cord-like due to similarity in RNA-seq) reduced mitochondrial activity, suggesting a role in metabolic quiescence. We also identified a novel specific subset of endodermal cells (allantois-like), which have not been recapitulated in any existing human stem cell-based multicellular model. These cells also induced metabolic quiescence.

[0439] To assess the necessity of physical proximity for niche function, we employed transwell inserts to separate CB cells from embryoid tissues. Althoughoverall expansion persisted, the frequency of phenotypic HSPCs (Lin’CD34+CD38“) dropped by approximately 50%, indicating that proximal cell-cell interactions are crucial for maintaining sternness.

[0440] IF staining also revealed clusters of expanding CB cells (marked by CD44) located in the close vicinity of endothelial cells (marked by CD34), with some CB cells making direct contact with the vasculature (FIGS. 47-48). Additionally, we observed a high density of endothelium in the area of the YS-WT interface (FIG. 49). Using scRNA-seq on day 16 heX-embryoids, we observe that heX-embryoids reconstitute the entire diversity of endothelial cells reported recently in the YS in vivo, including LYVE1 + STAB2+ sinusoidal endothelium, CXCR4+ GJA4+ arterial endothelium, VWF+ endothelial cells, and RUNX1 + hemogenic endothelium (FIGS.50A-50D).

[0441] IF analysis of heX-embryoids revealed the presence of both arterial endothelial cells, marked by CXCR4, and sinusoidal endothelial cells, marked by LYVE1 10, at the WT-YS interface (FIG. 51). However, co-staining these markers with CD34 to find the spatial positioning of the expanded CB cells revealed that these cells were mostly positioned in proximity to CXCR4+ arterial niches (FIGS. 52-53). Arterial endothelial cells also express notch ligands (DLL1 , DLL4, and JAG1 ), WNT5A, WNT5B, KITLG, IGFBP2 and IGFBP3 which are HSC maintenance and expansion factors (FIG. 54).

[0442] Overall, by utilizing heX-embryoids, our results reveal a previously unexplored role of the embryo-extraembryonic interface, including the stalk mesodermal cells, allantois, and potentially the arterial niches, in the maintenance and expansion of HSCs within a human yolk sac-like environment.

[0443] To systematically look at the interaction between cell types of the niche and CB-CD34+ cells, we performed scRNA-seq on day 20 of the heX-embryoids and CB coculture, at the end of the co-culture period, and analyzed patterns of cell-cell communication using CellPhoneDB33. This analysis revealed significant signaling contributions from mesoderm and endoderm cell type clusters, with 60 ligands originating from mesoderm and 54 from endoderm predicted to communicate with receptors within the CD34+ HSPC-enriched cluster in post-expansion CB cells. We also observed a high number of ligand-receptor pairings between this HSPC-enriched cluster and WT-derived populations, including amnion and neural ectodermal cells;however, most of these interactions were primarily redundant to those from mesoderm and endoderm.

[0444] We observed that YS endoderm, YS mesoderm, and YS endothelial cells provided both overlapping and distinct hematopoietic cues. These three lineages were all detected to be significant senders of insulin-like growth factor 2 (IGF2), key ligand for the induction of HSPC expansion; DLK1 , notch ligand important for HPSC maintenance; and prostaglandin E2, a known factor for enhancement of in vivo HSC expansion. Additionally, GAS6, known for supporting ex vivo expansion of HSCs, is also secreted by the endoderm and endothelial cell populations.

[0445] YS mesoderm and endoderm secrete a diverse range of canonical WNT signals (e.g., WNT2B, WNT3) that are implicated in promotion of expansion; these populations also secrete non-canonical WNTs (e.g., WNT5A, WNT1 1 ) and WNT antagonists (DKK1 ), which have been shown to contribute to HSPC maintenance. The role of WNT5A in HSC maintenance has also been reported previously both in vitro and in vivo

[0446] Endothelial cells and YS megakaryocyte strongly communicate via the maintenance factor TGF-[3, which is also prevalent in bone marrow megakaryocytes for HSC quiescence, suggesting shared functional mechanisms.

[0447] In addition to the automatic curation of likely ligand-receptor interactions produced by CellPhoneDB, we further noted the expression of important hematopoietic ligand-receptor pairs (on niche and CB CD34+ cells, respectively). These include BMP4-BMPR2, IGFBP2-PTPRB, THPO-MPL, EPO-EPOR, PTN- PTPRS, MDK-LRP1 , ANGPTL2-LILRB2, CXCL12-CXCR4, and SHH-PTCH1.

[0448] We conclude that the heX-embryoids can establish a multipronged signaling environment to direct HSC expansion ex vivo. Additionally, our experiments and analyses show the utility of heX-embryoids to identify previously unknown factors potentially involved in regulating HSC biology by human YS such as GAS6, prostaglandin E2, and WNT effectors.

[0449] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover modifications and equivalent arrangements that are within the spirit and scope of the appended claims. For example, it is to be understood that the presentinvention 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.

Claims

THE INVENTION CLAIMED IS1 . A method of producing an embryoid model comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; and culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components.

2. The method of claim 1 , further comprising co-culturing one or more third cells with the one or more first cells and the one or more second cells.

3. The method of claim 2, wherein the one or more third cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

4. The method of claim 1 , wherein expression of GATA6 is under the control of an inducible system, optionally a Dox-inducible system.

5. The method of claim 1 , wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

6. The method of claim 1 , wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

7. The method of claim 1 , wherein the GATA6 is endogenous to the cell.

8. The method of claim 1 , wherein the GATA6 is exogenous to the cell.

9. The method of claim 1 , wherein the GATA6 is human GATA6 or mouse GATA6.

10. The method of claim 1 , wherein the GATA6 is transiently expressed in the cell.1 1 . The method of claim 1 , wherein the embryoid secretes one or more of secreting stem cell growth factor (hSCF), thrombopoietin (TPO), and / or Fms- like tyrosine kinase 3 (FLT3L).

12. A method of expanding a population of CD34+ cells, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing one or more CD34+ cells with the embryoid in stem cell growth medium.

13. The method of claim 12, further comprising co-culturing one or more third cells with the one or more first cells and the one or more second cells.

14. The method of claim 13, wherein the one or more third cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.

15. The method of claim 12, wherein expression of GATA6 is under the control of an inducible system, optionally a Dox-inducible system.

16. The method of claim 12, wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

17. The method of claim 12, wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

18. The method of claim 12, wherein the GATA6 is endogenous to the cell.

19. The method of claim 12, wherein the GATA6 is exogenous to the cell.

20. The method of claim 12, wherein the GATA6 is human GATA6 or mouse GATA 6.

21. The method of claim 12, wherein the GATA6 is transiently expressed in the cell.

22. The method of claim 12, wherein the embryoid is capable of expanding CD34+ cells without the addition of a cytokine to the embryoid or the stem cell culture medium.

23. The method of claim 12, wherein the CD34+ cells are autologous or allogenic stem cells expressing one or more markers of a hematopoietic stem cell, for example, a CD34+ stem cell.

24. The method of claim 12, wherein the CD34+ cells are human cells.

25. The method of claim 24, further comprising delivering the expanded CD34+ cells to an immunocompromised mouse, wherein the expanded CD34+ cells engraft into bone marrow of the immunocompromised mouse.

26. The method of claim 25, wherein the engrafted human hematopoietic stem cells differentiate into leukocytes which generate immune chimerism in the lung, liver, spleen, kidney, brain, gonads, and / or peripheral blood of the transplanted mouse.

27. The method of claim 25, wherein the immunocompromised mouse is of NSG strain or NBSGW.

28. A method of rejuvenating a cell, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing one or more third cells with the embryoid or medium in which the embryoid is cultured, wherein the embryoid or the medium prevents exhaustion and aging of the third cells.

29. The method of claim 28, further comprising co-culturing one or more fourth cells with the one or more first cells and the one or more second cells.

30. The method of claim 29, wherein the one or more fourth cells expresses ETS variant transcription factor 2 (ETV2) or a variation or derivative thereof.31 . The method of claim 28, wherein expression of GATA6 is under the control of an inducible system, optionally a Dox-inducible system.

32. The method of claim 28, wherein the cell culture medium comprises 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and / or 0.1 -50 ng / mL of human thrombopoietin (hTPO).

33. The method of claim 28, wherein the first cells and the second cells are cultured for about 2-7 days, and wherein the embryoid structure is cultured for about 10-40.

34. The method of claim 28, wherein the GATA6 is endogenous to the cell.

35. The method of claim 28, wherein the GATA6 is exogenous to the cell.

36. The method of claim 28, wherein the GATA6 is human GATA6 or mouse GATA6.

37. The method of claim 28, wherein the GATA6 is transiently expressed in the cell.

38. The method of claim 28, wherein the CD34+ cells are autologous or allogenic stem cells expressing one or more markers of a hematopoietic stem cell, for example, a CD34+ stem cell.

39. The method of claim 28, wherein the one or more third cells are egg cells.

40. The method of claim 28, wherein the one or more third cells are sperm cells.41 . The method of claim 28, wherein the one or more third cells are obtained from a patient.

42. The method of claim 28, wherein the one or more third cells comprise a hematopoietic stem cell, a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglial cell, a blood cell and / or a blood cell derivative, a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased cell, and / or modified stem cell.

43. The method of claim 28, wherein the third cells are cultured with the embryoid in the presence of a target factor.

44. The method of claim 43, wherein the target factor is one or more of an exosome, an extracellular component, MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN, LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 , SELPLG, SIGLEC1 ,HGF, IGBP2, IGBP6, MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF1 1 , IGFBP4, PLTP, PROS1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IL1 1 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA, DHEA-S, CCL23, WNT3, LTE4, FGF2, KITLG, VTN, DKK1 , WNT1 1 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

45. A method of identifying one or more factors effective to rejuvenate a cell, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells;culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; culturing one or more third cells with the embryoid in stem cell growth medium comprising one or more factors; and identifying one or more of the one or more target factors that are capable of rejuvenating the one or more third cells.

46. The method of claim 45, wherein the one or more target factors are one or more of an exosome, an extracellular component, MDK, APP, FN1 , COL1 A1 , COL1 A2, MIF, IGF2, PTN, LAMB1 , DLK1 , JAG1 , PRSS3 F2, PLG, LGALS9, CDH1 , ITGB2, JAM1 , EFNB3, PECAM1 , RETN, ICAM2, TGFB1 , GDF15, THBS1 , SELPLG, SIGLEC1 , MYDGF, HDGF, TFPI, FABP5, TIMP1 , NAMPT, TIMP3, GDF1 1 , IGFBP4, PLTP, PROS1 , SPARC, IGBP3, IGFBP7, APOM, PDGFA, GRN, TIMP2, SERPINF1 , SERPINE2, CST3, CLU, ANG, EGFL7, CLEC1 1 A, FSTL1 , MANF, MGFE8, FAM3C, LGALS1 , LGALS3BP, GAS6, RBP4, BMP4, APOA1 , VEGFA, VEGFB, HMGB1 , PSAP, CREG1 , PDGFC, GDF6, ENPP2, NRTN, EGFL6, TGFB2, WNT5A, GPC6, BMP1 , BMP2, SFRP2, SFRP1 , IGFBP2, IGFBP6, IL1 1 RA, IL6ST, CCL3, IGF1 , TNFSF10, PGE2, INHA, DHEA-S, CCL23, WNT3, LTE4, FGF2, VTN, DKK1 , WNT11 , IGFBP3, CXCL14, WNT2B, WNT6, WNT4, WNT5B, CCL2, and / or SEMA3C.

47. The method of claim 45, wherein the one or more third cells comprise a hematopoietic stem cell, a skeletal muscle stem cell, a mesenchymal stem cell, a germ cell, a gamete, a microglial cell, a blood cell and / or a blood cell derivative, a megakaryocyte, a dendritic cell, a pancreatic islet cell, a diseased cell, and / or a modified stem cell.

48. A method of rejuvenating a cell, comprising administering to a cell in need of rejuvenation one or more target factors identified according to the method of claim 45.

49. A method of producing a macrophage or monocyte progenitor, comprising:co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and culturing the embryoid in a medium comprising glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage or monocyte progenitor from the culture.

50. The method of claim 49, wherein the macrophage or monocyte progenitors express CD68, CD14, and / or IBA1.51 . A method of generating extracellular vesicles (EVs) or exosomes with immunomodulatory or regenerative activity, comprising: culturing one or more macrophages generated according to claim 49 in a culture medium, thereby generating a conditioned medium; collecting the conditioned medium from the culture; and isolating EVs or exosomes from the conditioned medium with centrifugation, size-exclusion chromatography, or affinity-based capture.

52. The method of claim 51 , further comprising enriching the EVs or exosomes for those containing a cargo.

53. The method of claim 52, wherein cargo comprises mRNA, miRNA, and / or an anti-inflammatory or regenerative protein.

54. An embryoid model prepared according to claim 1 .

55. A culture of expanded CD34+ cells prepared according to claim 12.

56. A rejuvenated cell prepared according to claim 28.

57. A macrophage prepared according to claim 49.

58. An EV or exosome prepared according to claim 51 .

59. A method of treating a disease or condition in a patient, comprising administering to the patient an amount of CD34+ cells prepared according to claim 12 effective to treat the disease or condition.

60. A method of treating a disease or condition in a patient, comprising administering to the patient an amount of a rejuvenated cells prepared according to claim 28 effective to treat the disease or condition.61 . A method of preparing a patient for in vitro fertilization, comprising administering to the patient one or more rejuvenated cells prepared according to claim 28.

62. A method of generating an organ bud for transplantation, comprising expanding one or more rejuvenated cells prepared according to claim 28.

63. A method of producing a macrophage progenitor cell, comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; andculturing the embryoid in a medium comprising glutamine, glucose, sodium bicarbonate, 1 -100 ng / mL of human stem cell factor (hSCF), 1 -100 ng / mL of human FLT3L (hFLT3L), 1 -100 ng / mL of hepatocyte growth factor (HGF), 1 -100 ng / mL of Insulin-like growth factor-binding protein-2 (IGFBP-2), and 0.1 -50 ng / mL of human thrombopoietin (hTPO); and harvesting a macrophage progenitor cell from the culture.

64. A method of producing an embryoid model comprising: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells comprise induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells comprise non-engineered stem cells; culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that comprises embryonic and extraembryonic components; and isolating one or more regions of the heterogenous tissue, wherein the one or more isolated regions are subsequently used as a feeder layer, for expansion of one or more cells, for the production of a target factor, production of an exosome, production of extracellular matrix (ECM), and / or for rejuvenation of one or more cells.

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