Directing Human Embryonic Stem Cells to Generate Vascular Progenitor Cells

H Bai and ZZ Wang Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA

Pluripotent human embryonic stem cells (hESCs) differenti- it is also possible that endothelial cells and SMCs derived ate into most of the cell types of the adult human body, from hESCs can be used to engineer artiﬁcial vessels to including vascular cells. Vascular cells, such as endothelial repair damaged vessels and form vessel networks in cells and vascular smooth muscle cells (SMCs) are engineered tissues. Here we review the current status of signiﬁcant contributors to tissue repair and regeneration. In directing hESCs to differentiate to vascular cells. addition to their potential applications for treatment of Gene Therapy (2008) 15, 89–95; doi:10.1038/sj.gt.3303005; vascular diseases and stimulation of ischemic tissue growth, published online 16 August 2007

Keywords: human embryonic stem cells; endothelial cells; smooth muscle cells

Introduction which give rise to hematopoietic stem cells and vascular endothelial cells.10–12 Even both of them give rise to Some degenerative diseases are caused by the loss of hematopoietic and endothelial cells, it is unclear whether tissue-specific cells in organs, examples include: (i) type I hemangioblasts are the same as hemogenic endothelial diabetes, in which insulin-producing b cells are de- cells. For easy to discuss, we will use the term stroyed by an autoimmune disorder; (ii) Parkinson’s hemangioblast for both of them. disease, in which dopamine-producing neuronal cells are In the embryo proper, primitive vascular networks are destroyed; (iii) heart failure, which is caused by the formed de novo by the aggregation of embryonic massive loss of cardiomyocytes; and (iv) ischemic endothelial cells, a process termed vasculogenesis. In diseases, which is primarily caused by endothelial addition to originating from hemangioblasts, embryonic dysfunction. Cellular therapies to treat these diseases endothelial cells arise from restricted progenitor cells, are hampered by the lack of donor cells. Therefore, the called angioblasts that are derived from the mesoderm successful derivation of human embryonic stem cells and are committed to the endothelial lineage.13,14 The (hESCs)1 could provide an unlimited source of cells to initial vascular network, which consists of endothelial replace damaged or degenerated cells and tissues caused cells that form interconnected vessels, undergoes a by these diseases. succession of morphogenetic events involving sprouting, The endothelium is the first tissue to differentiate proliferation and migration of endothelial cells, and during vertebrate embryogenesis. Based on the close remodeling to generate large branched vessels, a process relationship of the vascular and hematopoietic systems called angiogenesis. Mural cells, including pericytes and during organogenesis, it was hypothesized that hemato- vascular smooth muscle cells (SMCs) are recruited to the poietic and endothelial cells are derived from a common newly formed vessels, causing the vascular networks to precursor, the hemangioblast.2–4 Commitment to the be mature and become stable.7,15 Controlling vascular hematopoietic and endothelial lineages within the yolk cells and angiogenesis, either positively or negatively, sac at mouse embryonic day (E) 7.5 results in the could be critical for the treatment of various diseases, development of blood islands that consist of an outer including cardiovascular diseases and cancers. layer of endothelial cells and an inner cluster of primitive Vasculogenesis and angiogenesis are largely depen- hematopoietic cells. Endothelial and hematopoietic cells dent on the functional changes of proliferation, differ- in blood islands are derived from aggregates of entiation and migration of endothelial cells, and current hemangioblasts.5–7 The structure of the yolk sac blood angiogenesis research relies primarily on mouse models. islands in the human embryo is similar to those in the Based on mouse models, the three families of receptor mouse embryo.8,9 The aorta-gonad-mesonephros region tyrosine kinases and their ligands found are: (i) vascular of the embryo proper also contains hemogenic endothe- endothelial growth factor receptors (VEGFRs) and lial cells (or called angio-hematopoietic progenitors), VEGFs; (ii) Tie receptors and angiopoietins; and (iii) Eph receptors and ephrins, which play major roles Correspondence: Dr ZZ Wang, Center for Molecular Medicine, in regulating vascular development.15 Members of the Maine Medical Center Research Institute, 81 Research Drive, fibroblast growth factor (FGF) family and the bone Scarborough, ME 04074, USA. E-mail: [email protected] morphogenetic protein (BMP) family influence mesoder- Received 27 April 2007; revised 13 June 2007; accepted 1 July 2007; mal lineage specification, therefore possibly facilitating published online 16 August 2007 vasculature formation.16,17 Generation of blood vessels from hESC H Bai et al 90 and proliferate in the presence of leukemia inhibitory factor (LIF), when MEF feeder cells are removed.24 The requirement for MEF feeder cells to support undifferentiated hESCs is supplanted by the addition of FGF-2 and other factors, but not LIF, to the culture medium.1,25 Under serum-free conditions, BMP signaling synergizes with LIF for mES cell self-renewal.26 In contrast to mES cells, suppression of BMP signaling by the antagonist noggin combined with FGF-2 sustains undifferentiation of hESCs.27 The differences of human and mouse ES cells clearly demonstrate that hESCs not only have great potential for clinical application, but also provide a novel opportunity to add significant understanding to the field of early human development, which is hampered by the lack of experimental systems and cannot be accomplished by Figure 1 Development of vascular cells from human embryonic mouse models. ES cells can be triggered to undergo stem cells. Hemangioblasts are bipotential precursors to hemato- spontaneous differentiation by forming 3-dimensional poietic cells and endothelial progenitor cells (EPCs). The origins of embryoid bodies (EBs) containing many differentiated smooth muscle cells (SMCs) are dependent on the location in the cell types. Although the EB is far less organized than an embryo. Vascular progenitor cells (VPCs) can further differentiate to embryo, it can partially mimic the spatial organization of mural cells (SMCs and pericytes) and endothelial cells. EPCs may 23 transdifferentiate into SMCs during vessel formation. EPCs from cells in an embryo. Similar to mES cells, hESCs cardiovascular progenitor cells may have hemogenic potential to differentiate into EBs in vitro, and the differentiation give rise to hematopoietic progenitor cells. process is somewhat directed by the culture environment.28,29 An enzymatic dissociation of hESC colonies into single cells leads to a significant decrease of ES cell propagation due to low efficiency of cell Because of ethical obstacles, the study of early attachment, which is not typically associated with mES vascular development in humans is hampered by the cells.30 Therefore, most of the EB induction experiments lack of experimental systems. Human umbilical vein from hESCs are performed as clumps from undiffer- endothelial cells are often used to study human entiated hESC colonies. Several cell types are character- endothelial cells. However, the signals and signaling ized during hESC differentiation, including cardiac pathways, which regulate embryonic endothelial cell fate tissue,31 neuronal tissue,32 b-islet pancreatic cells,33 and control endothelial cell migration and assembly into hematopoietic cells34 and endothelial cells.35,36 The hESC new vessels during embryonic development, remain differentiation process follows a reproducible temporal unknown in the human system. The study of embryonic pattern of development which, to some extent, resembles endothelial cells generated from hESCs may recapitulate early human embryogenesis. events seen in vascular network formation and increases the understanding of the molecular mechanisms of vascular development (Figure 1). Development of hemangioblasts from hESCs Human and mouse embryonic stem cells Zambidis et al.37 studied cellular kinetics of hESC Similar to mouse embryonic stem (mES) cells, hESCs are differentiation in serum-free clonogenic assays. When derived from the inner cell mass (ICM) of blastocysts or EB differentiation of hESCs were induced in 1% morula stage embryos.1,18,19 After removal from the ICM methylcellulose cultures containing fetal bovine serum and maintenance in culture, hESCs acquire an immortal (FBS), semiadherent mesodermal-hematoendothelial characteristic, that is, hESCs can grow indefinitely. They (MHE) cluster colonies emerged at days 6–9.37 The are capable of prolonged self-renewal and differentiation MHE colonies contained adherent and nonadherent into cells of all three germ layers in vivo and in vitro. cells. Nonadherent cells of MHE colonies expressed Mouse ES cells have been established and studied for CD45 and gave rise to hematopoietic colonies. Some more than 20 years.20–23 There is a substantial overlap adherent cells expressed endothelial markers, CD31, and between human and mouse ES cells in the expression of were capable of Dil-Ac-LDL uptake, whereas other key transcription factors, including Oct3/4, Nanog and adherent cells expressed the mesenchymal marker Sox2, which maintain pluripotency and self-renewal. vimentin. When the adherent cells from MHE colonies However, some fundamental differences were demon- were isolated and cultured in endothelial medium strated between human and mouse ES cells. For example, (EGM-2), they were expanded to the cells with endothe- the population-doubling time of hESCs (B36 h) is lial morphology, expressed the endothelial markers, significantly longer than that of mES cells (B12 h). CD31, VE-cad, vWF, and were capable of Dil-Ac-LDL Morphologically, hESCs form relatively flat and compact uptake.37 Because EB differentiation of this study was colonies. Undifferentiated hESCs express stage-specific initiated from hESC clumps, it is unclear whether MHE embryonic antigen (SSEA)-3 and SSEA-4, but lack SSEA-1; colonies are generated from single hESCs. The relation- whereas mES cells express SSEA-1 but lack SSEA-3 and ship of MHE colonies and bipotential hemangioblast SSEA-4.1 Significantly, mES cells remain undifferentiated remains unknown.

Gene Therapy Generation of blood vessels from hESC H Bai et al 91 The differentiation of embryonic endothelial cells VEGFR2+ cells from day 3 EBs were isolated and from restricted angioblasts and bipotential hemangio- cultured in 1% methylcellulose containing hematopoietic blasts is dependent on their location during embryo and endothelial growth factors, a small portion of development.13,14 The VEGF receptor VEGFR2 (or called VEGFR2+ cells (B2.4%) formed blast-forming colonies Flk1 or KDR) is the earliest marker expressed by (BL-CFCs). The BL-CFCs, which have both hematopoie- endothelial progenitor cells (EPCs) of avian and mouse tic and endothelial differentiation potentials, were embryos. VEGFR2+ cells in posterior mesoderm contain originally identified from mouse ES cells, and represent hemangioblasts, which are capable of differentiating into the equivalents of hemangioblasts in vitro.41 The kinetics endothelial and hemopoietic cells, whereas VEGFR2+ and frequency of BL-CFC formation from VEGFR2+ cells angioblasts from anterior mesoderm give rise to only were slightly different depending on hESC lines. The BL- endothelial cells.13 Embryonic endothelial cells can be CFCs have hematopoietic and endothelial differentiation derived from mouse, monkey and human ES cells.36,38,39 potential. After culturing BL-CFCs for 6 days, nonad- In mES cell differentiation, the endothelial markers, herent cells expressed hematopoietic genes and formed VEGFR2, CD31 (or called PECAM-1), Tie-2, Tie-1 and hematopoietic colonies, whereas adherent cells ex- VE-cadherin (VE-cad) are sequentially expressed in pressed the endothelial markers, VEGFR2, CD31, VE- spontaneously differentiated cystic EBs.40 ES-derived cad and P1HI2, and were capable of Dil-Ac-LDL endothelial cells acquire these markers in a time- uptake.46 The differentiation of VEGFR2+ cells generated dependent manner, which closely recapitulate endothelial two subpopulations, CD31+/VEGFR2+ cells and differentiation in vivo during embryonic development.40 CD31À/VEGFR2+ cells. All CD31+ cells also expressed A subpopulation of VEGFR2+ cells differentiated from CD34. Interestingly, CD31+/VEGFR2+ population, but mES cells contains hemangioblasts, which give rise to not CD31À/VEGFR2+ population, give rise to the hematopoietic cells and endothelial cells.41 Mouse hematopoietic colonies, suggesting that CD31 is not only embryos lacking VEGFR2 are embryonic lethal due to expressed in EPCs, but also in hematopoietic progenitor deficiency of hematopoietic and endothelial cells.42 cells.46 VEGFR2+ population from mouse ES cells contain Recently, Lu et al.48 used a two-step strategy to cardiovascular progenitor cell subpopulations that give generate functional hemangioblasts from human ESCs. rise to cardiomyocyte, endothelial cells and vascular They generated early-stage EBs, and then plated indivi- SMCs,43–45 therefore suggesting the existence of a multi- dual cells of dissociated EBs in serum-free semisolid potential VEGFR2+ mesoderm precursor. The properties blast-colony growth medium to generate blast cells (hES- of cardiovascular progenitor cells from hESCs have not BCs). The blast cells had characteristics of hemangio- been characterized. blasts giving rise to both multiple hematopoietic lineages Bipotential hemangioblasts were also identified dur- and to endothelial cells. Approximately 0.5% of hESC ing hESC differentiation. Wang et al.35 demonstrated that developed to hES-BCs, and 60% of hES-BCs gave both hematopoietic commitment occurred at around day 10 hematopoietic (nonadherent) and endothelial (adherent) during EB differentiation of hES cells, because CD45+ cells in the presence of hematopoietic and endothelial cells and hematopoietic colonies were not observed growth factors. When the blast cells were transplanted before day 10 EBs in differentiation medium containing into a rat model with diabetes or into a mouse model 20% FBS, BMP-4 and other cytokines. When CD31+/ with ischemia-reperfusion injury of the retina, they VEGFR2+/VE-cad+ cells were isolated from day 10 EBs, engrafted to the site of injury in the damaged vascu- they were capable of generating endothelial cells and lature, and appeared to participate in repair. Transplan- hematopoietic cells in the culture of endothelial or tation of the cells also reduced the mortality rate after hematopoietic medium, respectively. In the endothelial myocardial infarction and restored blood flow in hind medium, the cells generated from CD31+/VEGFR2+/ limb ischemia in mouse models.48 Interestingly, VEGFR2, VE-cad+ progenitor cells became phenotypically en- CD31 and CD34 were not expressed in hES-BCs, dothelial cells that expressed the endothelial markers, suggesting that hES-BCs have different properties from CD31, VE-cad, vWF and eNOS, and were capable of BL-CFCs in the study performed by Kennedy et al.46 Dil-Ac-LDL uptake. When single CD31+/VEGFR2+/ VE-cad+ cells were cultured in the presence of both hematopoietic and endothelial growth factors, approxi- Isolation of endothelial cells from hESCs mately 3.2% cells survived and formed endothelial or hematopoietic colonies, whereas a small portion of The hESC-derived endothelial cells were first identified survived cells (B0.18% of single cells) gave rise to and isolated in the Langer laboratory.36 Levenberg et al.36 colonies with both endothelial and hematopoietic cells, characterized the expression of endothelial-specific genes suggesting that CD31+/VEGFR2+/VE-cad+ cells contain during EB differentiation in the hESC culture medium a subset of cells with hemangioblast properties.35 It is without additional growth factors. Although VEGFR2, unclear whether CD31+/VEGFR2+/VE-cad+ cells before Tie2 and CD133 were expressed in undifferentiated day 10 EBs contain hemangioblast potential. hESCs, the genes of CD31, CD34 and VE-cad were not Kennedy et al.46 recently demonstrated that in serum- expressed in undifferentiated hESCs. The levels of CD31, free medium containing BMP-4, FGF-2 and VEGF, CD34 and VE-cad expression were increased during primitive erythroid progenitors were detected as early spontaneously EB formation, and reached a peak at day as 5 days EBs from differentiated hESCs. The kinetics of 13 to 15. The CD31+ cells (B2% of day 13 EB cells) were hematopoietic colony formation is different from pre- isolated from EBs by flow cytometry using anti-CD31 vious studies,35,47 suggesting that different hESC differ- antibodies, and expanded in endothelial medium. The entiation conditions and growth factors result in hESC-derived endothelial cells expressed endothelial different kinetics of hematopoiesis in vitro. When the markers, CD31, VE-cad, vWF and N-cadherin, and were

Gene Therapy Generation of blood vessels from hESC H Bai et al 92 capable of Dil-Ac-LDL uptake. The function of the endothelial morphology and expressed endothelial cell embryonic endothelial cells derived from hESCs was markers CD31, VE-cad and vWF. The hESC-derived assessed in severe combined immunodeficient (SCID) endothelial cells were capable of Dil-Ac-LDL uptake, and mice by subcutaneous implantation of the cells mixed in formed vascular network in Matrigel.53,54 When CD34+ a synthetic biopolymer scaffold. The sections of implants cells from hESCs were cultured in the presence of examined after 14 days showed microvessels expressing hematopoietic growth factors, they expressed CD45 and human CD31 and CD34.36 This study demonstrates that formed multilineage hematopoietic colonies on methyl- during EB induction, hESCs spontaneously differen- cellulose.54 These results suggest that CD34+/CD31+ tiated in embryonic endothelial cells, which were population contain hematopoietic and EPCs, although it isolated and maintained in vitro. However, the blood is not clear that this occurs at a clonal level. carrying function of the blood vessels generated by Whether there is a true hemangioblast population in embryonic endothelial cells is not well characterized CD34+/CD31+ cell fraction remains unknown. Highly in vivo. The long-term durability and quality of embryonic efficient generation of CD34+ cells from hESCs can be endothelial cell-derived microvessels and their integra- achieved by simply changing the differentiation medium tion into host vasculature is not well defined. The of the MEF feeder cells (Figure 2).54 In the presence of phenotype of EPCs that give rise to hESC-derived VEGF, FGF-2 and BMP-4, CD34+ progenitor cells can be endothelial cells needs further investigation. derived from hESCs in a serum-free medium with To investigate endothelial cell development and similar efficiency as the serum-containing medium.54 understand the mechanisms of embryonic vessel forma- The blood vessel forming function of hESC-derived tion in human early development, we intended to isolate endothelial cells was demonstrated by implantation of EPCs from hESCs. Studies from adult circulating cells green fluorescent protein (GFP)-expressing hESC-de- indicated that VEGFR2, CD133 and CD34 are expressed rived endothelial cells into SCID mice with the cranial on endothelial and hematopoietic progenitor cells in windows. Through the cranial window, the behavior of several human tissues, including peripheral blood and GFP+ cells can be constantly monitored. After 11 days of bone marrow.49–51 We chose CD34 as a possible marker to implantation, the hESC-derived endothelial cells aggre- isolate hESC-derived EPCs, because VEGFR2 and CD133 gated to form luminal structures. When rhodamine- are expressed in undifferentiated hESCs, whereas labeled dextran was injected into the tail veins, the CD34 is not expressed (or minimally expressed) in perfused vessels transported streaming red blood cells, undifferentiated hESCs.36,46,52 In our study, CD34+ cell suggesting the formation of functional engineered populations increased to approximately 10% at around vessels that are successfully integrated into the recipi- day 12–15 upon EB differentiation.36,53 The CD34+ cells ent’s vascular tree.54 at day 10 EBs were negative for hematopoietic marker The generation of endothelial cells from hESCs not CD45, whereas the majority of CD34+ cells were also only provides a cell resource for potential clinical CD31+ cells.53,54 Similar results were obtained by other applications, it also provides an excellent alternative to groups.35,36 After culture in endothelial cell medium, the study vasculogenesis and angiogenesis events in the isolated CD34+ progenitor cells gave rise to cells with human system. Using hESC-derived endothelial cells,

Figure 2 Generation of hESC-derived endothelial cells in 2-dimensional (2D) culture. The GFP+ hESCs were differentiated in monolayer (2D) or EB (3D) cultures. The CD34+ cells were isolated by MACS columns, and cultured in the presence of angiogenic or hematopoietic growth factors for endothelial and hematopoietic differentiation. The hESC-derived endothelial cells (green) in a collagen gel were implanted into cranial windows in SCID mice. After 11 days, rhodamine-dextran was injected into the tail vein to highlight perfused vessels (red). SCID, severe combined immunodeﬁcient.

Gene Therapy Generation of blood vessels from hESC H Bai et al 93 the molecular mechanism of blood vessel formation injury. However, there are scientific obstacles that need to during human embryonic development can be studied be overcome before clinical applications, such as animal in vitro. We demonstrated a role of the SDF-1/CXCR4 product contamination, tumorigenicity, immunocompat- signaling in human embryonic endothelial cell migra- ibility, isolation of desired cell types and availability of tion, branching and remodeling.53 suitable animal models to test cell function. For example, ES cells are pluripotent cells with self-renewal capability. When hESCs are injected subcutaneously, intramuscu- HESC-derived smooth muscle cells larly or into the testis, they form teratocarcinoma-like tumors in adult mice.1,18,59 These tumors contain differ- The majority of the studies of angiogenesis research entiated cells derived from all three germ layers. There- focus on the regulation of endothelium. Endothelial cells fore, any ES cell-based therapy has the risk of tumor alone cannot complete angiogenesis to form mature formation from undifferentiated ES cells. Studies of vasculature. Vascular SMCs play critical roles in struc- mouse ES cells indicated that differentiated ES cells tural and functional support of the vascular network by are less likely to generate teratomas after transplanta- stabilizing nascent endothelial vessels during vascular tion.60–62 It is an important step to minimize the development and blood vessel growth.55 Endothelial probability of teratomas by using highly differentiated cells and SMCs interact with each other to regulate not ES cells. Highly differentiated cells often have low only new vessel growth, but also multiple vascular proliferation potential. Isolation of progenitors from functions. In adult animals, SMCs regulate growth of differentiated hESCs may provide cells with potential blood vessels, vessel tone diameter, vascular permeabil- of proliferation and differentiation. It is a great challenge ity and blood flow distribution.15 A study of mouse ES to identify, characterize and isolate progenitor popula- cells by Yamashita et al.44 suggested that vascular tions that will not form tumors, and is capable of progenitor cells (VPCs) can differentiated into either proliferation and continually producing newly differen- endothelial cells or SMCs dependent on VEGF or PDGF- tiating cells in vivo. When hESC-endothelial cells from BB, respectively. SMCs originate from multiple cell CD34+ progenitor cells are implanted in SCID mice populations, including endothelial cells, neural crest under the cranial windows, stable blood conduits cells, mesenchymal cells, bone marrow precursors or form for more than 150 days without teratomas,54 macrophages. For example, coronary vein SMCs are suggesting that hESC-derived endothelial cells are safe derived from the atrial myocardium, whereas coronary for potential clinical applications. Whether or not there is artery SMCs originate in the epicardial layer that also the long-term tumorigenicity needs to be further in- gives rise to perivascular fibroblasts and intermyocardial vestigated. fibroblasts.56 Our study found that co-implantation of Unlike mouse ES cells, in which a single cell is able to hESC-derived endothelial cells and 10T1/2 cells (mouse form an EB in methylcellulose cultures,63 the formation embryonic fibroblasts) facilitated blood vessel formation of EBs from hESCs is inefficient and usually requires an in vivo.54 However, it is unclear whether 10T1/2 cells entire colony of hESCs. Dispersed single hESCs aggre- directly interact with hESC-derived endothelial cells or gate inefficiently to form EBs, and a large number secret soluble factors that enhance the hESC-derived of dispersed cells undergo cell death during EB endothelial cell vascular network. Recently, SMCs were formation.18 Due to spontaneous differentiation in EBs, characterized during hESC differentiation.57 Retinoic differentiated hESCs contain heterogeneous multiple cell acid has been used to induce mouse ES cells into SMCs.58 types simultaneously. Therefore, one of the major When the hESCs were cultured in differentiation challenges in stem cell research is to obtain sufficient medium containing all-trans retinoid acid (atRA), more and desired cell types for clinical applications. Selecting than 93% of the cells expressed SMC-markers, SM-MHC progenitor cells rather than mature cell type from and SM a-actin, and were capable of contraction.57 differentiated hESCs may be advantageous for in vitro More studies need to be carried out to address following expansion and in vivo function. Studies of endothelial questions: (i) is there a common vascular progenitor for cells from hESCs demonstrate that isolation of CD34+ human endothelial cells and SMCs? (ii) What is the EPCs is a suitable approach to generate endothelial cells. phenotype of such progenitor cells? (iii) Will SMCs The hESCs can be genetically manipulated. We and and endothelial cells from hESCs interact to facilitate others have used lentiviral vectors to expressed new blood vessel formation in vivo? (iv) Do hESC- transgenes.54,64,65 After implanting GFP-marked endothe- derived SMCs provide hemostatic control and protect lial cells, we are able to observe readily hESC-derived new endothelium-lined vessels against rupture or re- cells for at least 150 days.54 Such procedures of genetic gression? (v) Do hESC-derived SMCs stabilize nascent manipulation in hESCs could be used to correct genetic vessels by inhibiting endothelial proliferation and mutation in clinical gene therapy, to direct differentiation migration? (vi) Will embryonic endothelial cells from to a specific cell type, to reduce graft rejections by the hESCs transdifferentiate into SMCs under a proper modification of immune responses, or to trace hESC- condition? derived cells in vivo after transplantation.

Potentials and challenges in hESC Acknowledgements therapy I acknowledge our colleagues in the ﬁeld for their many The successful derivation of hES cells from blastocysts in outstanding studies. This work was supported by 1998 by Thomson et al.1 provides potential cell sources National Institute of Health Grants K01DK064696 and for cell-based therapies for many human diseases and P20RR018789.

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