Vascular progenitors and their methods of use

Abstract

There is provided herein a method of generating vascular progenitors from a starting population of human pluripotent stem cells (hPSCs) which had been differentiated to a mesothelial, preferably epicardial, fate, the method comprising: inducing epithelial to mesenchymal transition (EMT) in the population and expanding the population, each in a culture medium that is serum free with specific factors.

Description

[0001] VASCULAR PROGENITORS AND THEIR METHODS OF USE

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 668843, filed on July 9, 2024, the entire contents of which are hereby incorporated by reference.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to vascular progenitors, and more particularly to the generation thereof from human pluripotent stem cells and methods of using the same.

[0006] BACKGROUND OF THE INVENTION

[0007] The ability to vascularize tissue through the transplantation and engraftment of hPSC- derived vascular progenitors (VPs) offers unprecedented opportunities to develop novel cell-based therapies to treat a range of diseases resulting from vascular dysfunction and ischemia. This disclosure describes a method for generating engraftable VPs from hPSC-derived epicardial cells. The epicardial cells that form the epicardium that surrounds the developing heart originate from the septum transversum mesenchyme. Prior to wrapping the heart, these cells form a transient structure known as the pro-epicardial organ (PEO). Lineage tracing studies have shown that the PEO give rise to both endothelial cells and pericytes found in the adult heart.

[0008] SUMMARY OF THE INVENTION

[0009] In an aspect, there is provided a method of generating vascular progenitors from a starting population of human pluripotent stem cells (hPSCs) which had been differentiated to a mesothelial, preferably epicardial, fate, the method comprising: inducing epithelial to mesenchymal transition (EMT) in the population in a culture medium that is serum free and containing; a first factor for the induction of EMT through activation of the SMAD pathway; a second factor for the induction of EMT through activation of the SMAD2 / 3 pathway; a third factor for the induction of EMT through the promotion of cytoskeletal rearrangement; a fourth factor for the induction of EMT through the PI3K pathway; and expanding the population from the previous step in a culture medium that is serum free and containing; the third factor; the fourth factor; and a fifth factor for the inhibition of ALK5.

[0010] In an aspect, there is provided a population of vascular progenitors produced by the method described herein.

[0011] In an aspect, there is provided a population of hPSC-derived vascular progenitors.

[0012] In an aspect, there is provided the population of vascular progenitors described herein, for use in the revascularization of a patient in need thereof.

[0013] In an aspect, there is provided the population of vascular progenitors described herein, for use in the treatment of ischemia in a patient in need thereof.

[0014] In an aspect, there is provided the population of vascular progenitors described herein, for use in co-transplantation with cells or an organ in a subject in need of such transplantation.

[0015] In an aspect, there is provided the population of vascular progenitors described herein, for use in cell therapy. In an aspect, there is provided the population of vascular progenitors described herein, for use in the vascularization of engineered tissue in culture.

[0016] In an aspect, there is provided a method of revascularization in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0017] In an aspect, there is provided a method of treating ischemia in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0018] In an aspect, there is provided a method of co-transplanting cells or an organ in a subject in need of such transplantation, the method comprising co-administering the population of vascular progenitors described herein to the patient along with the cells or organ.

[0019] In an aspect, there is provided a method of cell therapy in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0020] In an aspect, there is provided a method of vascularizing engineered tissue in culture, the method comprising co-incubating the population of vascular progenitors described herein with the engineered tissue.

[0021] BRIEF DESCRIPTION OF FIGURES

[0022] These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

[0023] Figure 1 : Representative Day 4 ‘SHF’ Mesoderm profile. (A) Representative flow cytometry analyses of Aldefluor and CD235a / b in day 4 ‘SHF’ mesoderm.

[0024] Figure 2: Day 17, 23 QC using flow cytometry. (A) Representative flow cytometry analyses of Aldeflour (ALDH) in day 17 hPSC derived epicardial cells. (B) Left; Representative flow cytometry analysis of CD140b and CD13 in directly differentiated VPs derived from epicardial cells. Middle; Representative flow cytometry analysis of KDR in CD140b+ / CD13+ cells. Right; Quantification of percentage of CD140b+ / CD13+ / KDR+ cells within the whole population. (C) Left; Representative flow cytometry analysis of MYH11 within the VP population. Right; Quantification of MYH11+ cells within the VP population (N=5 biologically independent samples).

[0025] Figure 3. rt-qPCR panel of Day 23 cells for quality control of cell populations. (A)RT-qPCR expression analyses of smooth muscle (MYH11, SMTN, RGS5, CNN ), and extracellular matrix genes (COL1A1, COL3A1) genes in the starting epicardial population and stage matched VP or CF populations (N=4 biologically independent samples).

[0026] DETAILED DESCRIPTION

[0027] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.

[0028] There is described herein a protocol for the generation of a novel population of vascular progenitors from human pluripotent stem cells (hPSCs). These progenitors are distinct from all others described to date in that they are able to generate functional human capillaries following transplantation into different tissue sites in immunodeficient NSG recipients. With this potential, these cells offer a unique opportunity to develop novel cell-based therapies to re-vascularize ischemic tissues. Additionally, when transplanted together with different parenchymal cell types (pancreatic, liver, cardiac), these vascular progenitors may enhance their engraftment potential. Currently there are no other cell products in the commercial space that display this potential. The potential clinical market for this product is large and includes various ischemic pathologies as well as patients that will be treated with future cell-based therapies. One of the first markets targeted would be peripheral vascular disease, characterized by narrowed arteries that deliver blood to the limbs. This market extends globally and includes patients with diabetes as well as the aging populations worldwide.

[0029] There is also described herein a population of “vascular progenitor” cells generated from a well-defined hPSC-derived epicardial cell population. The population of vascular progenitor cells is defined by the co-expression of the cell surface protein antigens CD140B, CD13, and CD309. Upon injection these cells form perfusable human vessels.

[0030] Abbreviations

[0031] SOP: Standard Operating Procedure hPSC: Human Pluripotent Stem Cell

[0032] EB: embryoid body

[0033] PE: pro-epicardial cell

[0034] EPI: epicardial cell

[0035] VP: Vascular progenitor cell

[0036] B: BMP4

[0037] A: Activin A bFGF: basic fibroblast growth factor

[0038] FHF: first heart field

[0039] SHF: second heart field

[0040] In an aspect, there is provided a method of generating vascular progenitors from a starting population of human pluripotent stem cells (hPSCs) which had been differentiated to a mesothelial, preferably epicardial, fate, the method comprising: inducing epithelial to mesenchymal transition (EMT) in the population in a culture medium that is serum free and containing; a first factor for the induction of EMT through activation of the SMAD pathway; a second factor for the induction of EMT through activation of the SMAD2 / 3 pathway; a third factor for the induction of EMT through the promotion of cytoskeletal rearrangement; a fourth factor for the induction of EMT through the PI3K pathway; and expanding the population from the previous step in a culture medium that is serum free and containing; the third factor; the fourth factor; and a fifth factor for the inhibition of ALK5.

[0041] Methods to differentiate human pluripotent stem cells (hPSCs) to a mesothelial, preferably epicardial, fate are known in art. For example, these methods are described in WO / 2015 / 035506 and in Fernandes et al. (Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells, Nature Communications (2023) 14:8183).

[0042] In some embodiments, the first factor is TGFBi, TGFB2 / 3, Activin A, or Isoxazole 9. Preferably, the first factor is TGFBi.

[0043] In some embodiments, the second factor is EGF, NRG, TGFA, HBEGF, EREG, AREG, or NSC228155. Preferably, the second factor is EGF.

[0044] In some embodiments, the third factor is bFGF, FGF1 , FGF7, FGF8, FGF10, Colivelin, SUN11602, SC79, Neferine, or YS-49. Preferably, the third factor is bFGF.

[0045] In some embodiments, the fourth factor is PDGF-BB, PDGF-AA, PDGF-CC, PDGF- DD, 740 Y-P, or Recilisib. Preferably, the fourth factor is PDGF-BB.

[0046] In some embodiments, the fifth factor is SB431542, LY3200882, R-268712, TP0427736, RepSox, SB525334, GW788388, BIBF-0775, A-83-01 , Galunisertib, Vactosertib, or LY2109761. Preferably, the fifth factor is SB431542.

[0047] In some embodiments, the first factor is present in a concentration of 0.5-10 ng / ml. Preferably, the first factor is present in a concentration of 1 ng / ml. In some embodiments, the second factor is present in a concentration of 1-20 ng / mL. Preferably, the second factor is present in a concentration of 5 ng / mL.

[0048] In some embodiments, the third factor is present in a concentration of 1-100 ng / mL. Preferably, the third factor is present in a concentration of 10 ng / mL.

[0049] In some embodiments, the fourth factor is present in a concentration of 1-100 ng / mL. Preferably, the fourth factor is present in a concentration of 10 ng / mL.

[0050] In some embodiments, the fifth factor is present in a concentration of 0.1-10 uM. Preferably, the fifth factor is present in a concentration of 6 uM.

[0051] In some embodiments, the starting population is at least one of WT1+, HAND2+, BNC1+, GATA4 / 5+, TCF21+ and has little to no OSR1 mRNA.

[0052] In some embodiments, the starting population is all of WT1+, HAND2+, BNC1+, GATA4 / 5+, TCF21+ and has little to no OSR1 mRNA.

[0053] In an aspect, there is provided a population of vascular progenitors produced by the method described herein.

[0054] In an aspect, there is provided a population of hPSC-derived vascular progenitors.

[0055] In some embodiments, the vascular progenitors are CD140b+, CD13+, and CD309+.

[0056] In some embodiments, the vascular progenitors also contain little to no OSR1 mRNA.

[0057] In some embodiments, the vascular progenitors are additionally CD73+, CD105+, and / or CD44+.

[0058] In some embodiments, the population is engraftable.

[0059] In an aspect, there is provided the population of vascular progenitors described herein, for use in the revascularization of a patient in need thereof.

[0060] In an aspect, there is provided the population of vascular progenitors described herein, for use in the treatment of ischemia in a patient in need thereof. In an aspect, there is provided the population of vascular progenitors described herein, for use in co-transplantation with cells or an organ in a subject in need of such transplantation.

[0061] In some embodiments, the cells or organ is heart, liver or kidney. In an embodiments, the cells or organ, are islet cells.

[0062] In an aspect, there is provided the population of vascular progenitors described herein, for use in cell therapy.

[0063] In an aspect, there is provided the population of vascular progenitors described herein, for use in the vascularization of engineered tissue in culture.

[0064] In an aspect, there is provided a method of revascularization in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0065] In an aspect, there is provided a method of treating ischemia in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0066] In an aspect, there is provided a method of co-transplanting cells or an organ in a subject in need of such transplantation, the method comprising co-administering the population of vascular progenitors described herein to the patient along with the cells or organ.

[0067] In an aspect, there is provided a method of cell therapy in a patient in need thereof, the method comprising administering the population of vascular progenitors described herein to the patient.

[0068] In an aspect, there is provided a method of vascularizing engineered tissue in culture, the method comprising co-incubating the population of vascular progenitors described herein with the engineered tissue.

[0069] As used herein, “pharmaceutically acceptable carrier1' means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.

[0070] As used herein, “therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

[0071] The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

[0072] EXAMPLES

[0073] A standard operating procedure (SOP) describes in one embodiment a method to generate epicardial and derivative vascular progenitor cells from human pluripotent stem cells (hPSCs).

[0074] EB formation, day 0-1 hPSCs are grown on irradiated mouse embryonic fibroblast feeders in 6-well plates (Corning) in HES media supplemented with bFGF (line to line titration required). The hPSCs are harvested at approximately 80% confluency and dissociated into single cells by treatment with TrypLE (ThermoFisher). For hPSC dissociation, 1 mL of TrypLE is added to each well; the plates are returned to the 37°C hypoxic incubator for 2-3 minutes until the feeders are detached (small, rounded cells). The hPSC clusters are dissociated by pipetting and the resulting single-cell suspension is washed twice in Wash media (IM DM + 0.5% BSA). The dissociated hPSC population is filtered with 40 pm sterile cell strainers (Corning) and the cells harvested by spinning at 1000 rpm for 5 minutes. The collected single cells are then aggregated to form embryoid bodies (EBs) by culture in StemPro-34 media [Basal medium; (ThermoFisher) supplemented with 1% penicillin / streptomycin (ThermoFisher), 2mM L-glutamine (ThermoFisher), 150 pg / mL transferrin (ROCHE), 50 pg / mL ascorbic acid (Sigma) and 50 pg / mL monothioglycerol (Sigma) [Basal medium]], 5 pM ROCK inhibitor Y-27632 (TOCRIS) and 1 ng / ml rhBMP4 (R&D). To enhance EB formation, the cells are cultured on an orbital shaker (70 rpm) in 6 cm petri-grade dishes at a concentration of 2x106cells in 4 mL of media. The cultures are maintained in a low oxygen environment (5% CO2, 5% 02) for 18 hours.

[0075] Mesoderm specification, days 1-4

[0076] On day 1 , the EBs are filtered through 100 pm sterile cell strainers (Corning) and harvested by spinning at 600 rpm for 5 minutes. The harvested EBs are transferred to mesoderm induction medium consisting of base medium (StemPro-34), 5ng / ml_ rhbFGF (R&D) and various concentrations of rhBMP4 and rhActivinA. For HES2 hPSCs, 3 ng / mL rhBMP4 and 1 ng / mL rhActivinA (3B1A) are used to generate the second heart field (SHF) mesoderm that gives rise to and enriched pro-epicardial population. NOTE: careful titration of rhBMP4 and rhActivinA is required to generate the optimal SHF mesoderm population from different hPSC lines. Optimal mesoderm induction can be determined by flow cytometric analyses (Figure 1A). For this mesoderm induction step, the EBs are cultured (static) on polyheme coated 6-well plates in a low oxygen environment in the same volume of media as they are during days O to 1 (approximately 0.5 million cells / mL).

[0077] Pro-epicardial specification, day 4-8

[0078] On day 4 the EBs are harvested, collected in a 50ml_ conical tube(s) and dissociated enzymatically by treatment with TrypLE for 5 minutes at 37°C. This suspension is subsequently washed with wash medium (IMDM + 0.5% BSA) containing DNAse (or a base medium equivalent) and the cells counted. At this point, the mesoderm is analysed for expression of CD140a, CD235a as well as for ALDH activity. ALDH activity is measured using the commercial ALDEFLUOR assay. With this set of markers, the co-expression of ALDH and CD140a defines the second heart field mesoderm whereas expression of CD 140a, CD235a and lack of ALDH expression identifies the first heart field mesoderm (Figure 1). The second heart field mesoderm, the optimal mesoderm population for generating these cells is one that contains at least 25% CD140a+ALDH+cells with no more than 15% CD140ALDH’ (Figure 1). The day 4 cells are then plated as a monolayer at a concentration of 6x105- 8x105cells per mL on tissue culture plates (pre-coated with gelatin) in the appropriate volume of epicardial induction medium consisting of StemPro-34 + [2mM Glutamine + 50ug / ml_ Ascorbic Acid + 150ug / ml_ Transferrin + 400uM MTG] [Basel components] supplemented with 10ng rhBMP4, 1 uM CHIR, 2uM ROH and 6uM SB (ALK5 inhibitor). On day 6 the medium is changed to basal medium consisting of StemPro-34 + [2mM Glutamine + 50ug / ml_ Ascorbic Acid + 150ug / ml_ Transferrin + 400uM MTG] [Basel components] supplemented with 6uM SB (ALK5 inhibitor). The medium is changed every 48 hours by aspirating the entire well and replacing with the volume equivalent.

[0079] Epicardial development (days 8-12)

[0080] On day 8 the cells are harvested from the monolayer by treatment with Collagenase B for 1 hour at 37°C (It is normal for the monolayer to detach from the cell culture surface during this step). The cells are then washed and spun at 400 rpm for 5 minutes. The supernatant is then removed, and cells are further dissociated enzymatically using TrypLE at 37°C for 5 mins with the reaction being stopped by a 10-fold dilution. The cells are then replated at a concentration of 3 x 105cells per mL on a gelatin coated tissue culture dish. Cells are maintained until day 12 of culture in StemPro-34 + [2mM Glutamine + 50ug / mL Ascorbic Acid + 150ug / mL Transferrin + 400uM MTG] [Basel components] supplemented with 6uM SB (ALK5 inhibitor).

[0081] Epicardial expansion (days 12-17)

[0082] At day 12, the cells are harvested from the monolayer by treatment with Collagenase B for 1 hour at 37°C (It is normal for the monolayer to detach from the cell culture surface during this step). The cells are washed and spun at 400 rpm for 5 minutes. The supernatant is removed, and the cells are further dissociated enzymatically using TrypLE at 37°C for 5 mins with the reaction being stopped by a 10-fold dilution using ‘Wash’ Medium. Following dissociation, the cells are plated at a concentration of 1.5 x 105cells per mL on a gelatin coated tissue culture dish in StemPro-34 + [2mM Glutamine + 50ug / mL Ascorbic Acid + 150ug / mL Transferrin + 400uM MTG] [Basel components] supplemented with 6uM SB (ALK5 inhibitor). Induction of Vascular progenitor cells (Step 1, days 17-20)

[0083] On day 17, the monolayer is dissociated by treatment with Collagenase B for 1 hour at 37°C. The detached monolayer of cells is washed, spun at 400 rpm for 5 minutes and further dissociated enzymatically using TrypLE at 37°C for 5 mins. The reaction is stopped by a 10-fold dilution with ‘Wash’ Medium. The single cell suspension is then replated onto gelatin-coated culture dishes in StemPro-34 + [2mM Glutamine + 50ug / ml_ Ascorbic Acid + 150ug / ml_ Transferrin + 400uM MTG] [Basel components] supplemented with 1 ng / ml_ TGFB, 5ng / ml_ EGF, 10ng / ml_ bFGF and 10ng / ml_ PDGFBB at a concentration 3 x 105cells per mL and cultured for 72 hours.

[0084] Induction of Vascular progenitor cells (Step 2, days 20-23)

[0085] Following the 72-hour culture step, the medium is fully aspirated from the newly formed adherent monolayer and replaced with, StemPro-34 + [2mM Glutamine + 50ug / ml_ Ascorbic Acid + 150ug / ml_ Transferrin + 400uM MTG] [Basel components] supplemented with 10ng / ml_ bFGF + 10ng / ml_ PDGFBB and 6uM SB (ALK5 inhibitor). At day 23, the cells can be enzymatically dissociated by treatment with TrypLE at 37°C for 5 mins. The resultant single cell suspension can then be analyzed by flow cytometry for the cell surface markers CD140b, CD13 and CD309 as shown in Figure 2B. This is the VP population used for transplantation. At this stage cells are frozen for downstream applications.

[0086] Quality Control

[0087] Figure 2 shows the day 17, 23 QC using flow cytometry. Figure 3 shows rt-qPCR panels of Day 23 cells for quality control of cell populations.

[0088] Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

Claims

CLAIMS:

1. A method of generating vascular progenitors from a starting population of human pluripotent stem cells (hPSCs) which had been differentiated to a mesothelial, preferably epicardial, fate, the method comprising:(a) inducing epithelial to mesenchymal transition (EMT) in the population in a culture medium that is serum free and containing; a first factor for the induction of EMT through activation of the SMAD pathway; a second factor for the induction of EMT through activation of the SMAD2 / 3 pathway; a third factor for the induction of EMT through the promotion of cytoskeletal rearrangement; a fourth factor for the induction of EMT through the PI3K pathway; and(b) expanding the population from step (a) in a culture medium that is serum free and containing; the third factor; the fourth factor; and a fifth factor for the inhibition of ALK5.

2. The method of claim 1 , wherein the first factor is TGFBi, TGFB2 / 3, Activin A, or Isoxazole 9.

3. The method of claim 2, wherein the first factor is TGFBi.

4. The method of any one of claims 1-3, wherein the second factor is EGF, NRG, TGFA, HBEGF, EREG, AREG, or NSC228155.

5. The method of claim 4, wherein the second factor is EGF.

6. The method of any one of claims 1-5, wherein the third factor is bFGF, FGF1 , FGF7, FGF8, FGF10, Colivelin, SUN11602, SC79, Neferine, or YS-49.

7. The method of claim 6, wherein the third factor is bFGF.

8. The method of any one of claims 1-7, wherein the fourth factor is PDGF-BB,PDGF-AA, PDGF-CC, PDGF-DD, 740 Y-P, or Recilisib.

9. The method of claim 8, wherein the fourth factor is PDGF-BB.

10. The method of any one of claims 1-9, wherein the fifth factor is SB431542,LY3200882, R-268712, TP0427736, RepSox, SB525334, GW788388, BIBF- 0775, A-83-01 , Galunisertib, Vactosertib, or LY2109761.

11. The method of claim 10, wherein the fifth factor is SB431542.

12. The method of any one of claims 1-11 , wherein the first factor is present in a concentration of 0.5-10 ng / ml.

13. The method of claim 12, wherein the first factor is present in a concentration of 1 ng / ml.

14. The method of any one of claims 1-13, wherein the second factor is present in a concentration of 1-20 ng / mL.

15. The method of claim 14, wherein the second factor is present in a concentration of 5 ng / mL.

16. The method of any one of claims 1-15, wherein the third factor is present in a concentration of 1-100 ng / mL.

17. The method of claim 16, wherein the third factor is present in a concentration of 10 ng / mL.

18. The method of any one of claims 1-17, wherein the fourth factor is present in a concentration of 1-100 ng / mL.

19. The method of claim 18, wherein the fourth factor is present in a concentration of 10 ng / mL.

20. The method of any one of claims 1-19, wherein the fifth factor is present in a concentration of 0.1-10 uM.21 . The method of claim 20, wherein the fifth factor is present in a concentration of 6 uM.

22. The method of any one of claim 1-20, wherein the starting population is at least one of WT1+, HAND2+, BNC1+, GATA4 / 5+, TCF21+ and has little to no OSR1 mRNA.

23. The method of any one of claim 1-20, wherein the starting population is all of WT1 + , HAND2+, BNC1+, GATA4 / 5+, TCF21+ and has little to no OSR1 mRNA.

24. A population of vascular progenitors produced by the method of any one of claims 1-23.

25. A population of hPSC-derived vascular progenitors.

26. The population of claim 21 or 22, wherein the vascular progenitors are CD140b+, CD13+, and CD309+.

27. The population of claim 26, wherein the vascular progenitors are additionally CD73+, CD105+, and / or CD44+, and preferably contain little to no OSR1 mRNA.

28. The population of any one of claims 24-27, wherein the population is engraftable.

29. The population of vascular progenitors of any one of claims 24-28, for use in the revascularization of a patient in need thereof.

30. The population of vascular progenitors of any one of claims 24-28, for use in the treatment of ischemia in a patient in need thereof.

31. The population of vascular progenitors of any one of claims 24-28, for use in co-transplantation with cells or an organ in a subject in need of such transplantation.

32. The population of vascular progenitors for use of claim 31 , wherein the cells or organ, are islet cells.

33. The population of vascular progenitors for use of claim 31 , wherein the cells or organ is heart, liver or kidney.

34. The population of vascular progenitors of any one of claims 24-28, for use in cell therapy.

35. The population of vascular progenitors of any one of claims 24-28, for use in the vascularization of engineered tissue in culture.

36. A method of revascularization in a patient in need thereof, the method comprising administering the population of vascular progenitors of any one of claims 24-28 to the patient.

37. A method of treating ischemia in a patient in need thereof, the method comprising administering the population of vascular progenitors of any one of claims 24-28 to the patient.

38. A method of co-transplanting cells or an organ in a subject in need of such transplantation, the method comprising co-administering the population of vascular progenitors of any one of claims 24-28 to the patient along with the cells or organ.

39. The method of claim 38, wherein the cells or organ, are islet cells.

40. The method of claim 38, wherein the cells or organ is liver or kidney.

41. A method of cell therapy in a patient in need thereof, the method comprising administering the population of vascular progenitors of any one of claims 24-28 to the patient.

2. A method of vascularizing engineered tissue in culture, the method comprising co-incubating the population of vascular progenitors of any one of claims 24-28 with the engineered tissue.

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