Endodermal progenitor cells
View Patent ↗The present invention relates to endodermal progenitor cells and methods of isolation, culture, differentiation and use thereof.
1. A method of obtaining endodermal progenitor cells comprising:
(a) culturing a cell population obtained from an adult endodermal tissue to induce cluster formation in culture medium;
(b) seeding the clusters in a vessel coated with one or more attachment factors, wherein cells comprising the clusters adhere to the one or more attachment factors and form a monolayer;
(c) culturing the monolayer clusters in culture medium until a population of endodermal progenitor cells emerges; and
(d) isolating the endodermal progenitor cells, wherein the isolated endodermal progenitor cells do not express smooth muscle actin or PDX-1,
wherein a function of a cell in the tissue is not reduced by application of an exogenous stressor prior to harvesting the tissue, wherein the exogenous stressor is adverse chemicals, nutritional stressors, cell depletion or physical stress.
2. The method of claim 1 , wherein the tissue is derived from liver, stomach, intestine, pancreas, lung, colon, bladder or thyroid.
3. The method of claim 1 , wherein the tissue is pancreas.
4. The method of claim 1 , wherein the clusters areisolated prior to culturing in step (b).
5. The method of claim 1 , wherein the one or more attachment factors comprise one or more of collagen type I, collagen type II, collagen type IV, fibronectin, chondroitin sulfate, vitronectin, thrombospondin or matrigel.
6. The method of claim 1 , wherein the culture medium of step (b) is changed every two to three days for at least about 7 days.
7. The method of claim 6 , wherein after about 7 days the culture medium of step (b) is not changed prior to isolating the endodermal progenitor cells.
8. The method of claim 1 , wherein the culture medium of step (a) and/or (b) comprises serum.
9. The method of claim 8 , wherein the serum content is about 0.5% to about 5%.
10. The method of claim 1 , wherein the culture media of step (a) and/or (b) comprises one or more of epidermal growth factor (EGF), leukemia inhibitory factor (LIP), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF).
11. The method of claim 1 , wherein the cell population is subjected to continuous density gradient centrifugation prior to step (a).
12. The method of claim 1 , wherein the tissue is disassociated prior to step (a).
13. The method of claim 12 , wherein the tissue is disassociated enzymatically.
14. The method of claim 13 , wherein the enzyme comprises one or more of collagenase, trypsin, dispase I, hyaluronidase, therinolysin, neutral protease, liberase RI, DNase I, pancreatin, or pronase.
15. The method of claim 1 , where n the tissue is obtained from a mammal.
16. The method of claim 15 , wherein the mammal is a human, swine, mouse or rat.
17. The method of claim 1 further comprising differentiating the endodermal progenitor cells to yield an endodermal cell type.
18. The method of claim 17 , wherein the differentiated endodermal cull type is selected from the mono consisting of pancreatic, islet, intestinal, thyroid, lung, colon, bladder and liver cell types.
19. The method of claim 17 , wherein the endodermal progenitor cells are differentiated in the presence of one or more differentiation factors comprising β-cellulin, glucagon-like peptide-1 (GLP-1), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), nicotinamide, transforming growth factor-α(TGF-α), transforming growth factor-β(TGF-β), activin, cyclopamin, bone morphogenetic protein 4 (BMP4), sonic hedgehog (SHH) antibody, Oncostatin M, dexamethasone, exendin4, growth differentiation factor 11 (GDF11) or a combination thereof.