Cardiac progenitor cells and methods of use therefor
Provided are pharmaceutical compositions that include a pharmaceutically acceptable carrier and isolated post-natal cardiac progenitor cells (CPCs) and/or progeny cells thereof that are SSEA3-positive and c-kit-negative. Also provided are methods for preparing cells capable of repairing damaged myocardium, methods for isolating populations of SSEA3-positive/c-kit-negative CPCs from cardiac tissue samples, methods for preparing an isolated cell population enriched in post-natal SSEA3-positive/c-kit-negative CPCs, therapeutic methods for using the presently disclosed cells and populations of cells to treat subjects in need thereof, and cell cultures that contain the presently disclosed cells and populations of cells.
1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and isolated SSEA3-positive/c-kit-negative cardiac progenitor cells (CPCs), in vitro expanded SSEA3-positive/c-kit-negative progeny cells thereof, or a combination thereof, wherein the isolated SSEA3-positive/c-kit-negative CPCs are isolated from post-natal myocardium.
2. The pharmaceutical composition of claim 1 , wherein the CPCs are isolated from human post-natal myocardium.
3. The pharmaceutical composition of claim 1 , wherein the concentration of CPCs is about 1×10 5 cells/ml to about 1×10 9 cells/ml in the pharmaceutical composition.
4. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition further comprises one or more cell types selected from the group consisting of non-cardiac-derived SSEA3-positive cells, SSEA4-positive cells, c-kit-positive cardiac stem cells, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), bone marrow cells (BMCs), aldehyde dehydrogenase positive (ALDH-positive) cells, very small embryonic like cells (VSELs), cardiosphere-derived cells (CDCs), or a combination thereof.
5. The pharmaceutical composition of claim 1 , wherein the pharmaceutically acceptable carrier is acceptable for use in a human.
6. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition further comprises one or more growth factors, cytokines, or a combination thereof.
7. The pharmaceutical composition of claim 6 , wherein the one or more growth factors and the one or more cytokines are selected from the group consisting of insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), stromal-derived growth factor-1 (SDF-1), vascular endothelial growth factor (VEGF), a bone morphogeneic protein (BMP), platelet derived growth factor (PDGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), transforming growth factor-β (TGF-β), and stem cell factor (SCF).
8. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is formulated as an injectable.
9. The pharmaceutical composition of claim 8 , wherein the CPCs, the in vitro expanded progeny cells thereof, or both are also CD34-negative, CD45-negative, or both CD34-negative and CD45-negative.
10. A cell culture comprising a population of post-natal SSEA3-positive/c-kit-negative cardiac progenitor cells (CPCs) and/or progeny cells thereof growing on a surface, wherein at least about 50% of the population of post-natal CPCs and/or the progeny cells thereof present in the cell culture are SSEA3-positive/c-kit-negative.
11. The cell culture of claim 10 , wherein the SSEA3-positive/c-kit-negative post-natal CPCs and/or the progeny cells thereof are maintained as subconfluent in the cell culture.
12. The cell culture of claim 10 , wherein the surface upon which the SSEA3-positive/c-kit-negative CPCs and/or the progeny cells thereof are growing comprises a growth-promoting medium or substrate selected from the group consisting of poly-lysine, gelatin, MATRIGEL®, fibronectin, vitronectin, an extracellular matrix component, and a scaffold, or any combination thereof.
13. The cell culture of claim 10 , wherein the SSEA3-positive/c-kit-negative CPCs and/or the progeny cells thereof express at least one cardiac-specific marker selected from the group consisting of Nkx2.5, Gata4, Mef2c, Isl1, and Gata6; one or more pluripotency-associated markers selected from the group consisting of Oct3, Oct4, Nanog, and Sox2; one or more other markers selected from the group consisting of SSEA1, CD105, CD73, CD90, CD29, CD44, CD166, SSEA5, ALDH, and alkaline phosphatase; or any combination thereof.
14. The cell culture of claim 10 , wherein the post-natal SSEA3-positive/c-kit-negative cardiac progenitor cells (CPCs) and/or the progeny cells thereof are also CD34-negative, CD45-negative, or both CD34-negative and CD45-negative.
15. A method for preparing an isolated cell population enriched in post-natal SSEA3-positive/c-kit-negative cardiac progenitor cells (CPCs), the method comprising:
(a) disrupting a cardiac tissue sample obtained from a post-natal subject to obtain tissue fragments and/or single cells;
(b) culturing the tissue fragments and/or single cells in a culture medium for a time and under conditions sufficient to generate 10 4 -10 9 cells;
(c) harvesting all or at least a portion of the adhered tissue fragments and/or single cells from the culture;
(d) purifying one or more subpopulations selected from the group consisting of a subpopulation of SSEA3-positive/c-kit-negative cells, a subpopulation of SSEA3-positive/c-kit-negative/CD34-negative cells, a subpopulation of SSEA3-positive/c-kit-negative/CD45-negative cells, and a subpopulation of SSEA3-positive/c-kit-negative/CD34-negative/CD45-negative cells from the single cell suspension; and
(e) expanding the one or more subpopulations in culture for a time and under conditions sufficient to generate at least about 10 4 SSEA3-positive/c-kit-negative cells that are also optionally CD34-negative and/or CD45-negative,
wherein an isolated cell population enriched in post-natal SSEA3-positive/c-kit-negative cardiac progenitor cells (CPCs) that are also optionally CD34-negative and/or CD45-negative is prepared.
16. The method of claim 15 , wherein the cardiac tissue sample comprises tissue and/or cells isolated from and/or expanded from cardiac right atrial appendage tissue, left atrial appendage tissue, cardiac ventricular tissue, cardiac valvular tissue, cardiac vascular tissue, and/or endomyocardial biopsy tissue.
17. The method of claim 15 , wherein cells of the one or more subpopulations express one or more markers selected from the group consisting of ISL1, GATA4, NKX2.5, MEF2C, GATA6, BRACHYURY, MESP1, OCT3, OCT4, NANOG, and SOX2.
18. The method of claim 15 , wherein the tissue fragments and/or single cells are cultured in the absence of exogenously added feeder cells.
19. The method of claim 15 , wherein the culturing is on a support that comprises a growth-promoting medium selected from the group consisting of poly-lysine, gelatin, MATRIGEL®, fibronectin, laminin, collagen, vitronectin, an extracellular matrix component, and a natural and/or synthetic scaffold.
20. The method of claim 15 , wherein the culturing is in a medium that comprises at least one growth promoting factor selected from the group consisting of IGF-1, FGF, a BMP, EGF, SCF, PDGF, and VEGF.
21. The method of claim 15 , further comprising disrupting the harvested cells to produce a single cell suspension prior to the purifying step.