Enriched stem cell and progenitor cell populations, and methods of producing and using such populations
The present invention provides a novel method to isolate and expand pure progenitor/stem cells from a primary tissue explant, which produces a population enriched in multipotent functional progenitor/stem cells free of contaminating fibroblasts and other cell types. Cardiac progenitor/stem cells isolated by this method maintain their self-renewal and clonogenic character in vitro and differentiate into normal cells in myocardium, including cardiomyocytes, endothelial cells, and smooth muscle cells, after transplantation into ischemic hearts. The present invention also includes substantially pure populations of multipotent progenitor/stem cells, e.g., cardiac progenitor/stem cells, and their use to treat and prevent diseases and injuries, including those resulting from myocardial infarction.
1. A method of preparing a purified isolated cell population enriched in cardiac stem cells or cardiac progenitor cells, comprising:
(a) obtaining a cardiac tissue sample that comprises (1) cardiac stem cells or cardiac progenitor cells and (2) fibroblasts; then
(b) culturing said cardiac tissue sample for a time sufficient to allow the fibroblasts to adhere to the culture dish; then
(c) collecting cells from the culture dish that migrate above the adhered fibroblasts of step (c); then
(d) separating Sca-1-expressing cardiac stem cells or Sca-1-expressing cardiac progenitor cells from the collected cells; then
(e) culturing the separated Sca-1-expressing cardiac stem cells or Sca-1-expressing cardiac progenitor cells in a cardiac fibroblast-conditioned medium in the absence of feeder cells;
thereby obtaining a purified isolated cell population enriched in cardiac stem cells or cardiac progenitor cells.
2. The method of claim 1 , wherein said cardiac tissue sample is an adult mammalian cardiac tissue sample.
3. The method of claim 1 , wherein said cardiac tissue is subjected to enzymatic dissociation.
4. The method of claim 1 , wherein said cardiac tissue is cultured for at least 10 days.
5. A method of preparing an isolated population of differentiated cells comprising:
(a) preparing a purified isolated cell population enriched in cardiac stem cells or cardiac progenitor cells according to the method of claim 1 ; and
(b) inducing said cardiac stem cells or cardiac progenitor cells to differentiate to cardiomvocytes, cardiac endothelial cells, cardiac smooth muscle cells, or cardiac neural crest cells, thereby obtaining an isolated population of differentiated cells.
6. The method of claim 5 , wherein said cardiac stem cells are induced to differentiate by contacting said cardiac stem cells or cardiac progenitor cells with transforming growth factor β, bone morphogenic protein 2, bone morphogenic protein 4, and activin A.
7. A method of preparing a purified isolated cell population enriched in cardiac stem cells or cardiac progenitor cells, comprising:
(a) obtaining cardiac tissue sample that comprises (1) cardiac stem cells or cardiac progenitor cells and (2) fibroblasts; then
(b) culturing said cardiac tissue sample for a time sufficient to allow the fibroblasts to adhere to the culture dish; then
(c) collecting cells from the culture dish that migrate above the adhered fibroblasts of step (c); then
(d) separating Sca-1-expressing cardiac stem cells or Sca-1-expressing cardiac progenitor cells from the collected cells; then
(e) cloning one or more cells isolated in step (e) to produce one or more clonogenic populations; then
(f culturing said clonogenic populations in a cardiac fibroblast-conditioned medium in the absence of feeder cells;
thereby obtaining a purified isolated cell population enriched in cardiac stem cells or cardiac progenitor cells.