Methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof
The present invention is directed to pluripotent fetal stem cells derived from chorionic villus, amniotic fluid, and placenta and the methods for isolating, expanding and differentiating these cells, and their therapeutic uses such as manipulating the fetal stem cells by gene transfection and other means for therapeutic applications.
1. A method of proliferating a population of cells enriched for human pluripotent fetal stem cells comprising:
(a) selecting at least one c-kit positive cell from a human amniotic fluid sample, wherein said cell is c-kit, SSEA3 and SSEA4 positive, and immunonegative for SSEA1;
(b) introducing said at least one selected cell to a culture medium; and
(c) proliferating said at least one selected cell in the culture medium.
2. A method of obtaining a composition enriched for human pluripotent fetal stem cells, said cells being c-kit, SSEA3 and SSEA4 positive, and SSEA1 negative, comprising the steps of:
(a) cryopreseverving a specimen of the amniotic fluid;
(b) thawing the cryopreserved specimen at a later date; and
(c) selecting for c-kit positive cells.
3. A method of producing a composition enriched for human pluripotent fetal stem cells, said cells being c-kit, SSEA3 and SSEA4 positive, and SSEA1 negative comprising,
(a) isolating c-kit positive cells from a sample of amniotic fluid; and
(b) proliferating said cells in culture medium.
4. A method of obtaining a composition enriched for human pluripotent fetal stem cells that are c-kit, SSEA3 and SSEA4 positive, and SSEA1 negative comprising selecting c-kit positive cells from a human amniotic fluid sample.
5. The method of claim 4 , wherein the selecting c-kit positive cells is performed using an antibody against c-kit.
6. The method of claim 5 , wherein the antibody against c-kit is a monoclonal antibody.
7. The method of claim 5 , wherein the monoclonal antibody against c-kit is a mouse monoclonal IgG against an antigenic epitope of human c-kit.
8. The method of claim 5 , wherein the antibody against c-kit is fluorochrome conjugated.
9. The method of claim 5 , wherein the antibody against c-kit is conjugated to magnetic particles.
10. The method of claim 4 , wherein the selecting is by flow cytometry.
11. The method of claim 4 , wherein the selecting is by fluorescence activated cell sorting or high gradient magnetic selection.
12. The method of claim 4 , wherein the amniotic fluid sample is cryopreserved prior to the selection step.
13. The method of claim 4 , further comprising cyropreserving the c-kit, SSEA3 and SSEA4 positive, and SSEA1 negative cells.
14. A method of producing a composition enriched for human pluripotent fetal cells comprising:
(a) selecting at least one c-kit positive cell from a human amniotic fluid sample, wherein said cell is c-kit, SSEA4, and telomerase positive;
(b) introducing said at least one selected cell to a culture medium; and
(c) proliferating said at least one selected cell in the culture medium.