Substantially pure human retinal progenitor, forebrain progenitor, and retinal pigment epithelium cell cultures and methods of making the same
Methods for producing substantially pure cultures of human neural retinal progenitor cells, forebrain progenitor cells, and retinal pigment epithelial cells are disclosed. In addition, the successful differentiation of human embryonic stem cells and human induced pluripotent stem cells through the major developmental stages of human retinogenesis is disclosed.
1. A cell culture comprising: a human vesicular neurosphere, wherein the human vesicular neurosphere includes a hollow center and an outer ring-like laminar layer comprising human retinal progenitor cells oriented radially outwards relative to the neurosphere center, wherein the outer ring-like laminar layer appears phase-bright and golden in color when observed using bright-field microscopy, wherein the human vesicular neurosphere is capable of generating cells of neural retina or retinal pigment epithelium (RPE), and wherein at least 80% of the cells within the human vesicular neurosphere are human retinal progenitor cells.
2. The cell culture of claim 1 , wherein the human vesicular neurosphere comprises the genetic background of a subject having a retinal degenerative disease.
3. The cell culture of claim 2 , wherein the retinal degenerative disease is an inherited retinal degenerative disease.
4. The cell culture of claim 3 , wherein the inherited retinal degenerative disease is gyrate atrophy.
5. The cell culture of claim 1 , further comprising exogenous activin, wherein the vesicular neurosphere in the presence of the exogenous activin expresses a higher level of Mitf, RPE65, or Bestrophin, and a lower level of Chx10, Crx, or Brn3B than the vesicular neurosphere in the absence of the exogenous activin.
6. The cell culture of claim 1 , wherein at least 90% of the cells within the human vesicular neurosphere are human retinal progenitor cells.
7. The cell culture of claim 1 , wherein the human retinal progenitor cells are Chx10-positive.
8. The cell culture of claim 1 , further comprising a serum-free culture medium.
9. A method for generating a cell culture comprising a population of cells wherein at least 90% of the cells in the population are human retinal progenitor cells, comprising:
(i) culturing human neuroepithelial colonies in suspension in retinal differentiation medium until neurospheres are formed;
(ii) identifying retinal progenitor cell (RPC) neurospheres having a vesicular morphology and neurospheres having a non-vesicular morphology, wherein the RPC neurospheres having a vesicular morphology include a hollow center and an outer ring-like laminar layer comprising human retinal progenitor cells oriented radially outwards relative to the neurosphere center, and wherein the outer ring-like laminar layer appears phase-bright and golden in color when observed using bright-field microscopy; and
(iii) isolating the identified neurospheres having a vesicular morphology to obtain a cell culture comprising isolated RPC neurospheres comprising a population of cells wherein at least 90% of the cells in the population are human retinal progenitor cells, wherein after three months of maintenance, the human retinal progenitor cells are capable of producing multiple retinal cell types.
10. The method of claim 9 , wherein the culturing is serum free.
11. The method of claim 9 , wherein the cultured human neuroepithelial colonies are derived from human pluripotent stem cells.
12. The method of claim 11 , wherein the human pluripotent stem cells are human induced pluripotent stem cells.
13. The method of claim 12 , wherein the human induced pluripotent stem cells were generated from a subject having a retinal degenerative disease or at risk of an inherited retinal degenerative disease.
14. The method of claim 13 , wherein the retinal degenerative disease is an inherited retinal degenerative disease.
15. The method of claim 9 , further comprising differentiating the isolated RPC neurospheres into a population comprising cell types of the neural retina.
16. The method of claim 9 , further comprising extended culturing of the isolated RPC neurospheres in the presence of Activin until retinal pigment epithelium (RPE) neurospheres are formed.
17. The method of claim 16 , further comprising culturing of the RPE neurospheres on a laminin-coated culture substrate.