Generation and cryopreservation of pluripotent stem cell-derived clinical grade corneal endothelial cells
Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived clinical-grade corneal endothelial cells (CECs) generation and cryopreservation through the neural crest cell (NCC) lineage using peripheral blood as a donor source and hESCs maximizes the availability of these vital cells for treating the corneal endothelial disease as an alternative to donor corneas for corneal endothelium (CE) transplantations and other applications.
1 . A method of producing clinical-grade corneal endothelial cells (CECs) comprising:
i) isolating peripheral blood mononuclear cells (PBMCs) from a subject,
ii) reprogramming of the PBMCs to produce peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs),
iii) differentiating the peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs), by culturing the iPSCs on human protein and cell culture medium,
iv) replacing the cell culture medium with a differentiation medium, to produce differentiating corneal endothelial cells (CECs),
v) transferring the differentiating CECs to human protein-coated plates comprising a cornea cell culturing medium,
whereby steps i)-iv) produce clinical-grade corneal endothelial cells (CECs) in about 20 days or less.
2 . The method of claim 1 , wherein the differentiation media comprises one or more growth factors, inhibitors of transforming growth factor (TGF) or combinations thereof.
3 . The method of claim 1 , wherein the iPSCs cells expressing pluripotent markers are cultured in a protein or gelatinous protein mixture with one or more factors to induce differentiation of the cells.
4 . The method of claim 3 , wherein the cells cultured in the gelatinous protein mixture differentiate into corneal endothelial cells (CECs) as determined by the expression of corneal endothelium (CE)-associated markers or combinations thereof.
5 . The method of claim 1 , wherein upon cryopreservation of the cryopreserved clinical-grade corneal endothelial cells (CECs), the cryopreserved CECs retain their viability and/or function when thawed.
6 . The method of claim 1 wherein peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs) are differentiated into clinical-grade corneal endothelial cells (CECs).
7 . The method of claim 1 , wherein the differentiating corneal endothelial cells (CECs) are replated on a culture dish comprising human protein and cornea medium.
8 . The method of claim 1 , wherein the iPSCs are cultured a differentiation medium for about 1-3 days.
9 . The method of claim 7 , wherein the differentiating corneal endothelial cells (CECs) are cultured on new plates comprising human protein and cornea medium for about 7-14 days.
10 . A method of producing clinical-grade corneal endothelial cells (CECs), the method consisting of:
i) isolating peripheral blood mononuclear cells (PBMCs) from a subject,
ii) reprogramming of the PBMCs to produce induced pluripotent stem cells (iPSCs),
iii differentiating peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs),
iv) culturing the differentiating iPSCs,
v) replacing the cell culture medium with a differentiation medium, to produce differentiating corneal endothelial cells (CECs),
thereby producing clinical-grade corneal endothelial cells (CECs).
11 . The method of claim 1 , wherein the step of differentiating peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs) comprises culturing the iPSCs on human protein and cell culture medium for about 3-6 days.
12 . The method of claim 1 , wherein the culture medium comprising one or more inhibitors of cell signaling pathways is replaced with cornea medium after about 2-4 days.
13 . The method of claim 12 , wherein the cornea medium comprises human growth factors, and B27 supplements.
14 . The method of claim 12 , wherein the differentiating CECs are transferred to a new human protein coated plate comprising cornea medium.
15 . The method of claim 14 , wherein the differentiating CECs are cultured for about 10 to 15 days.
16 . The method of claim 1 , wherein purification of differentiating cells is not required.
17 . The method of claim 1 , wherein the iPSCs are cultured on human protein and cell culture medium for a period exceeding 1 day prior to culture in media comprising one or more inhibitors of cell signaling pathways.
18 . The method of claim 1 , wherein the iPSCs are cultured on human protein and cell culture medium for a period of about 4 days prior to culture in media comprising one or more inhibitors of cell signaling pathways.
19 . The method of claim 10 , wherein the iPSCs are cultured on human protein and cell culture medium for a period exceeding 1 day prior to culture in media comprising dual Smad inhibitors.
20 . The method of claim 10 , wherein the iPSCs are cultured on human protein and cell culture medium for a period of about 4 days prior to culture in media comprising dual Smad inhibitors.