Cgmp compliant differentiation of ipscs to iecs

A method of generating endothelial cells from induced pluripotent stem cells (iECs), comprising

• (i) differentiating induced pluripotent stem cells (iPSCs) into mesoderm cells by:
• removing cGMP feeder-free iPSC maintenance medium from plated induced pluripotent stem cells (iPSCs) and adding mesoderm induction medium, and
• culturing the iPSCs for about 1.5-2.5 days in about 36-38 C and in the presence of about 4-6% CO2 to generate mesoderm cells;
• (ii) differentiating the mesoderm cells into vascular progenitor cells by:
• removing the mesoderm induction medium and adding vascular progenitor medium, and
• culturing the cells for about 1.5-2.5 days in about 36-38 C and in the presence of about 4-6% CO2 to generate vascular progenitor cells;
• (iii) differentiating the vascular progenitor cells into iECs by:
• dissociating the vascular progenitor cells by using a first dissociation enzyme for about 4-6 minutes at 36-38 C and in the presence of about 4-6% CO2, wherein the dissociation enzyme comprises an animal origin free enzyme that cleaves peptide bonds on the C-terminal sides of lysine and arginine;
• neutralizing the vascular progenitor cells by using basal endothelial cell growth medium MV2 plus about 9-11% human AB serum, spinning the vascular progenitor cell solution, removing the basal endothelial medium, and resuspending the vascular progenitor cells in GMP-grade iEC differentiation medium, wherein the human AB serum comprises human serum lacking antibodies against A and B blood-type antigens that was converted from Pooled Plasma (Human), Solvent/Detergent Treated;
• plating the vascular progenitor cells at about 9,000-11,000 cells/cm2 with iEC differentiation medium onto a dish that was previously coated with 4-6 ug/ml human recombinant laminin 421 in PBS Mg+/Ca+ for about 1.5-2.5 hours at about 36-38 C and in the presence of about 4-6% CO2,
• culturing the vascular progenitor cells at about 36-38 C and in the presence of about 4-6% CO2 for about 6-8 days, feeding about every other day with iEC differentiation medium to generate iECs;
• and
• (iv) further culturing the iECs by:
• dissociating the iECs by using animal origin free enzyme that cleaves peptide bonds on the C-terminal sides of lysine and arginine (1×) for about 4-6 minutes at about 36-38 C and in the presence of about 4-6% CO2;
• neutralizing the iECs using by using basal endothelial cell growth medium MV2 plus 9-11% Human AB serum, spinning the iEC solution, removing the basal endothelial medium, and resuspending the iECs in GMP-grade iEC differentiation medium, wherein the human AB serum comprises human serum lacking antibodies against A and B blood-type antigens that was converted from Pooled Plasma (Human), Solvent/Detergent Treated;
• plating the iECs at about 9,000-11,000 cells/cm2 with iEC differentiation medium onto a dish that was previously coated with Sug/ml human recombinant laminin 421 in PBS Mg+/Ca+ for about 1.5-2.5 hours at about 36-38 C and in the presence of about 4-6% CO2,
• culturing the iECs at about 36-38 C and in the presence of about 4-6% CO2 for about 9-11 days, feeding about every other day with iEC medium, thereby generating iECs expressing CD31 and CD144.