Derivation of human microglia from pluripotent stem cells
The present invention relates to methods for deriving human hematopoietic progenitors, primitive macrophages, and microglial cells from human pluripotent stem cells. In particular, provided herein are highly efficient and reproducible methods of obtaining human primitive macrophages and microglia from human pluripotent stem cells, where the primitive macrophages and microglia can be suitable for clinically relevant therapeutic applications.
1. A method of obtaining human hematopoietic precursor cells, comprising culturing human pluripotent stem cells under normoxic conditions for about 24 hours, wherein the pluripotent stem cells are cultured on a substrate that promotes cell adhesion and in a culture medium consisting essentially of L-ascorbic acid-2-phosphate magnesium, sodium selenium, transferrin, insulin, NaHCO 3 , fibroblast growth factor 2 (FGF2), transforming growth factor beta 1 (TGFβ1), and a Rho kinase (ROCK) inhibitor, whereby the cultured pluripotent stem cells differentiate into human hematopoietic precursor cells (HPCs) expressing CD45 with a portion co-expressing CD34.
2. The method of claim 1 , wherein the substrate that promotes cell adhesion comprises Tenascin-C.
3. The method of claim 2 , wherein the Tenascin-C is recombinant human Tenascin-C.
4. The method of claim 1 , wherein the ROCK inhibitor is selected from the group consisting of Y-27632 and Blebbistatin.