JPWO2020252477A5 - - Google Patents

Claims (36)

An in vitro method for differentiating induced pluripotent stem cells (iPSCs), comprising:
(a) culturing said iPSCs on a positively or negatively charged surface in the absence of extracellular matrix proteins to produce hematopoietic progenitor cells (HPCs) ; An in vitro method comprising differentiating into meristrial stem cells (MSCs) or microglia . 2. The method of claim 1, wherein the charged surface is a positively charged surface. 3. The method of claim 2, wherein the positively charged surface is an amine surface or a poly-L-lysine surface. 3. The method of claim 2, wherein the positively charged surface comprises nitrogen-containing functional groups. 2. The method of claim 1, wherein the charged surface is a negatively charged surface. 6. The method of claim 5, wherein said negatively charged surface is a carboxyl surface. 6. The method of claim 5, wherein the negatively charged surface comprises oxygen-containing functional groups. A method according to any preceding claim, wherein the charged surface is a polymer surface. 9. The method of claim 8 , wherein the polymer surface is a polystyrene surface. The method of any one of claims 1-9 , wherein the iPSCs are cultured in a serum-free defined medium. 11. The method of claim 10 , wherein differentiating comprises culturing in the presence of blebbistatin or a ROCK inhibitor. 12. Any one of claims 1 to 11 , wherein said culturing is performed in the absence of laminin, fibronectin, vitronectin, MATRIGEL™, tenascin, entactin, thrombospondin, elastin, gelatin and/or collagen. The method described in section. 13. The method of any one of claims 1-12 , further comprising engineering said iPSCs to have disrupted expression of TREM2 , SNCA, or MeCP2 prior to step (a). 14. The method of any one of claims 1-13 , comprising differentiating said HPCs into endothelial cells. Step (a) comprises culturing on an amine surface to produce HPCs and step (b) comprises culturing on a carboxyl surface in the presence of an endothelial differentiation medium to produce endothelial cells. 15. The method of claim 14 . 16. The method of any one of claims 1-15 , further comprising differentiating said endothelial cells into brain microvascular endothelial cells (BMEC) or lymphoid endothelial cells . 16. The method of any one of claims 1-15 , comprising differentiating said HPCs into MSCs. 18. The method of claim 17 , wherein differentiating comprises culturing said HPCs on an amine surface in the presence of MSC medium. 19. The method of claim 18 , wherein said MSCs are positive for CD73, CD44 and CD105. 19. The method of claim 18 , wherein at least 90% of said differentiated cells are positive for CD73. 18. The method of claim 17 , further comprising differentiating said MSCs into pericytes. 22. The method of claim 21 , wherein said MSCs are cultured in the presence of pericyte medium in the absence of extracellular proteins. 22. The method of claim 21 , wherein said pericytes are positive for NG2, PDGFR[beta] and CD146. 24. The method of claim 23 , wherein said method comprises differentiating said HPCs into microglia. 25. The method of claim 24 , wherein differentiating comprises culturing said HPCs on a neutrally charged surface or ultra-low attachment surface in the presence of a microglial differentiation medium. 26. The method of claim 25 , wherein said microglial differentiation medium comprises IL34, TGF and /or MCSF. 27. The method of claim 26 , wherein said microglia are positive for CD45, CD11b and CD33. 28. The method of claim 27 , wherein at least 50% or 90% of said differentiated cells are positive for CD11b. 29. The method of any one of claims 1-28 , wherein step (b) of the method does not comprise purification of the cells. 30. The method of any one of claims 1-29 , wherein the method complies with Good Manufacturing Practices (GMP). The method of any one of claims 1-30 , which is carried out under hypoxic conditions. 32. The method of any one of claims 1-31 , wherein each of steps (a)-(d) is performed under xeno-free, feeder-free and/or conditioned medium-free conditions. A composition comprising a microglial cell population that is at least 90% positive for TREM2, P2RY12, TMEM119, IBA-1 and/or CX3CR1, wherein said microglial cell population is differentiated from an iPSC. A co-culture comprising microglia produced by the method of any one of claims 1-32 and endothelial cells, pericytes, astrocytes and/or neural progenitor cells. 35. Use of the co-culture according to claim 34 for mimicking human brain development. 2. The method of claim 1, wherein the culturing of step (b) is further defined as two-dimensional culturing.