We observed that exposure to undifferentiated neural progenitor conditioned medium maintains the rounded phenotype of immature microglia and promotes their proliferation. We investigated whether maturation of pMGLs in the presence of human pluripotent stem cell-derived mature neural cells would further refine their molecular signature (Fig. 6a, top panel). Principal component analysis of the transcriptome revealed that primary fetal microglia and pMGLs cluster tightly together along PC1, and segregate away from differentiated neural progenitors cultured in the same conditions (Fig. 6b). The difference between NPCs and pMGLs along PC1 may reflect the main myeloid vs neural identity, accounting for ~77% of sample variance, while the variation along PC2 (~12% of sample variance) may be refined by tissue residency, ECM and cell/cell interactions. To directly test this notion, we grew pMGLs in conditioned medium from differentiating neural cultures (defined before conditioning, with no other variation in recombinant growth factor concentrations). Fig. 6b shows that, after 2 weeks, the pMGLs partially shifted their signature towards that of primary microglia along the PC2 axis.
