Cux1/Cux2, cut-like homeobox 1/2, are upper layer-specific markers for neurons, which participate in the fundamental regulation of the late neuronal differentiation, spine formation, dendritic branching, and synaptogenesis in upper-layer (L2-3) neurofs of the cortex [ 33, 34].
Antibodies against the markers for neurons- β-III tubulin (1∶1000, Promega, Madison, USA) and astrocytes-GFAP (1∶500, Dako, Glostrup, Denmark) were used.
In contrast, the expression of several genes considered to be markers for neurons, oligodendrocytes or microglia [23] was low or absent (Fig. 2), confirming that the samples were indeed free from contaminating mRNAs from other cell types.
Transcripts for nestin (an NSC specific marker), neurofilament triplet proteins (NF-L, NF-M and NF-H, cell type-specific markers for neurons), glial fibrillary acidic protein (GFAP, a specific marker for astrocytes) and Akt1 are all expressed by both F3 and F3.Akt1 cells.
Next, we evaluated the differentiation of IhNSC-Ps into specific neuronal and glial phenotypes following transplantation into the ischemic environment by analyzing the colocalization of selective markers for neurons, astroglial and oligodendroglial cells with the anti-human specific antibody anti-huN.
NeuN and glial fibrillary acidic protein (GFAP) were used as markers for neurons and astrocytes, respectively.
IRF8+ cells were not double-labeled with markers for neurons (NeuN, MAP2, and NF200) or astrocytes (GFAP).
Following incubation in differentiation medium, cells displayed a sharp increase in markers for neurons and neural precursors.
Double-staining with markers for neurons, microglia, astrocytes, and oligodendrocytes revealed co-localization with microglia, but not with neurons or other glia.
FABP7 was not colocalized with markers for neurons including NeuN (Supplementary Fig. 2a) and MAP2 (Supplementary Fig. 2b), or with makers for microglia including CD11b (Supplementary Fig. 2c).
