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. 2019 Feb 12;13:26. doi: 10.3389/fncel.2019.00026

Figure 1.

Figure 1

Endogenous expression of integrins, growth-promoting proteins and deep layer cortical neuron markers in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs). Endogenous α9 (A) and β1 (B) integrin subunit expression in wild type (WT) human NPCs (hNPCs) was confirmed using western blotting (WB), with faint bands observed at approximately 115 kDa (α9 integrin) or 120 kDa (β1 integrin). This was analyzed alongside retinal pigmented epithelial (RPE) cell lysates as positive controls for both integrin subunits. Blots were also stained with β-actin (approximately 42 kDa) to confirm equal protein loading. Immunocytochemistry (ICC) analysis further confirmed a low level of endogenous α9 (C,D) and β1 (E,F) integrin subunit expression within hNPCs. Furthermore, ICC analysis demonstrated hNPCs express tenascin-C (TN-C) as observed in punctate dots (white arrows, G,H) around the cell nucleus (identified using DAPI). The hNPCs also express a number of growth-promoting proteins including the progenitor cell marker doublecortin (DCX), observed in both the cell body and neurite projections (white arrows, I), which co-labeled with βIII-tubulin (white arrows, J). Expression of the neurotrophic factor brain-derived neurotrophic factor (BDNF; white arrows in K,L) was also identified as well as expression of its receptor, Trk B (white arrows in M,N). The hNPC cultures largely contained βIII-tubulin-positive cells (white arrows in O), however some glial fibrillary acidic protein (GFAP)-positive cells (white arrows in P) were also detected. No myelin basic protein (MBP)-positive cells were detected following 21 days in culture (Q). Expression of deep-layer cortical neuron markers T-box brain 1 (Tbr1; white arrows, R–T) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) interacting protein 2 (Ctip2; white arrows, U–W) was detected following ICC within the DAPI-stained nucleus of hNPCs. Cells were co-stained with βIII-tubulin and DAPI. Scale bar in (C–N,T,W) = 20 μm; (O–S,U,V) = 100 μm.