Tuesday, August 30, 2016

In Depth Analysis of Differentiating Neural Stem Cells

Foundation for Future Therapies

Here we share an example of this analysis. Results from worldwide research is now being made available via hPSCreg: Ilyas Singec, Andrew M. Crain, Junjie Hou, Brian T.D. Tobe, Maria Talantova, Alicia A. Winquist, Kutbuddin S. Doctor, Jennifer Choy, Xiayu Huang, Esther La Monaca, David M. Horn,5 Dieter A. Wolf, Stuart A. Lipton, Gustavo J. Gutierrez, Laurence M. Brill, and Evan Y. Snyder. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling. Stem Cell Reports (2016), http://dx.doi.org/10.1016/j.stemcr.2016.07.01.

Images: MDK Expression in Pluripotent and Differentiated Cells and Disruption of Neural Conversion by a Monoclonal Antibody against MDK (A) Representative immunocytochemical analysis of MDK expression by hESCs (WA09), skin fibroblasts (FB, line HS27), and fibroblasts (HS27) after reprogramming to hiPSCs. Scale bars represent 100 μm. (B) Western blot showing that MDK was not detected in HS27 fibroblasts but was expressed in WA09 hESCs and hiPSCs from HS27 fibroblasts, and was upregulated in differentiating cells (EBs and hNSCs from hESCs). Probing for GAPDH demonstrated similar loading of the lanes. (C) A monoclonal antibody against MDK did not impair pluripotent self-renewal of hESCs. Antibody concentrations are shown. (D) The percentage of pluripotent cells expressing OCT4 and NANOG did not differ significantly between anti-MDK and isotype control antibody treatment (3.0 μg/mL). Error bars represent SEM; n = 5. (E) qRT-PCR showed that a monoclonal antibody against MDK (1.5 μg/mL) inhibits neural gene expression during 6-day DAP treatment. Error bars represent mean + SEM, n = 3. ∗p less than  0.05. (F) Western blot analysis confirmed the inhibitory effect of anti-MDK on early neural marker expression (OTX1, PAX6), despite DAP treatment, in contrast to the isotype control. (G) Immunocytochemical evidence that neural conversion is inhibited by the anti-MDK antibody (1.5 μg/mL) during 6-day DAP treatment. Few cells induced PAX6 expression in the presence of anti-MDK antibody (right panel). Scale bar represents 200 μm. (H) Western blot analysis and time course of hESCs differentiated for 6 days with recombinant MDK alone (100 ng/mL) or a combination of the DAP cocktail and MDK (25 or 100 ng/mL) suggest that MDK specifically promotes neural commitment of hPSCs. SOX17, MIXL1, and Brachyury were not induced by MDK. In contrast, controls treated with fetal bovine serum (FBS) for 6 days, which stimulates multi-lineage differentiation of hPSCs, induced these non-neural markers.
This level of basic research yield knowledge that is important for the development of future cell based therapies for Neuro-related Diseases. We will continue to post updates here.

No comments: