I am reprogramming hematopoietic stem cells to iPSCs. Ideally I would like to have a few different methods available for confirming pluripotency that could be either used in conjunction for more in-depth analysis or independently for a quick screen. Any thoughts?

Answer

Several methods are commonly used for PSC characterization {Marti et al (2013) Characterization of Pluripotent Stem Cells, 8, 223-253}. For initial screening of emerging iPSC colonies, Alkaline Phosphatase Live Stain (http://www.lifetechnologies.com/order/catalog/product/A14353) can be used. This is a method of identifying pluripotent stem cells without impacting cell survival or characteristics {Singh et al (2012) Stem Cell Rev. 8, 1021-1029}.

Alternately, immunostaining can be carried out on live cells using antibodies against pluripotent specific surface antibodies such as SSEA4, TRA-1-60 and TRA-1-80. (http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/pluripotent-stem-cell-detection/pluripotent-stem-cell-antibodies.html?icid=cvc-stemcelldetection-c2t1)

The final confirmation is to check for trilineage differentiation potential either using in vitro embryoid body formation or in vivo teratoma assays. Recently molecular assays have been developed as a comprehensive high-throughput method for characterization of pluripotent stem cells based on gene expression signatures. TaqMan® hPSC Scorecard™ Assay is a qRT-PCR based method that quantitatively measures the expression level of 93 genes comprised of a combination of self-renewal and lineage specific markers, against validated reference standards.
(http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/taqman-hpsc-scorecard-panel.html) This method can be used to analyze undifferentiated and differentiating PSCs to not only confirm biomarker expression in an undifferentiated state but also show expression of markers specific for the three germ layers during differentiation, thus confirming functional pluripotency.