We recently finished our Ask the Expert discussion with Options for Identification and Characterization of Pluripotent Stem Cells. This week we had several interesting questions and helpful suggestions. Specific information was provided for methods in reprograming, confirming pluripotency, selecting colonies for expansion, alternatives to morphological selection, identifying undifferentiated cells, phenotypic characterization, isolating cells, utilizing cell surface signatures and flow cytometry, and enzyme-free isolation.

The vast advances in technologies for the efficient generation of footprint-free induced pluripotent stem cells (iPSC) have led to the creation of several iPSC lines from varying sources, genetic backgrounds, and derivations in different medias and growth conditions, thus necessitating thorough characterization of the resulting cell lines.

One critical step in establishing iPSC lines involves the early identification of true iPSC clones and their subsequent characterization to ensure functional pluripotency. Various methods of characterization, ranging from visual morphological observation to the use of differentially expressed biomarkers are utilized for the initial identification of pluripotent cells. Dyes such as Alkaline Phosphatase Live Stain enable the early detection of emerging iPSC colonies that can be used in combination with morphological assessment to pick the right iPSC clone for further expansion. Established clones are further subjected to a combination of in vitro and in vivo cellular analysis to confirm functional pluripotency based on expression of self-renewal markers and trilineage differentiation potential. While such traditional methods have been successfully used, there is a need for a uniform standardized method for comprehensive characterization. Recently, the TaqMan® hPSC Scorecard™ Panel, a real-time PCR gene expression assay, provides a rapid molecular method to generate quantitative transcriptome data for the confirmation of functional pluripotency.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Dr. Uma Lakshmipathy. Dr. Lakshmipathy has been involved in the field of stem cells for nearly a decade. Her doctoral degree in Molecular Biophysics and subsequent postdoctoral experience in DNA repair brought new perspective and led her to the area of stem cell research with focus on developing ex vivo gene repair systems. As a junior faculty at the Stem Cell Institute, University of Minnesota, she identified efficient gene delivery methods into stem cells to enable repair of adult stem cells from monogene disorders. She moved to Invitrogen, Life Technologies, in 2005 and was involved in the development of novel technology platforms for creating labeled stem cells. Her current research interests at Life Technologies, now a part of Thermo Fisher Scientific, are regulation of stem cell maintenance, development of technologies for generation, identification, characterization of pluripotent stem cells, and modification their derivatives.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – Options for Identification and Characterization of Pluripotent Stem Cells.

Question:

Many colonies of different shapes and sizes seem to express some surface markers of self renewal, how do I identify the best colonies to select and expand?

The Answer:

The use of a negative marker in conjunction with a positive marker is the most effective method of identifying fully reprogrammed colonies prior to expansion and selection. Somatic tissues like CD34+ blood cells, PBMCs, and human Fibroblasts express the surface marker CD44 at high levels. Expression of this marker has been demonstrated to be significantly down-regulated in fully reprogrammed cells. The combination of CD44 antibody staining with either SSEA4, Tra-1-60 or Tra-1-81 will more clearly distinguish fully reprogrammed colonies from partially reprogrammed colonies by the absence of CD44 and presence of self-renewal marker expression.

For a complete list of PSC markers, please visit lifetechnologies.com/stemcellantibodies

Question:

I have selected my reprogrammed colonies and am expanding them, how can I further confirm that they are still pluripotent?

The Answer:

The continued proliferation of the clones with good morphology is a good indicator, but further characterization is necessary. The continued expression of self-renewal markers, such as SSEA4, Tra-1-60 and Tra-1-81, can be supplemented by looking at intracellular markers  of self-renewal like OCT4, SOX2, KLF4 and Nanog via antibody staining.

For a complete list of PSC markers, please visit lifetechnologies.com/stemcellantibodies

Question:

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?

The 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.