Options for Identification and Characterization of Pluripotent Stem Cells

Sponsored by: Life Technologies
Session ends: May 16th, 2013, 3:00pm MST
Answers by: Dr Uma Lakshmipathy , Principal Scientist , at  Thermo Fisher Scientific


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.

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This session is sponsored by Life Technologies

Join Uma Lakshmipathy, Principal Scientist at Thermo Fisher Scientific, to explore various options for identification and characterization of pluripotent stem cells!

Questions & Answers

We are working with mesenchymal stem cells and need to measure their ability to suppress T-cell proliferation. We were outsourcing this, but would like to bring it in-house. Any advice? Is it better to keep outsourcing?

A simple in vitro protocol such as the Mixed Lymphocyte Reaction assay can be used to measure immunosuppression of MSC and AdSCs. An example protocol can be found in this publication: Kruisbeek et al., (2004) Proliferative Assays for T Cell Function. Current Protocols in Immunology 3.12.1-3.12.20. http://www.currentprotocols.com/WileyCDA/CPUnit/refId-im0312.html» Read More

Without giving away too much detail, we are working with cardiomyocytes and are looking for a more efficient way to isolate on a larger scale. Currently we prep the tissue sample, plate cells and grown them in selection media. Bound cells are then transferred into culture media and expanded. The trouble is that this can be a very time consuming part of the process and we have had some challenges in consistency. Is there a process, kit, product, that you could recommend to improve our process, particularly time-investment when increasing scale?

It is not clear if the cardiomyocytes referred to in your question are from a primary source or derived from iPSCs. If it is derivation from iPSC, the process requires (A) expansion of the stem cell, (B) efficient differentiation into cell type of choice. This is indeed a challenge for generating large-scale production of cells. […]» Read More

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?

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 […]» Read More

I am looking for an enzyme-free method of isolating my PSCs.

Isolation of iPSCs from a master reprogramming plate must be done manually. Colonies should be selected through the use of a 27-gauge needle and a pipettor and then transferred to individual vessels for clonal expansion. Once the PSCs have been expanded they may then be split through future passages by the same manual selection (using […]» Read More

In my lab we work with cord blood and we are looking for the fastest, simplest way to take a mixed population of cells from cord blood and identify, isolate and characterize the cells. Do you have any recommendations?

Dynabeads® CD34 Positive Isolation kit can be used for rapid isolation of CD34+ cells from a heterogeneous population of mononuclear cells from cord blood, bone marrow or peripheral blood. For more information, please refer to the protocol below: (http://www.lifetechnologies.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/cell-separation-methods/human-cell-separation-protocols/dynal-cd34-progenitor-cell-selection-system.html)» Read More

I have been tasked with developing a protocol for identifying and isolating different cell types from a mixed population of neural cells using flow cytometry. I have been working on using cell surface signatures, but it has been challenging. Could you recommend any materials or methods for review?

The Gibco® Neurobiology Protocol handbook provides a comprehensive list of methods and protocols to support neural cell research. The handbook includes protocols with topics including neural cell culture and differentiation, cell analysis, molecular characterization, and transfection. (http://www.lifetechnologies.com/us/en/home/global/forms/gibco-neurobiology-handbook.html) In addition to marker expression, reagents such as fluorescent probes can be used to study neural health, anatomy […]» Read More

I am hoping you can help with a verification issue. We are currently using telomerase to identify any undifferentiated PSCs. We would like to add a second method for verification purposes. Which method do you think is best?

Elimination of pluripotent stem cells from differentiated cells is critical prior to downstream in vivo experiments. The most simple method would involve using bead-conjugated antibodies against the pluripotent surface marker SSEA4 (see link below). http://www.lifetechnologies.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/cell-separation-methods/embryonic-stem-cell-isolation-protocols/dynabeads-ssea-4.html Using flow cytometry, SSEA4 negative population can be further assessed for the presence of cells expressing SSEA4 and other pluripotent […]» Read More

We have been working to reduce serum in our stem cell culture while maintaining pluripotent characteristics. Our standard media contains 10% FBS and we have not been able to reduce this amount successfully without the culture suffering. We recently were able to use human serum instead at 1.25% while maintaining similar pluripotent characteristics. However, we have concerns about adopting a process that uses human serum in a potential human therapy. Do you have any recommendations on supplements that might work to help us reduce the FBS down to a similar level as what we have achieved when using human serum?

The gold standard for feeder-dependent culture of pluripotent stem cells is KnockOutTM SR (serum replacement) containing media and the use of FBS is becoming less and less common. The added advantage of using this system is that in addition to robust culture system of ESC and iPSCs, KnockOutTM SR Xenofree enables culture of cells in […]» Read More

Our lab has isolated mesenchymal stem cells from tissue and we have shown differentiation into adipose, osteo and condro, but we still need to demonstrate their multipotency using phenotypic characterization. Do you have any suggestions on the best way to do this? Thanks

As per the minimal criteria defined by ISCT in 2006, in addition to in vitro differentiation of multipotent mesenchymal stromal cells (MSC) into adipocytes, osteoblasts and chondroblasts, cells must adhere to plastic and show positive expression of CD105, CD73, CD90 and absence of CD45, CD34, CD14, CD19 and HLA-DR {Dominici et al., Cytotherapy (2006) 8, […]» Read More

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

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 […]» Read More

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 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 […]» Read More

My reprogramming experiment is at Day 21 post induction and I am ready to select colonies for clonal expansion, how do I know which to pick?

At the time of selecting colonies to expand, the use of morphology can also be aided by pluripotent markers such as Alkaline Phosphatase Live Stain, which can be used to select robustly, uniformly expressing clones. The dye can be used to transiently identify fully reprogrammed colonies without affecting survival or cell growth once transferred to […]» Read More

My iPSC have demonstrated their self-renewal and differentiation potential, but how do I know they measure up to other established ESCs or iPSCs?

An alternative to looking at marker expression via antibody staining is to look at transcription of genes associated with self-renewal and differentiation potential. The Taqman® hPSC Scorecard™ Assay allows you to determine this through a panel of self-renewal and pluripotency markers. The panel enables qPCR analysis of either undifferentiated or differentiated cells with a single […]» Read More

I am in the process of reprogramming, but am new to stem cell morphology, is there anything I can use to determine how my reprogramming is progressing?

Emerging iPSC colonies do not typically have the same morphologies as established iPSCs. Pluripotent Marker expression can aid in determining pluripotency. Alkaline Phosphatase activity is one of the first markers that is highly expressed during reprogramming. We recommend using the Alkaline Phosphatase Live Stain. This allows you to monitor AP activity via a transient fluorescent […]» Read More