Options for Identification and Characterization of Pluripotent Stem Cells
Introduction
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.
Question 1
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 signal which is non-destructive in nature. Its use on the master reprogramming plate will not compromise your cells and can be used again and again throughout the process.
See sample data using the Alkaline Phosphatase Live Stain
Question 2
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 assay. It can tell you whether your differentiated iPSC have the hallmark self-renewal gene expression profile and whether your differentiated samples have tri-lineage potential or skew towards particular lineages. In addition, the cloud-based software compares your results against a panel of established reference cell lines.
Question 3
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 new culture dishes.
Question 4
Are there any alternatives to morphological selection that is more specific to fully reprogrammed cells?
The use of antibodies against surface markers of self-renewal is a convenient method to distinguish reprogrammed colonies. Typically the robust expression of SSEA4, Tra-1-60, and Tra-1-81 on the cell membranes is a more specific method of characterizing your cells.
For a complete list of PSC markers, please visit lifetechnologies.com/stemcellantibodies
Question 5
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 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 ifetechnologies.com/stemcellantibodies
Question 6
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 markers, please visit lifetechnologies.com/stemcellantibodies
Question 7
My iPSC clones appear to demonstrate the markers of self-renewal, is this enough to confirm pluripotency?
No, self-renewal is a key measure of pluripotency, but confirmation of differentiation potential is also required. The use of in vitro differentiation via EB formation allows the iPSC to spontaneously differentiate into cells that are characteristic of the three germ layers. These differentiated cultures can be probed with antibodies against SMA, beta 3 Tubulin, and AFP for mesoderm, ectoderm, and endoderm respectively.
For a complete list of PSC and tri-lineage markers, please visit lifetechnologies.com/stemcellantibodies
Question 8
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, 315-317}. Phenotypic characterization can be carried out via flow cytometric analysis of antibody stained cells.
Question 9
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 media containing human derived and human recombinant proteins. This media and has been successfully used for the derivation and expansion of hiPSC {Rodriguez-Piza et al., Stem Cells (2010) 28, 36-44}.
In addition, several feeder-free media systems are also available as xeno-free formulations. For more information on different media options for pluripotent stem cells, click on the link below: http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/ipsc-selection-expansion.html
Question 10
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 markers such as TRA-1-60 and TRA-1-180.
Recently, alternate methods are emerging that are summarized in a recently publication in Nature Protocols {Ben-David & Benvenisty Nature Protocols (2014) 9, 729-740}.
Question 11
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 and function. See an overview of products used for neural cell health at http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/neuroscience.html
Question 12
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)
Question 13
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 a needle) or bulk passaged using a tool like the StemPro® EZPassage™ Disposable Stem Cell Passaging Tool.
Another option for non-enzymatic expansion of PSCs is to use UltraPure™ EDTA to dissociate the cells from the plate. This is only applicable when cells are cultured in a feeder-free media like Essential 8™ medium on either vitronectin or Geltrex® coated TC dishes.
Learn more about Essential 8™ medium
Question 14
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 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.
Question 15
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. There are several culture systems for generation and expansion of pluripotent stem cells.
Use this link to learn more about PSC expansion.
http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/ipsc-selection-expansion.html
Newer methods for efficient differentiation of PSCs into cardiomyocytes are being reported. A recent study by researchers at Nottingham recently reported a new stem cell microenvironment that allows self-renewal of stem cells and their subsequent differentiation into cardiomyocytes. {Dixon et al. (2014) doi/10.1073/pnas.1319685111}
Question 16
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
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