Analysis of Stem Cells



Characterization of stem cell cultures falls into two main categories: first, monitoring the genomic integrity of the cells and second, tracking the expression of proteins associated with pluripotency. Genomic analysis is necessary to ensure stem cells maintained in culture for long periods of time have not become unstable through chromosomal loss or duplication, or changes in their epigenetic profiles. Proteomic analysis ensures that the cells are expressing the factors necessary to maintain pluripotency. Finally, analysis of the differentiated state by analyzing key genetic and protein markers will ensure identification and propagation of the proper cell type This section provides an overview of different analysis methods for stem cells such as karyotyping, single nucleotide polymorphism (SNP) analysis, epigenetic profiling, flow cytometry and immunocytochemistry, RT-PCR, RT-qPCR, western blotting, and teratoma formation.

Related Topics: Isolation and Maintenance of Stem Cells, Differentiation of Stem Cells, and Transfection of Stem Cells.

Stem Cell Characterization

A variety of factors affect stem cell cultures

Medium composition
Feeder type/density
Growth factors/additives
Feeder free culture
Passage method
Freezing Method

Possible changes
Molecular Signature
Genetic stability
Epigenic stability
Tumorigenic potential

Three main genomic techniques are used for stem cell characterization: karyotyping, SNP analysis, and epigenetic profiling. Healthy stem cells also express cell surface and cytoplasmic proteins associated with pluripotency. Flow cytometry, immunocytochemistry, and western blot analysis are techniques commonly used for confirming pluripotency-related protein expression.

Analyzing gene expression, epigenetic changes, and protein expression profiles are crucial during stem cell differentiation and during the creation of induced pluripotent stem cells (iPSCs). A stem cell that has been differentiated into a motor neuron, for example, will have a fundamentally different profile than its progenitor cell, which is also committed to differentiate into a neuron, but is less differentiated. Similarly, somatic cells being induced into pluripotency must be screened for expression of stem cell markers.

The following section provides an overview of techniques important for stem cell research. Detailed protocols for stem cell analysis are provided on the WiCell Research Institute and National Stem Cell Bank website.


Karyotyping is the examination of chromosome number and appearance in a cell. Exposure of stem cell populations to stressful circumstances, such as subculturing or removal of feeder cells, can lead to chromosomal abnormalities and significant changes in cellular functions (Inzunza et al. 2004). Thus, it is crucial to monitor a culture for the accumulation of chromosomal abnormalities. It is recommended that a stem cell line be karyotyped every 10–15 passages to ensure chromosomal duplication, deletions, or translocations have not occurred. Traditional karyotyping uses a dye (for example, giemsa or quinacrine) to stain the chromosomes of a metaphase cell in distinct banding patterns (G-banded karyotyping). These banding patterns are assessed to identify any abnormalities. Recently, spectral karyotyping (SKY) has become popular. SKY is a hybridization-based combinatorial technique that uses fluorescently-labeled DNA probes specific for each chromosome. This method can detect translocations within and between chromosomes more accurately than traditional karyotyping (Liehr et al. 2004).

Normal karotype of the human iPSC clone. Image courtesy of Dr Miguel Esteban.

SNP Analysis

Single nucleotide polymorphisms, or SNPs, are the most common type of genetic variation (Brookes 1999). A SNP is a single base pair mutation within a region of DNA. SNPs are heritable and can be used for tracing lineage. SNPs may accumulate in a stem cell population over time and can lead to phenotypic changes that influence survival or growth. These genetic changes may result in unwanted outcomes such as loss of pluripotency or gained tumorigenicity. SNP genotyping with high-density oligonucleotide arrays (Gunderson et al. 2005) can specifically identify a stem cell line's origin and monitor its genomic integrity. SNPs can be identified using PCR, microarrays, or DNA sequencing.

Epigenetic Profiling

A stem cell's pluripotency is dictated to a large extent by its epigenetic profile (Bibikova et al. 2006). DNA methylation and histone modification regulate the accessibility of DNA to the transcriptional machinery and thus regulate gene expression (Jaenisch and Bird 2003).

Analysis of DNA methylation patterns is performed by treating sheared DNA with bisulfite under controlled conditions such that all cytosines are converted to uracils while leaving methyl cytosines unchanged. After the conversion, the DNA can be analyzed for global methylation patterns using either a chip array or DNA sequencing. Chromatin immunoprecipitation (ChIP) analyzes patterns of histone modifications by cross-linking the DNA to histones. The cross-linked chromatin is then sheared and purified using antibodies against a specific protein or histone modification. Analysis of the cross-linked regions can be performed using qPCR, microarrays, or DNA sequencing. New stem cell lines can be initially characterized by examining the epigenetic state of a few key genes such as H19, Xist, Oct4, Notch 1, Dlk1/MEG3, and PWS/AS (Loring et al. 2007).

Quantitative assessment of chromatin structure in cultured cells can also be performed using specific epigentics tools such as the EpiQ™ chromatin analysis kit from Bio-Rad. In EpiQ kit, the chromatin is digested in the presence or absence of nuclease and then the genomic DNA is purified and quantified. Chromatin structure is assessed via real-time qPCR by comparing results against an epigenetically silenced (reference) gene. Using these kits, it is possible to discriminate open, actively transcribed chromatin regions from closed, transcriptionally silent regions. This helps in quantifying the impact of epigenetic events, such as DNA methylation and histone modification, on gene expression regulation through chromatin state changes.

DNA methylation profile of the Oct4 and Nanog proximal promoters in the indicated cell types. Image courtesy of Dr Miguel Esteban.

Flow Cytometry and Immunocytochemistry

Flow cytometry with cell sorting is a method for sorting a heterogeneous population of cells based on their light scattering characteristics (Watson 2004). Fluorescently tagged monoclonal antibodies are used to distinguish a specific cell population. Stem cells express a number of cell surface proteins that are considered to be markers of pluripotency and many antibodies are available for their detection (Adewumi et al. 2007). Glycolipid antigens SSEA-3 and SSEA-4 and the keratin sulfate antigens TRA-1-60 and TRA-1-81 are commonly used antigens for identifying and sorting live stem cell populations. Fixed stem cells can also be sorted based on the expression of the transcription factors Oct-3/4 and NANOG. As stem cells differentiate, the loss of these markers and expression of new markers can be used to track the progress and overall condition of a stem cell population. For instance, Nestin is a protein marker for neural progenitor cells, BNP (brain natriuretic peptide) is a marker for cardiomyocytes, and CD31, CD34, and CD45 are markers for hematopoietic cell types (see Deb et al. 2008, and references therein).

Immunocytochemistry analyses protein expression in fixed cells without disturbing the cell culture as a whole. Similar to flow cytometry, cells are stained with fluorescently labeled antibodies targeting specific proteins. Immunohistochemistry enables the visualization of an individual cell within the context of a stem cell colony and the evaluation of the subcellular localization of the molecular markers (Myers 2006).

iPSC clone generated from the human fibroblast cell line IMR90 using medium containing vitamin C (Vc). Phase contrast photographs and immunofluorescence microscopy for the human ESC surfact markers TRA1-60, TRA-1-81, SSEA-3, SSEA-4, and the transcription factor Nanaog. Nuclei are shown in blue. Image courtesy of Dr Miguel Esteban.


Reverse-transcription-quantitative PCR (RT-qPCR) provides a rapid, sensitive, and quantitative method for monitoring the gene expression profile of a cell population. (For a comprehensive review of real-time PCR see Real-Time PCR section). RT- qPCR first requires isolation of the stem cell RNA. The RNA is then converted into cDNA using the enzyme reverse transcriptase (RT). The cDNA for a gene of interest is amplified by the addition of gene-specific DNA primers and using PCR, enabling the detection and quantitation of the levels of gene expression in a given cell population. Stem cells express a number of unique genes that are used as markers for pluripotency. These genes are mainly transcription factors that are involved in creating and maintaining the undifferentiated state. Loss of expression of these genes correlates with a loss of pluripotency. Some of the best known transcription factors expressed in stem cells are Oct-3/4, NANOG, FOX2D, SOX2, and UTF-1 (Bhattacharya et al. 2004). Other important genes expressed in stem cells are LIN28, a regulator of translation and microRNA production (Büssing et al 2008) and human telomerase reverse transcriptase (hTERT) (Ju and Rudolph 2006).

RT-qPCR is also a valuable tool for evaluating the state of differentiated cells. For example, glial fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP-2) are markers for neural progenitor cells (Gage 2000). RT-qPCR is such a widely used technique for analysis of stem cells as well as differentiated cells that primers have been designed and validated for nearly every gene of interest. The qPrimer Depot ( and the RTPrimerDB ( are excellent resources for finding validated primers for nearly every human gene.

Real-time PCR analysis of endogenous OCT4, SOX2, and NANOG and markers for the three germ layers in EBs from two periosteum cell lines. Image courtesy of Dr Miguel Esteban.

Western Blotting

Western blotting is an analytical technique for detecting a specific protein in a complex sample. The proteins are separated using 1-D gel electrophoresis and then transferred to a membrane (nitrocellulose or PVDF). Antibodies specific to the protein of interest are then used to probe the membrane. Detection is through a two-step process which utilizes a reporter system consisting of an enzyme linked to an antibody that interacts with a colorimetric substrate to produce a band on the membrane. For stem cell scientists studying the mechanism of differentiation, western blotting is especially useful in determining the success of transfection experiments. When a gene is either introduced into the cell or knocked down using RNAi, detecting and quantitating its protein level through a western blot procedure coupled with measuring the transcript level by RT-qPCR is a common way to assess transfection efficiency. More importantly, western blots can be used to study the effects of transfection on downstream protein expression during differentiation or culture maintenance (Rubinson et al. 2003).

Western blot for p21

Western blot for p21 shows reduced senescence in MEFs transduced with SKO factors and treated with vitamin C (Vc) compared to control. Images courtesy of Dr Miguel Esteban.

Teratoma Formation

Teratoma formation is another way of analyzing stem cells in vivo. Teratomas are tumor-like formations containing tissues and organs derived from all three germ layers. Because of this unique composition containing differentiated and undifferentiated cells from all germ layers they are often used as indicators of stemness when analyzing the pluripotecy of stem cells. Stem cells are introduced into immunocompromised mice, which do not have a functionally active immune system. Therefore, stem cells are not considered as foreign and are not rejected, but will retain the ability to grow and differentiate into multiple cell types.

Teratomas composed of tissues derived from the three germ layers

Teratomas composed of tissues derived from the three germ layers produced after injection of the same cell line into immunosuppressed mice. Image courtesy of Dr Miguel Esteban.


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