Figure 4.4. Live cell imaging of iPSCs. iPSC colony cultured on mouse embryonic feeder layer and stained using Alexa Fluor conjugated antibodies for fibroblast marker CD44 and PSC marker TRA-1-60. Imaging was performed after replacing the staining medium with FluoroBrite DMEM imaging medium.
PSC Handbook – Characterization of Pluripotent Stem Cells
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With recent technological advances, iPSCs can now be derived from various somatic cells using different reprogramming methods and can be cultured with different media and matrices. As diverse PSC lines are derived and cultured under different conditions, there is a need for reliable characterization methods to confirm the quality of the PSCs.
Current PSC characterization practices consist of a panel of assays primarily testing functional pluripotency and detecting abnormalities that can affect cell behavior and safety.
Figure 4.1. PSC characterization practices. Characterization is performed to check the functional pluripotency of newly derived PSC lines.
During the derivation of iPSCs and ESCs, characterization is performed to confirm that a pluripotent line has truly been obtained (Figure 4.1). During routine maintenance and after significant manipulations like gene editing, the goal of characterization is to ensure that the fundamental properties of the PSCs have not changed. In this section, basic and commonly used PSC characterization practices are described in the context of reprogramming and the derivation of a new iPSC line. Note that new iPSC lines require karyotyping and often undergo cell banking in addition to the characterization described. Scale up of the culture is necessary in order to generate enough cells for all of these processes. An example scale-up scheme and the allotment of cells is shown in Figure 4.2.
Figure 4.2. Generation of undifferentiated and differentiated cells for karyotyping, cell banking, and characterization. It is recommended that newly derived iPSC lines be scaled up to about seven 6 cm dishes and a T25 flask in order to maintain the culture while karyotyping, performing marker analyses, and creating cell banks. The same approach can be used for PSCs grown on feeders or under feeder-free conditions.
Live staining methods
Undifferentiated PSCs and their differentiated derivatives can be identified through careful observation of cellular morphology. For example, elongated fibroblasts subjected to reprogramming protocols transform into more compact PSCs that have high nucleus-to-cytoplasm ratios and form three-dimensional colonies with well-defined edges when grown on feeders.
Ideally, each of the colonies picked for further culture and analysis contains only fully reprogrammed or bona fi de pluripotent cells. However, in reality, the colonies that emerge include both partially and fully reprogrammed cells that can appear indistinguishable even to the well-trained eye. The visualization of PSC markers increases the likelihood of obtaining a fully reprogrammed iPSC line. These PSC markers can be identified through the detection of PSC-specific enzymatic activity, through live cell immunofluorescence against PSC surface markers or through fixed cell immunocytochemistry using intracellular PSC markers.
Live alkaline phosphatase (AP) staining
AP is an enzyme that is upregulated in PSCs. AP expression can be detected using the Invitrogen Molecular Probes Alkaline Phosphotase Live Stain, which consists of a substrate that selectively fluoresces as a result of AP activity1. This method for differential AP staining is both quick and reversible and helps preserve the viability of the cells AP Live Stain can thus be used to discriminate stem cells from feeder cells or parental cells during reprogramming (Figure 4.3).
Figure 4.3. Detection of AP in live PSCs. Feeder-free PSCs were analyzed using the AP Live Stain (green), with counterstaining performed using an antibody for the PSC marker SSEA4 (red).
Live cell immunostaining
More specific cell staining can be achieved using antibodies against established markers. Surface proteins like the positive PSC markers SSEA4, TRA-1-60, TRA-1-81, and the negative PSC markers CD44 and SSEA1 are particularly useful because they can be stained quickly while keeping cells in culture2,3. Of the positive PSC markers, TRA-1-60 is thought to be the most stringent because it is upregulated later on during reprogramming4. On the other hand, CD44 is found on many differentiated cell types, but is absent from PSCs. The presence of CD44 on fibroblasts and partially reprogrammed cells as opposed to PSCs, increases confidence in picking colonies for expansion during reprogramming, especially when it is combined with a positive PSC marker3 (Figure 4.4).
Both TRA-1-60 and CD44 can be detected using live imaging kits that are designed to maximize the signal-to-noise ratio and allow continued culture of cells through the use of live-qualified Molecular Probes Alexa Fluor dye-conjugated antibodies and optically clear Gibco FluoroBrite DMEM (Figures 4.5 and 4.6). Both are available with three different fluorophores to accommodate commonly used fluorescence filters and can be used for two applications:
- Monitoring the reprogramming process and distinguishing between partially reprogrammed and fully reprogrammed colonies when picking colonies for expansion
- Detection of self-renewal or pluripotency markers for routinely cultured hESCs and hiPSCs
Figure 4.5. Standard media contain phenol red and other sources of background fluorescence.
Figure 4.6. FluoroBrite DMEM included in the live imaging kits exhibits optical clarity similar to PBS and supports cell survival post-staining.
While the staining and imaging approaches described are qualitative, flow cytometry provides a quantitative measure of how many cells are expressing the markers and at what level, revealing any downregulation of the markers or heterogeneity in the population. It is most common to perform flow cytometry using surface markers such SSEA4 and TRA-1-60 (Figure 4.7). Antibodies that can be used for this purpose include a monoclonal SSEA4 antibody conjugated to Alexa Fluor 647 and an unconjugated monoclonal TRA-1-60 antibody used with a secondary antibody like Alexa Fluor 594 Goat Anti-Mouse IgG (H+L) Antibody.
Figure 4.7. Flow cytometry analysis of a feeder-free PSC culture. Isotype controls (gray) are used to determine the percentage of cells expressing TRA-1-60 (green) and SSEA4 (magenta). Dual staining permits the quantification of the number of cells in the culture that are expressing both markers. Typically, >95% SSEA4+/TRA-1-60+ cells are expected. Flow cytometry was performed using the Attune NxT Cytometer with blue/red lasers.
Fixed cell immunostaining
To increase confidence in the quality of an iPSC clone, it is recommended to confirm the expression of not just one or two, but multiple PSC markers. Well-established markers include the transcription factors OCT4 and SOX2, which are known to play key roles in maintaining pluripotency2 (Figure 4.8). Since these are intracellular proteins, staining for these markers requires fixation and permeabilization, which necessitates termination of the culture while a duplicate culture of the clone is maintained. The Invitrogen Molecular Probes PSC Immunocytochemistry. Kits enable optimal image-based analysis of up to four key markers of hPSCs: OCT4, SOX2, SSEA4, and TRA-1-60. These immunocytochemistry kits include a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for optimized staining of fixed PSCs. The antibodies included in the kit have been validated for high performance and multiplexing ability, allowing the specific and simultaneous assessment of two markers at a time.
Figure 4.8. Analysis of intracellular markers in fixed PSCs. iPSCs derived from CD34+ cord blood were grown under feeder-free conditions using Essential 8 Medium in wells coated with vitronectin. The cells were stained for pluripotency markers SOX2 (green) and TRA-1-60 (red) using the PSC4-Marker Immunocytochemistry Kit.
Analyzing iPSCs and confirming the presence of self-renewal gene products or the absence of parental somatic gene products is important, but not sufficient, for verifying the functional pluripotency of a newly derived iPSC line. The other critical test is to confirm trilineage potential or the ability of the iPSCs to differentiate into cells of the three embryonic germ layers: ectoderm, mesoderm, and endoderm. This can be done in vivo through teratoma formation or, more commonly, through embryoid body (EB) formation in culture.
Teratoma formation involves injecting PSCs into mice and allowing them to proliferate and differentiate into the three lineages over 6–30 weeks, depending on the protocol. On the other hand, EB formation involves culturing PSC aggregates in suspension, in the absence of bFGF. These aggregates are allowed to spontaneously differentiate over 7 to 21 days and are typically transferred into adherent cultures after the first few days. Although the differentiation of EBs occurs under non-physiological conditions, EB formation has advantages over the teratoma formation not only because it takes much less time, but also because it is less laborious and EBs are easier to analyze.
Common markers for analyzing differentiation in EBs include smooth muscle action (SMA) for mesoderm, alpha-fetoprotein (AFP) for endoderm, and beta-III tubulin (TUBB3/TUJ1) for ectoderm (Figure 4.9)3.These three markers can be detected using the Invitrogen Molecular Probes 3-Germ Layer Immunocytochemistry Kit , which includes a complete set of high-performance primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment.
Figure 4.9. Cellular analysis of EBs. EBs from H9 ESCs were allowed to spontaneously differentiate for 23 days prior to staining beta-III tubulin (TUJ1, yellow), smooth muscle actin (SMA, red), and alpha-fetoprotein (AFP, green) against a DAPI nuclear counterstain (blue) using the 3-Germ Layer Immunocytochemistry Kit.
Cellular analyses like immunostaining are low-throughput methods that are limited to the detection of markers for which antibodies are available. In contrast, molecular analyses may allow the quantitative analysis of many markers at one time, thereby complementing the cellular data. Such molecular analyses are best done using both undifferentiated and differentiated cells (Figure 4.10). qPCR is currently the most popular method for molecular analysis, and predesigned Applied Biosystems TaqMan® Assays offer ideal tools for rapid screening and analysis of gene expression.
Figure 4.10. Generation of undifferentiated and differentiated PSCs for molecular analysis. When performing molecular analyses, it is best to simultaneously check the purity and quality of the PSCs, as well as confirm the differentiation of the EBs through flow cytometry. Scaling up to about four 6 cm dishes generates sufficient cells for both molecular and flow analyses.
The Applied Biosystems TaqMan® hPSC Scorecard Panel utilizes qRT-PCR, but offers a higher throughput analysis by employing a panel of 93 gene expression assays, including 9 self-renewal genes, 74 germ layer–specific genes, 10 housekeeping genes, and even an assay to confirm clearance of the SeV backbone from iPSCs after reprogramming with the CytoTune-iPS Sendai Reprogramming Kit (Figure 4.11). The assay utilizes bioinformatics analysis based on the work of Bock et al 5 The analysis software facilitates interpretation of the data by statistically comparing the gene expression profile to a reference set of well-characterized ESC and iPSC lines. The software then scores the expression of self-renewal genes and trilineage markers (Figure 4.12).
As such, this permits the analysis of not only undifferentiated PSCs, but also the derivative EBs to determine functional pluripotency6. By providing a more comprehensive and sophisticated analysis of EBs rather than just the confirmation of a few differentiation markers via immunostaining, the TaqMan® hPSC Scorecard assay enables a more reliable and consistent method for quantifying the differentiation potential of PSCs, and makes EB formation an increasingly attractive alternative to time-consuming and laborious teratoma formation assays.
Colors correlate to the fold change in expression of the indicated gene relative to the undifferentiated reference set.
Figure 4.11. TaqMan® hPSC Scorecard analysis of an EB formation time course using H9 ESCs. 93 genes are analyzed as part of the TaqMan® hPSC Scorecard Panel . Colors correlate to the fold change in expression relative to the reference set. Markers of the undifferentiated state are downregulated over the course of EB formation, shown by the blue shading. Markers of the three germ layers are upregulated over the course of EB formation, shown by the red shading.
Figure 4.12. TaqMan® hPSC Scorecard assay results. H9 ESCs and H9 ESC-derived EBs were analyzed using the TaqMan® hPSC Scorecard assay. The comparison of self-renewal and germ layer marker expression against the reference standards is summarized in box plots and in simple pass/fail scores.
- Live AP staining: AP is a differential marker that is expressed more robustly in undifferentiated cells relative to unreprogrammed parental fibroblast cells or fibroblasts in the feeder layer. The washing steps in the staining protocol are critical for observing the differential staining pattern Note that AP is not an ideal method to distinguish between undifferentiated and early differentiating cells.
Live cell and fixed cell immunostaining: Feeder-free cultures need to be handled with caution while staining and washing. Excessive or harsh washes can dislodge the cells from the plate, leading to peeling away of the cells.
- Flow cytometry: It is recommended that cultures be harvested as single cells using TrypLE Express Enzyme prior to antibody staining. Alternatively, live monolayer feeder-free cultures can be stained with antibodies; after images are captured, cells can be harvested using TrypLE Express Enzyme for flow analysis.
- Evaluating differentiation potential: Obtaining the right size of EBs is essential for achieving successful trilineage differentiation. Since feeder-dependent PSCs grow in thick three-dimensional colonies, while feeder-free PSCs grow in flatter colonies, it is important to use the appropriate harvesting procedures that will result in the optimal EB size.
- The TaqMan® hPSC Scorecard Panel measures the potential for self-renewal and trilineage differentiation.
- Since PCR is a global analysis, it is critical to ensure the high quality of the undifferentiated cells and EBs at day 7 of differentiation. If the medium or differentiation method deviates from the proposed method, it is recommended to evaluate a time course with 2 to 3 time points of differentiation (e.g., days 7, 10, and 14).
- MEFs constitute a small percentage (~20%) of feeder-dependent PSC cultures. Although selective harvesting using collagenase reduces MEF carryover, and the gene panel is specific to human genes with minimal cross-reactivity with mouse cells, it is recommended that feeder-dependent cells be cultured for one passage on Geltrex matrix and MEF-conditioned medium to eliminate MEFs prior to analysis If cells are cultured on human feeders, at least two passages are necessary to ensure complete removal of human feeders, which can alter the gene expression signature.
- One well of a 6-well dish or about 0.5 million cells are recommended for the standard RNA extraction method using Invitrogen Ambion TRIzol Reagent . However, as few as 100,000 cells can be used with TRIzol Reagent or as few as 15,000 cells can be used with the Ambion Cells-to-CT Kit or the Ambion CellsDirect One-Step RT-qPCR Kit.
- When reprogramming, it is recommended that stable and homogeneous iPSC clones at passage 8 or higher be used for molecular analysis.
- Early-passage iPSC clones reprogrammed with SeV can be assessed for dilution of the virus by detecting the presence of SeV (the SeV backbone). It is recommended to include parental somatic cells as a control to eliminate SeV cross-reactivity with the parental line. TaqMan® hPSC Scorecard results for the somatic primary cells would show a low self-renewal score, with one or more lineages showing high expression. Results that indicate the presence or absence of SeV in established iPSC clones do not have an impact on pluripotency.
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- Takahashi K, Tanabe K, Ohnuki M, et al (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872.
- Singh U, Quintanilla RH, Grecian S, et al (2012). Novel live alkaline phosphatase substrate for identification of pluripotent stem cells. Stem Cell Rev 8(3):1021–1029.
- International Stem Cell I, Adewumi O, Aflatoonian B, et al (2007). Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25(7):803–816.
- Quintanilla RH, Asprer JS, Vaz C, et al (2014). CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLos One 9(1):e85419.
- Bock C, Kiskinis E, Verstappen G, et al (2011). Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144(3):439–452.
- Fergus J, Quintanilla R, Lakshmipathy U (2014).Characterizing pluripotent stem cells using the TaqMan® hPSC Scorecard Panel. Methods Mol Biol 1307:25–37.
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