Induced pluripotent stem cells (iPSCs) are valuable tools for disease modeling, drug discovery, and cell therapy. As new iPSC lines are generated through somatic reprogramming, a battery of assays are employed to confirm that they exhibit the hallmark characteristics of pluripotent stem cells (PSCs), including PSC marker expression and the ability to generate cells from the three embryonic germ layers. Asprer and coworkers recently published a review of a broad set of molecular and cellular methods for the comprehensive characterization of human PSCs . Here, we distill the most essential steps in the cellular analyses that are performed on newly reprogrammed iPSCs.
During the process of reprogramming, the emergence of colonies is initially monitored based on morphological changes and the appearance of embryonic stem cell (ESC)–like colonies. For example, elongated fibroblasts transform into more compact PSCs that have high nucleus-to-cytoplasm ratios. In feeder-dependent systems, these cells form three-dimensional colonies with well-defined edges. By days 21 to 28 after the initiation of reprogramming, the colonies are usually large enough to be picked and transferred to new culture dishes.
Visualize PSC markers in live cells
The emerging colonies can consist of partially or fully reprogrammed cells that sometimes appear indistinguishable, even to the well-trained eye. The visualization of PSC markers increases the likelihood of obtaining a fully reprogrammed iPSC line. However, the markers must be detected without compromising the viability and pluripotency of the colonies, which will be expanded to establish new PSC lines. This marker detection can be achieved through live alkaline phosphatase staining and live-cell immunostaining.
Live alkaline phosphatase (AP) staining. AP is an enzyme that is up-regulated in PSCs and can be detected using a substrate that selectively fluoresces as a result of AP activity . This differential staining method for AP activity is quick and reversible, and it preserves the viability of the cells. Thus, it can be used to discriminate stem cells from feeder cells or parental cells during reprogramming (Figure 1).
Live-cell immunostaining. More specific cell staining can be achieved using antibodies for established markers. Surface proteins like the positive PSC markers, SSEA4, TRA‑1‑60, and TRA‑1‑81, and the negative PSC markers, CD44 and SSEA1, are particularly useful because they can be stained quickly while keeping cells in culture [3,4]. Of the positive PSC markers, TRA‑1‑60 is thought to be most stringent because it is up-regulated later in reprogramming . In contrast, the negative PSC marker CD44 is found on many differentiated cell types as well as partially reprogrammed cells but is absent from PSCs. Confirming the absence of CD44 expression increases confidence in picking colonies for expansion, especially when it is combined with a positive PSC marker  (Figure 2).
Figure 1. Reversible alkaline phosphatase staining of live human pluripotent stem cells (hPSCs). Invitrogen™ Alkaline Phosphatase (AP) Live Stain (green) robustly stains a hPSC colony (left panel). The fluorescent signal is lost from the cells by 90 min after removal of the dye from the medium (right panel).
Figure 2. Live-cell immunostaining of human pluripotent stem cells (hPSCs). Live feeder-dependent hPSCs were stained with Invitrogen™ Alexa Fluor™ 555 anti–TRA-1-60 antibody, supplied in the TRA-1-60 Alexa Fluor™ 555 Conjugate Kit and Invitrogen™ Alexa Fluor™ 488 anti-CD44 antibody, supplied in the CD44 Alexa Fluor™ 488 Conjugate Kit (middle panel); the right panel shows the merged image. Images were acquired using the Invitrogen™ EVOS™ FL Imaging System.
Visualize PSC markers in fixed cells
When emerging iPSC colonies are still being picked, two to three markers may be analyzed at once. This is sufficient for a quick screen, but once the cells have been expanded, more markers need to be analyzed to increase confidence in the identity and quality of an iPSC clone. Indeed, many PSC markers are intracellular proteins that can only be stained when cultures are fixed and permeabilized; thus, there must be enough cells for duplicate cultures before these markers can be used. OCT4 and SOX2 are two such well-established intracellular PSC markers (Figure 3); both are transcription factors known to play key roles in maintaining pluripotency .
Figure 3. Fixed-cell immunostaining of human pluripotent stem cells (hPSCs). Feeder-dependent hPSCs were fixed and stained with primary and fluorescent secondary antibodies from the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit. The anti-OCT4 (red, left panel) and anti-SOX2 (green, middle panel) antibodies were used to stain nuclei of hPSCs. DAPI (blue) served as a nuclear counterstain and can be seen in the merged image (right panel).
Evaluate differentiation potential
Analyzing iPSCs and confirming the presence of self-renewal markers or the absence of original somatic markers is important but not sufficient for verifying the functional pluripotency of a newly derived iPSC line. It is critical to also confirm the iPSCs’ ability to differentiate into the three germ lineages: ectoderm, mesoderm, and endoderm.
The most physiological method for testing this in human iPSCs is to perform teratoma formation, which is labor-intensive, takes around 6 to12 weeks to complete, and is associated with a high animal-testing burden. The most common alternative to teratoma formation is embryoid body (EB) formation, an in vitro assay involving the spontaneous differentiation of PSCs into the three germ lineages over 7 to 21 days. Although differentiation occurs under nonphysiological conditions, EB formation has the advantage of being shorter, less laborious, and easier to analyze. Common markers for analyzing differentiation in EBs include β-III tubulin (TUJ1) for ectoderm, smooth muscle actin (SMA) for mesoderm, and α-fetoprotein (AFP) for endoderm (Figure 4). Even more markers can be quantitatively and simultaneously analyzed using the Applied Biosystems™ TaqMan® hPSC Scorecard™ Assay, which is described in "An Improved qPCR-Based ScoreCard Assay".
Figure 4. Immunostaining of embryoid bodies (EBs). Day 21 EBs were stained using primary and fluorescent secondary antibodies provided in the 3-Germ Layer Immunocytochemistry Kit. The markers shown are (A) TUJ1 (yellow), (B) SMA (red), and (C) AFP (green), which are merged in (D). DAPI (blue) served as a nuclear counterstain.
Newly derived iPSC lines are initially characterized through marker analysis, both in the undifferentiated and differentiated states. Live AP staining and live-cell immunostaining facilitates the selection of colonies for expansion, while additional fixed-cell staining allows the investigation of many more PSC or germ-layer markers. Marker choice, antibody specificity, and reagent quality are critical considerations for the successful characterization of new iPSC lines. Thermo Fisher Scientific offers cell analysis tools for each of these characterization steps, including the Alkaline Phosphatase Live Stain, PSC Immunocytochemistry Kits, and 3-Germ Layer Immunocytochemistry Kit.
- Asprer JS, Lakshmipathy U (2015) Current methods and challenges in the comprehensive characterization of human pluripotent stem cells. Stem Cell Rev 11:357–372.
- Singh U, Quintanilla RH, Grecian S et al. (2012) Novel live alkaline phosphatase substrate for identification of pluripotent stem cells. Stem Cell Rev 8:1021–1029.
- Adewumi O, Aflatoonian B, Ahrlund-Richter L et al. (2007) Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25:803–816.
- 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:e85419.
- Chan EM, Ratanasirintrawoot S, Park IH et al. (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol 27:1033–1037.
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