Table 2.
Recommendations for iPSC quality control from primary tissue to reprogramming, banking, routine QC, and gene editing.
| # |
Guidance 2. iPSC QC recommendations |
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|---|---|---|---|---|---|
| Assay | Primary tissue | Early reprogrammed clone(s) | iPSC line(s): When generating master or working banks | iPSC line(s) monitoring during routine culture | |
| 2.1 | Cell line identity (STR allele profile recorded) | Required | Required | Required | Every 6–8 weeks or 10–12 passages on lines in culture (competence of users and use of facility should be taken into account here. Novice users and/or heavily used laboratory spaces can increase risk of cell line identity switches/contamination, so routine screening should be implemented on a more frequent basis). When cell lines have been accessed from an external source, always ask for the STR profile to use as a reference point. |
| 2.2 | Genetic stability (such as G banding, SNP, or aCGH) | Preferable | Required | Every 6 weeks or 10 passages if extended culture is required, after any significant selection event such as single cell cloning, or if morphology or growth rate alters in culture | |
| 2.3 | Negative result for bacteria, yeast, and fungi screening using TSB and FTM inoculation | Required | Required | Required | Visual, daily. a more sensitive screen, such as TSB and FTM inoculation, should be performed prior to sharing any cultures to other researchers and when receiving cultures from external sources |
| 2.4 | Negative result for Mycoplasma screening using high-sensitivity method | Required | Required | Required | Every 3–4 weeks or 5–6 passages if extended culture is required. Perform if morphology or growth cycle alters in culture. From a practical perspective, this could be a monthly screen of every in vitro cell line in culture within a single laboratory. This should also be performed prior to sharing any cultures with other researchers and when receiving cultures from external sources |
| 2.5 | Negative result for human viral pathogens (HIV1, HIV2, HBV, and HCV) screening | Required (if not performed on the sample donor) | Only if not done on primary tissue/donor | ||
| 2.6 | Morphology | Required | Required | Required | Visual, ideally daily or whenever cultures are checked |
| 2.7 | Viability and recovery post thaw should be assessed and specific recovery requirements, such as high-density seeding or the temporary use of rho-kinase inhibitors, recorded | Required | Required | ||
| 2.8 | Clearance or silencing of reprogramming vector | Required | Only if not done on earlier clones | ||
| 2.9 | Expression of markers associated with undifferentiated hPSCs, assessed using flow cytometry. Recommended to include both transcriptional regulators (e.g., POU5F1) and surface markers (e.g., SSEA-1, SSEA-4) | Required | Recommended to be performed prior to initiation of differentiation experiments. Perform if morphology or growth cycle alters in culture |
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| 2.10 | Pluripotency: assessment of differentiation potential through in vitro trilineage differentiation and germ layer marker expression | Required (scientific consensus agrees that a functional differentiation assay is the most robust way to assess pluripotency. Solely assessing expression of markers such as POU5F1 and TRA-1-60 does not take into account mutations and or accurately indicate functional pluripotent efficacy; Andrews and Stacey, 2015; Stacey et al., 2019) | Perform on early master stocks, long-term cultures established for experimental purposes, if morphology or growth cycle alters in culture or if issues with established differentiation protocols or similar are observed | ||
| 2.11 | Confirmation of genetic lesion for disease-relevant lines | Preferable | Preferable (mandatory for gene-edited lines, see Guidance 3) | Confirmation of genetic lesion should be included as a routine cell line identity check, if using multiple gene-edited lines from the same donor | |
| Guidance 3. Additional qualification for iPSC lines that have been genetically edited | |||
|---|---|---|---|
| # | Assay | Parental iPSC line(s) | Genetically modified iPSC line(s) |
| 3.1 | Genomic array such as G banding, SNP, or aCGH | Required | Required |
| 3.2 | Sequence of target locus | Required | Required |
| 3.3 | Impact on protein expression where appropriate (e.g., gene knockouts, insertion of reporter or inducible gene expression systems) | Required | |
| 3.4 | Assessment of off-target effects (assess potential off-target effects using database prediction tools and sequence most likely affected loci; e.g., NGS or WGS) | Required | |
| 3.5 | Assessment of OnTEs (e.g., quantitative-genotyping PCR) | Required | |
aCGH, array comparative genomic hybridization FTM, fluid thioglycolate medium; hPSCs, human pluripotent stem cells; NGS, next-generation sequencing; TSB, tryptone soya broth; WGS, whole-genome sequencing.