Banking, Characterization & Quality Control
Banking
From all hiPSC lines derived by the Core and from gene edited subclones cryopreserved cell banks are produced. These banks are robustly characterized and quality controlled.
Furthermore the production of quality controlled cell banks (e.g. from lines derived by external parties) can be requested as a service.
Characterization & QC
At CUSCO we have established all characterization and QC methods that are standard in the field. By default all hPSC banks are analyzed regarding their genomic integrity, identity, sterility, morphology and viability (Basic CQC panel). Depending on the individual needs, we offer additional characterization and QC analysis (Optional CQC analysis).
| Basic CQC panel |
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| Virtual Karyotyping using a SNP Array |
| G-Banding Karyotyping |
| Cell Line Identity |
| Sterility and Mycoplasma testing |
| Morphology and Viability |
| Optional CQC analysis |
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| Marker for the undifferentiated state of hPSC (FACS or IF) |
| Differentiation capacity of hPSC into cells of the three germ layers |
| Testing for human pathogens (HIV I, HIV II, HBV, HCV) |
| Confirmation of disease genotype / gene editing |
Genomic Stability
Genomic integrity of hiPSC lines is assessed by karyotyping using G-banding and copy number variation (CNV) analysis using single nucleotide polymorphism (SNP) microarray analysis.
SNP array based CNV analysis
To identify submicroscopic (<5Mb) genomic abnormalities, so called copy number variations (CNV) data from SNP arrays can be used. Therefore, genomic DNA from hiPSC are run on Illumina Infinium Global Screening Array-24 BeadChips. Data is subsequently analysed for CNVa. While molecular karyotyping detects genomic variations including duplications, deletions, unbalanced translocations and aneuploidies as well as copy neutral loss of heterozygosity and >20% mosaicism, it cannot identify balanced translocations, insertions or inversions. Therefore we combine this analysis with G-Banding krayotyping.
G-banding Karyotyping
For G-banding, cells in exponential growth phase are arrested in metaphase using colcemid, harvested, fixed and stained using Giemsa. Per each cell line, 20 metaphase spreads are analysed.
G-banded karyotyping can detect microscopic genomic abnormalities >5-10Mb including inversions, duplications, deletions, balanced and unbalanced translocations as well as aneuploidies. Mosaicism >10% can also be discovered.
Cell Line Identity
To authenticate a hPSC line, i.e. to assure that a newly generated line matches the original donor sample or that an engineered hPSC line is isogenic to its parental line, STR analysis or SNP array data can be used. We recommend STR fingerprinting as it is the standard method for cell line authentication.
Short Tandem Repeat (STR) Analysis
For STR analysis a multiplex PCR from genomic DNA generates a molecular fingerprint of alleles at various genomic loci. As the STR pattern is unique to each human individual, it can be used to identify cell lines from different sources or passages as long as a reference STR profile (parental line or donor sample) is available for comparison.
Sterility and Mycoplasma testing
To check for potential contaminations (bacterial or fungal) in hPSC cultures, cells are maintained in antibiotic free medium and monitored daily for morphological changes or other conspicuous features such as colour change in medium, particle formation or movement. In addition, the presence of Mycoplasma spp., a common contaminant in cell cultures, is evaluated via testing of the used culture medium for the presence of mycoplasma DNA via qPCR analysis.
what it detects: 96 species of mycoplasma contamination from stem cell cultures. Sensitivity (5-100 CFU/ml)
what it does not detect: this system does not allow the amplification of DNA originating from other sources, such as bacteria.
Morphology and Viability
hPSC show a typical morphology in cell culture which can be used as a marker for their undifferentiated state, culture quality and hPSC identity. To test morphology and viability, a sample vial from a cell bank is thawed and taken into cell culture. Cell recovery and morphology is recorded for five days microscopically.
Markers for undifferentiated state of hPSC
Undifferentiated human PSC typically express pluripotency markers including the transcription factors octamer-binding transcription factor 4 (OCT4) and Homeobox protein NANOG as well as the cell surface markers stage specific embryonic antigen-4 (SSEA-4) and tumor rejection antigen 1-60 (TRA-1-60). Expression of these markers can be analysed both via immunofluorescence staining microscopy and via flow cytometry (FACS).
Marker for undiff hiPSC
Differentiation capacity of hPSC into cells of the three germ layers
Directed differentiation
For the determination of the capacity of hPSCs to differentiate into representative cell types of the three germ layers - ectoderm, mesoderm and endoderm - specific differentiation protocols are being applied. Lineage-specific morphological changes are monitored and differentiation efficiencies are assessed by flow cytometry analysis via germ layer specific marker expression.
Ectoderm: The neural transcription factor PAX6 is specific for ectoderm differentiation. In addition, SOX2 promotes differentiation of hPSC into neural ectoderm while inhibiting differentiation into mesoderm.
Endoderm: The transcription factor SOX17 plays an important role in the determination of endoderm formation. Additionally, expression of the cell surface marker CXCR4 is characteristic of endoderm cell populations.
Mesoderm: The cell surface markers CD144 (VE-Cadherin) and CD140b are specific for cells differentiated along the mesodermal lineage.
