hPSC Characterization & QC Assays Available in the PSCF

Characterizing iPSC lines using a standard series of quality control metrics is critical before investing resources in project-specific iPSC analyses. The PSCF has implemented all the standard assays used for basic iPSC quality assessment.

Morphology

Cultured undifferentiated hPSCs have a stereotypical morphology including formation of characteristic colonies with defined borders containing tightly packed cells exhibiting a high nuclear:cytoplasmic ratio and prominent nucleoli.

View cells cultured in feeder-free culture conditions for 15 passages.

 

Typical hESC-like morphology of an iPSC line derived from human fibroblasts using non-integrating episomal plasmids. These cells have been cultured in feeder-free culture conditions for 15 passages. Note the identical colony and cellular morphology compared to H1 hESCs cultured under these conditions.

Mycoplasma detection

Several mycoplasma species are commonly found in cultured mammalian cells, including hPSC cultures. Unlike other microbial contamination of cell cultures, mycoplasma contamination is visually undetectable. However, mycoplasma can profoundly impact the biology of the cultured cells. Consequently, frequent testing of cultured cells to demonstrate that they are mycoplasma-free is required. The PSCF routinely performs mycoplasma detection on all new cell lines cultured in the lab, as well as at the completion of all iPSC generation projects, and whenever a cryopreserved bank of cells is prepared. Mycoplasma testing is available as a service in the PSCF.

Short Tandem Repeat (STR) analysis

Cultured cell lines in the laboratory are susceptible to mislabeling, cross contamination and other mistakes that can result in misidentified cell lines being used in experiments. Consequently, authentication of the identity of cultured mammalian cells is an essential part of the cell culture workflow to ensure valid and reproducible results. Several methods exist for validating cell line identity. The PSCF utilizes STR analysis to assess 16 polymorphic regions of the human genome to obtain a DNA profile that can be compared to a known sample for identity validation.

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STR profile of iPSC 91_11 cells, generated by reprogramming patient-derived fibroblasts by episomal plasmid mediated Yamanaka factor expression. This STR profile was identical to the STR profile obtained from the donor fibroblasts, authenticating the identity of the iPSC line.

Sterility analysis

To rule out latent microbial contamination of cultured cells, sterility analysis should be performed. This is particularly important to do when preparing cryopreserved banks of cultured cells for future use. The PSCF offers assessment of sterility by analysis of microbial growth in trypticase soy broth (TSB) as a service.

Thaw viability analysis

When preparing cryopreserved banks of important cells, it is advisable to test thaw a vial to ensure successful recovery from cryopreservation. The PSCF offers assessment of thaw viability of cryopreserved hPSCs as a service. NB: hPSCs must be cultured in the mTeSR1/Matrigel system.

Stemness Marker Expression

Undifferentiated hPSCs have a characteristic protein expression profile. Many methods exist for assessing the presence of these markers. The PSCF uses flow cytometry to detect the presence of the cell surface markers Tra_1_60 and SSEA4.

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An iPSC line derived from PBMCs using Sendai virus vectors was assessed for expression of stemness Tra_1_60 and SSEA4 by flow cytometry. >90% of iPSC285_1 cells in the analyzed culture were double positive for both markers.

Tri-lineage differentiation (functional pluripotency)

Differentiation into each of the three embryonic germ layers is a defining property of hPSCs. The PSCF uses the Scorecard assay to assess functional pluripotency. hPSCs are differentiated into embryoid bodies (EBs). Total RNA from undifferentiated cells and EBs is assessed for expression of a range of stemness and germ layer-specific markers and compared to a reference set of validated pluripotent hPSCs.

Learn more about functional pluripotency.

 

Functional pluripotency of H1 hESCs was determined by the Taqman hPSC Scorecard assay (ThermoFisher). Total RNA was harvested from undifferentiated H1s and from H1-cells differentiated in embryoid bodies (EBs) for 14 days. RNA was reverse transcribed to cDNA which was subjected to Scorecard assay to quantitatively assess trilineage differentiation potential by real-time qPCR analysis of markers of undifferentiated cells and of each embryonic germ layer (endoderm, ectoderm and mesoderm). (A) Scorecard results confirmed trilineage differentiation potential of H1s. In the undifferentiated H1s, the expression levels of the self-renewal markers match or exceed that of an undifferentiated reference set derived from 13 hPSC lines. For the EB differentiated H1s, the marker expression profile for each germ layer has a high probability of matching or exceeding that of day five EB samples derived from the 13 reference lines. (B) Fold change between undifferentiated and EB differentiated samples for each gene analyzed in the Scorecard assay.

Karyotype Analysis

Like most cultured mammalian cells, hPSCs are subject to acquisition of karyotypic abnormalities during in-vitro culture. Multiple recurrent chromosomal abnormalities are selected for in hPSCs that elicit unpredictable behaviors including prevention of robust directed differentiation. Consequently, it is critical to ensure that cultured hPSCs exhibit a normal karyotype. The PSCF uses the Cincinnati Children's cytogenetics core for standard metaphase spreads and G-banded karyotype analysis.

Learn more about b-banded karyotype analysis of PBMC-iPSCs demonstrating normal karyotype.

G-banded karyotype analysis of PBMC-iPSCs demonstrating normal karyotype.

Directed Differentiation

We have successfully performed directed differentiation of iPSC lines into several cell types including intestine, neuroepithelia and cardiomyocytes.

Genomic iPSC Characterization

Additional iPSC characterization metrics focusing on genomic analyses (e.g. RNA-seq, micro-RNA, methylation analysis, DNA fingerprinting, copy number variation) are available through the DNA Sequencing and Genotyping Core.

Preparing master banks and working banks of human iPSCs

A well-characterized bank of the most important iPSC clones for your research is essential to ensure a consistent supply of high-quality cells for study. The PSCF recommends the establishment of two banks for priority cell lines to ensure this supply. A smaller, well-characterized small-scale master bank can be stored separately from, and utilized for, the supply of a working bank, which is typically a larger bank for more frequent access. In this system, the master bank should be accessed only for the generation of additional working banks. Once an investigator receives the clones from an iPSC generation the PSCF recommends the following steps:

1.  Differentiate all clones to the cell type of interest to identify those clone(s) that most robustly generate these cells using standard protocol(s). Typically, the identified clone(s) should have the highest priority for banking, quality-control and experimentation.

2.  Prepare a master bank of 8-10 vials for each identified clone.

3.  For each clone, pool the material before aliquoting into cryovials to ensure preparation of a homogeneous master bank.

4.  When you set up cultures to cryopreserve the master bank, also set up enough wells to perform the following quality control assays (available as a service from the PSCF)

a. STR (identity testing)
b. Sterility
c. Karyotype
d. Thaw viability

5.  Prepare a working bank for clone(s) with the highest priority, accessing the master bank to do so. The scale of the working bank should be determined by the needs of each research group. Factors to consider include experimental plans, the number of investigators who will use the cells, the available storage space, etc. For a clone that will only be used by one individual a working bank of 25-30 vials will typically last for several years. The goal should be to balance the need for new vials of high-quality, well-characterized cells with the cost of replacing and quality-controlling each new working bank, while also minimizing the need to frequently access the master bank.

6.  For each clone, pool the material before aliquoting into cryovials to ensure preparation of a homogeneous working bank.

7.  When you set up cultures to cryopreserve the working bank, also set up enough wells to perform the following quality control assays (available as a service from the PSCF):

a. STR (identity testing)
b. Karyotype
c. Sterility
d. Thaw viability
e. Stemness marker expression
f. Functional pluripotency

8.  After thawing from the working bank, the PSCF recommends that human PSCs are not maintained in culture for >20 additional passages. After 20 passages thaw a new vial from the working bank.

9.  To avoid catastrophic loss, split bank storage into more than one location. We recommend using the Cincinnati Children's Biobank; at minimum separate freezers in different physical locations.