Once hiPSC or hESC lines have been generated, what validation is needed? How should quality control be established and maintained?
1. Establish hPSC line – Generate hiPSC or hESC
2. For all cell cultures, implement a regular mycoplasma testing schedule.
- See Bionique Laboratories FAQ about mycoplasma contamination in cell cultures, and why screening is important.
- Why does over use of antibiotics result in higher mycoplasma contamination rates?
- Abstract: Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture.
- The UConn Stem Cell Core does mycoplasma testing as a service for UConn Health and UConn labs. We use the Myco-Alert kit from Lonza.
To submit samples:
- Please fill out the mycoplasma testing submission form.
- Write the name of the samples clearly on your tube(s). Your sample names written on the form should match what is written on your tube(s).
- Submit the order through the UConn Stem Cell Core CORES System.
- When you are ready to submit your samples, contact us to set up details for drop off.
Details about sample preparation:
- Two mls of cell culture supernatant should be submitted to the stem cell core. Cell culture supernatant for this assay can be stored 2°C-8°C for 1 week or -20°C for up to 2 months.
- Please submit culture medium from cells that have been grown without antibiotics such as Pen-Strep for at least 1 week.
- You may see the attached manufacturer’s FAQ for the Mycoalert kit, which addresses additional questions about how to prepare samples for this assay.
3. Validate Morphology
- General Cell Morphology
- Examples of hPSC Morphology: Nature (Robinton and Daley)
- Phase Images
- Pathology Images
4. Ability to attach and proliferate (cloning efficiency) – when passaged or thawed
5. Cryopreservation, including test thaw validation
6. Scale Up and Cell Bank – Good cell banking practices
7. Identity – DNA profile
- For iPSCs, validation of match with source cell line.
- Stem Cell Line Authentication and Contamination Detection
- Cell Line Authentication
8. Assess pluipotency and self-renewal/stemness
There are a variety of options to demonstrate pluripotency. Some options are listed below:
- Functional Assays for Human Embryonic Stem Cell Pluripotency
- Undifferentiated PSC – assess they have PSC and self-renewal properties
- What Is Stemness? – Stemness – Stemness-2
- Immunocytochemistry (ICC) for hPSC Analysis
- Immunocytochemistry and Immunohistochemistry
- Differentiate the PSC to assess they can generate three germ layers.
- in vivo trilineage assay: Teratoma – In vivo Pluripotency Sssay
- In vitro trilineage assay: Embryoid bodies
- Creation of Embryoid Bodies from iPSCs using Complete KnockOut™ Serum Replacement Feeder-Free Medium
- Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency
- Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency (The Harvard community has made this article openly available)
- Functional Pluripotency Assay – Three Germ Layer Direct Differentiation and Staining 2D differentiation to three lineages in separate wells. Assess by ICC.
- Molecular Gene Expression – To detect linage potency (and bias) in in vitro differentiation
- Taqman Scorecard quantitative analysis of trilineage differentiation potential – The TaqMan® hPSC Scorecard™ Panel assesses pluripotency and trilineage differentiation potential using real-time qPCR assays and intuitive data analysis software.
- Promega Monitoring Stem Cell Differentiation and Pluripotency Using the StemElite™ Gene Expression System
- AMSBiO – GlobalStem’s PluriPCR™ Kit is designed as a quantitative, easy to use, and reliable assay of five genes strongly specific to pluripotency. These genes: Oct-3/4, Nanog, DNMT3b, Dppa4, and Rex1 are expressed by human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC); all are sharply down-regulated during differentiation.
- Illumina TruSeq Targeted RNA Expression Stem Cell Panel – A targeted RNA sequencing panel for studying various stem cell types.
- Pluritest Array – Comparison of gene expression to novel undifferentiated hPSCs to reference standards
9. Evaluate and monitor Genetic Stability
- Karyotyping, FISH, and cGH done by the UConn Chromosome Core
- Karyotype: Dr. Judy Brown at UConn
- FISH: UConn Chromosome Core
- cGH: UConn CGI Institute
- Array-Comparative Genomic Hybridization Characterization of Human Pluripotent Stem Cells – Also see more references list below (*)
10. Epigenetic Stability
- Epigenetic Profiling
- Methods of Epigenetic Analysis
- Techniques for Epigenetic Analysis – Andrea Baccarelli, Harvard
- Understanding epigenetic modifications and their impact on gene regulation – Illumina
- hPSC epigenetic instability discussion
11. Monitor and re-test hPSC cultures as needed, including:
- When transferred or received in a new lab
- Prior and completion experiments
- For publication
- Before and after genetic modification –
Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells
Gene Editing of human PSCs by the UConn Human Genome Editing Core
- Regular intervals during culture lifespan
- When frozen/cryopreserved banks are created
12. More references
(*) see these references for more overviews of hPSC characterization: