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

• Standard DMSO and FBS
• Defined Conditions
• Protocol with Rock Inhibitor

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:

• Current Methods and Challenges in the Comprehensive Characterization of Human Pluripotent Stem Cells
• Cellular Characterization of Human Pluripotent Stem Cells
• Characterization of Human Pluripotent Stem Cells
• Characterization of Pluripotent Stem Cells
• Analysis of Stem Cells