You may see the attached manufacturer\u2019s FAQ for the Mycoalert kit<\/a>, which addresses additional questions about how to prepare samples for this assay.<\/li>\n<\/ul>\n3. Validate Morphology<\/p>\n
\n- General Cell Morphology<\/a><\/li>\n
- Examples of hPSC Morphology:\u00a0Nature (Robinton and Daley)<\/a><\/li>\n
- Phase Images<\/a><\/li>\n
- Pathology Images<\/a><\/li>\n<\/ul>\n
4. Ability to attach and proliferate<\/a> (cloning efficiency) – when passaged or thawed<\/p>\n5. Cryopreservation, including test thaw validation<\/p>\n
\n- Standard DMSO and FBS<\/a><\/li>\n
- Defined Conditions<\/a><\/li>\n
- Protocol with Rock Inhibitor<\/a><\/li>\n<\/ul>\n
6.\u00a0Scale Up and Cell Bank<\/a> – Good cell banking practices<\/p>\n7. Identity – DNA profile<\/a><\/p>\n\n- For iPSCs, validation of match with source cell line.<\/li>\n
- Stem Cell Line Authentication and Contamination Detection<\/a><\/li>\n
- Cell Line Authentication<\/a><\/li>\n<\/ul>\n
8. Assess pluipotency<\/a> and self-renewal\/stemness<\/p>\nThere are a variety of options to demonstrate pluripotency. Some options are listed below:<\/p>\n
\n- Functional Assays for Human Embryonic Stem Cell Pluripotency<\/a><\/li>\n
- Undifferentiated PSC – assess they have PSC and self-renewal properties<\/li>\n
- What Is Stemness?<\/a> – Stemness<\/a> – Stemness-2<\/a><\/li>\n
- Immunocytochemistry (ICC) for hPSC Analysis<\/a><\/li>\n
- Immunocytochemistry and Immunohistochemistry<\/a><\/li>\n
- Differentiate the PSC to assess they can generate three germ layers<\/a>.<\/li>\n
- in vivo<\/em> trilineage assay: Teratoma – In vivo<\/em>\u00a0Pluripotency Sssay<\/a><\/li>\n
- In vitro<\/em> trilineage assay: Embryoid bodies<\/li>\n
- Creation of Embryoid Bodies from iPSCs using Complete KnockOut™ Serum Replacement Feeder-Free Medium<\/a><\/li>\n
- Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency<\/a><\/li>\n
- 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)<\/a><\/li>\n
- Functional Pluripotency Assay – Three Germ\u00a0Layer Direct Differentiation and Staining<\/a> 2D differentiation to three lineages in separate wells. Assess by ICC.<\/li>\n
- Molecular Gene Expression<\/a>\u00a0– To detect linage potency (and bias) in in vitro<\/em> differentiation<\/li>\n
- Taqman Scorecard<\/a>\u00a0quantitative analysis of trilineage differentiation potential – The TaqMan\u00ae hPSC Scorecard™ Panel assesses pluripotency and trilineage differentiation potential using real-time qPCR assays and intuitive data analysis software.<\/li>\n
- Promega<\/a> Monitoring Stem Cell Differentiation and Pluripotency Using the StemElite™ Gene Expression System<\/li>\n
- AMSBiO<\/a> – 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.<\/li>\n
- Illumina TruSeq Targeted RNA Expression Stem Cell Panel<\/a>\u00a0– A targeted RNA sequencing panel for studying various stem cell types.<\/li>\n
- Pluritest Array<\/a> – Comparison of gene expression to novel undifferentiated hPSCs to reference standards<\/li>\n<\/ul>\n
9. Evaluate and monitor Genetic Stability<\/p>\n
\n- Karyotyping, FISH, and cGH done by the UConn Chromosome Core<\/a><\/li>\n
- Karyotype:\u00a0Dr. Judy Brown at UConn<\/a><\/li>\n
- FISH:\u00a0UConn Chromosome Core<\/a><\/li>\n
- cGH:\u00a0UConn CGI Institute<\/a><\/li>\n
- Array-Comparative Genomic Hybridization Characterization of Human Pluripotent Stem Cells<\/a>\u00a0– Also see more references<\/em><\/strong> list below (*)<\/li>\n<\/ul>\n
10.\u00a0Epigenetic<\/a> Stability<\/p>\n\n- Epigenetic Profiling<\/a><\/li>\n
- Methods of Epigenetic Analysis<\/a><\/li>\n
- Techniques for Epigenetic Analysis – Andrea Baccarelli, Harvard<\/a><\/li>\n
- Understanding epigenetic modifications and their impact on gene regulation – Illumina<\/a><\/li>\n
- hPSC epigenetic instability discussion<\/a><\/li>\n<\/ul>\n
11. Monitor and re-test hPSC cultures as needed, including:<\/p>\n
\n- When transferred or received in a new lab<\/li>\n
- Prior and completion experiments<\/li>\n
- For publication<\/li>\n
- Before and after genetic modification –
\nEfficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells<\/a>
\nGene Editing of human PSCs by the UConn Human Genome Editing Core<\/a><\/li>\n- Regular intervals during culture lifespan<\/li>\n
- When frozen\/cryopreserved banks are created<\/li>\n<\/ul>\n
12. More references
\n(*) see these references for more overviews of hPSC characterization:<\/p>\n
\n- Current Methods and Challenges in the Comprehensive Characterization of Human Pluripotent Stem Cells<\/a><\/li>\n
- Cellular Characterization of Human Pluripotent Stem Cells<\/a><\/li>\n
- Characterization of Human Pluripotent Stem Cells<\/a><\/li>\n
- Characterization of Pluripotent Stem Cells<\/a><\/li>\n
- Analysis of Stem Cells<\/a><\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"
Once hiPSC or hESC lines have been generated, what validation is needed?\u00a0How 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 […]<\/p>\n","protected":false},"author":39,"featured_media":0,"parent":10,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"acf":[],"publishpress_future_action":{"enabled":false,"date":"2026-05-13 16:49:02","action":"change-status","newStatus":"draft","terms":[],"taxonomy":""},"_links":{"self":[{"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/pages\/37"}],"collection":[{"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/users\/39"}],"replies":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/comments?post=37"}],"version-history":[{"count":15,"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/pages\/37\/revisions"}],"predecessor-version":[{"id":229,"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/pages\/37\/revisions\/229"}],"up":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/pages\/10"}],"wp:attachment":[{"href":"https:\/\/health.uconn.edu\/stem-cell-core\/wp-json\/wp\/v2\/media?parent=37"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}