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January 29, 2020

Stemming (pun intended) from the fundamental question in developmental biology around whether cellular differentiation could be reversed – like many transformative scientific discoveries – identification of induced pluripotent stem cells (iPSCs) was a curiosity-induced accident. In their foundational 2006 study, Takahashi and Yamanaka determined that fully differentiated adult skin cells could be reprogrammed back into stem cells by the over-expression of four genes (Oct3/4, Sox2, Klf4, c-Myc).

iPSCs are reprogrammed differentiated somatic cells that exhibit properties similar to embryonic stem cells, including:

  • Proliferation
  • Self-renewal
  • Expression of pluripotency markers
  • Ability to differentiate into cells from all three germ layers
  • Ability to contribute to mouse embryonic development when injected into blastocysts

Why reprogram cells to iPSCs?

Reprogramming somatic cells to iPSCs has transformed the fields of biology and regenerative medicine. The top three application areas for iPSCs include:

  1. Drug discovery/development: Physiological relevance of iPSCs makes them an ideal tool to test the effects of experimental drugs and target validation.
  2. Developmental biology: Cultured iPSCs from an individual can act as mechanistic tools to study normal and disease progression.
  3. Cell/tissue replacement: With the ability to be differentiated into any cell of interest, iPSCs have been instrumental in providing new avenues as replacement/therapy for cells damaged by trauma or compromised by disease. Numerous autologous (patient-specific) cell replacement therapies with iPSCs have been reported in many different clinical trials for indications like spinal cord injury, blindness, cardiovascular and neurodegenerative disease.

Essentials of iPSC reprogramming:  

Reprogramming many different somatic cell types is possible. The two mostiPSC Reprogramming commonly used cell types for reprogramming are human fibroblasts (skin-derived cells) and peripheral blood cells. Reprogramming is achieved by delivery of and subsequent cellular expression of reprogramming factors. These factors can be delivered as viruses, DNA or mRNA. The reprogramming process takes approximately three weeks and is followed by clonal selection, cell expansion, banking and characterization.

Characterization of clonal iPSC lines requires important quality control tests, such as:

  • Assessment of pluripotency status AND differentiation potential (e.g. by flow cytometry and qRT-PCR)
  • Karyotype analysis
  • Cell line identity testing
  • Cell viability assessment
  • Transgene/vector clearance testing
  • Sterility

Creating GMP-ready iPSCs: 

Reprogramming iPSCs remains an open and manual process, carried out using conventional basic research techniques. To advance cell and gene therapy (CGT) and to get products derived from iPSCs to the clinic, generation and maintenance of cells for therapeutics in a Good Manufacturing Practices (GMP) setting becomes paramount.

So, how is this achieved?

A critical aspect of manufacturing GMP-grade iPS cell lines is identification of GMP-grade reagents that will not compromise reprogramming efficiency. All reagents required for reprogramming (e.g. cell culture media, reprogramming factors, passaging reagents, or cell adherence reagents) will need to be tested and optimized for successful cell conversion and growth. Early integration of such GMP-grade reagents will prevent bottlenecks later in development. 

Other important considerations for GMP reprogramming that one might not consider when working in a research environment are sourcing donor samples with the appropriate consent, appropriate testing of the samples to be considered GMP-compliant and a commercial source, and finding out what commercial or therapeutic licenses are needed based on the chosen method for reprogramming.

In conclusion, while iPSC reprogramming has become common, it remains a technique that requires specialized expertise. Reprogramming from a scale-up and manufacturing perspective in a time-efficient and cost-effective manner can be challenging but is achievable.

CCRM has supported numerous academic and industry partners with reprogramming and iPSC scale-up services, as well as process development for GMP reprogramming.

Need help with reprogramming? Ask us how here.

Stay tuned for the second part of this post, which will cover gene editing for iPSCs.

Image credit: Bouquet of Cells by Lalit Antonyan.

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