A simplified step-by-step process for the generation of induced pluripotent stem cells (iPSCs) using the Yamanaka factors, which are a set of transcription factors, named after Shinya Yamanaka, the scientist who discovered their ability to transform somatic cells into a pluripotent state. The factors are Oct3/4, Sox2, Klf4, and c-Myc.
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Remember that this process should be carried out in a lab setting, following all safety and ethical guidelines, and it requires a deep understanding of cell biology and genetic engineering techniques.
Materials Needed:
- Somatic cells (such as fibroblasts)
- Viral vectors or other means of gene delivery (like lentivirus, Sendai virus, plasmids, or electroporation)
- Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) cloned into the chosen vectors
- Stem cell culture medium (e.g., DMEM supplemented with fetal bovine serum (FBS), nonessential amino acids, GlutaMAX, and β-mercaptoethanol)
- Pluripotency test (such as alkaline phosphatase staining, immunostaining for pluripotency markers, or teratoma formation assay)
Procedure:
1. Culture of Somatic Cells: Isolate and culture the somatic cells according to standard tissue culture protocols until they reach the desired confluence, usually about 70-90%.
2. Transduction/Transfection of Yamanaka Factors: Introduce the Yamanaka factors into the somatic cells. This is typically done by transfection or transduction using a viral vector such as a lentivirus or Sendai virus, or through non-viral methods like electroporation.
3. Incubation and Expression of Yamanaka Factors: After successful transfection/transduction, incubate the cells to allow for the expression of the Yamanaka factors. This is often for a period of 24 to 48 hours.
4. Change of Medium: After a suitable incubation period, change the medium to a stem cell culture medium.
5. Monitoring of Cell Colonies: Monitor the cells daily, refreshing the culture medium as needed. Within 2-3 weeks, colonies resembling pluripotent stem cells should begin to appear.
6. Picking and Expansion of Colonies: Once sufficiently large, pick the colonies that resemble pluripotent stem cells and transfer them to new culture plates for expansion.
7. Verification of Pluripotency: The colonies need to be verified as pluripotent stem cells through a variety of assays, such as alkaline phosphatase staining, immunostaining for pluripotency markers (like NANOG, SSEA-4), or a teratoma formation assay.