Introduction:
Induced pluripotent stem cells (iPSCs) are stem cells generated form individual somatic cells by exogenous expression of several transcirption factors to initiate the reprogramming process. (Itoh, “Generation of 3D skin equivalents fully reconstituted form human induced pluripotent stem cells (iPSCs) PLOS ONE, October 2013, 8(10))
Somatic cells can be reprogrammed by transferring their nuclear contents into oocytes or by fusion with ES cells, indicating that unfertilized eggs and ES cells contain factors that can confer totipotency or pluripotency to somatic cells.
In 2006, Keyoto University’s Kazutoshi Takahashi, PhD and Shinya Yamanaka, MD, PhD, reported that they had used defined factors to reprogram differentiated cells to an embryonic like state. This work led to induced pluripotent stem cells (iPSCs) that made it much easier to conduct stem cell research.
The “induced” part of iPSC comes from knowing the cell’s history: One starts with a differentiated somatic cell (for example, a fibroblast), reprogram it—classically using factors such as OCT4, SOX2, KLF4 and c-MYC—and then demonstrate that the resulting cells have acquired pluripotent characteristics.
Characterization of iPSCs:
iPSCs are obtained form adult donor somatic cells. The cells are reprogrammed to pluripotency using a combination of genetic elements delivered into the cells by viral or other means. IPSCs proliferate indefinetly and maintain the potential to differentiate to nearly any functional cell type in the body. Thus, the cells can be gene edited, expanded and characterized to create master cell banks that can be used for every prouct batch. Because of those attributes, IPSCs have become one of the preferred starting materials for cell theary development.
One generally dooes rely on a single test to establish that reprogrammed cells are iPSCs. Researchers characterize them using several complementary indicators of pluripotency and identity.
Pluripotency markers. The cells express characteristic factors such as OCT4 (POU5F1), SOX2, NANOG, and surface markers such as TRA-1-60, TRA-1-81, SSEA-3/SSEA-4. These can be assessed by immunostaining, flow cytometry, RT-qPCR, etc.
Cell/colony morphology. Properly cultured human iPSCs typically form compact colonies with cells having relatively large nuclei and little cytoplasm. This is useful but isn’t sufficient by itself.
Ability to differentiate. A major functional demonstration is showing that the cells can generate derivatives representing all three germ layers—ectoderm, mesoderm, and endoderm.
Patient-Derived iPSC Disease Modeling: See also Organoids
Introduction: In recent years, patient-specific iPSCs ahve been derived from patietns with several human diseases to investigate unkown disease mechanisms and perform pre-clinical testing in varous models. Autologous PS-iPSCs have the potential to provdie an unlimited source of cells for gene and cell therapies for specific human diseases, since they are beleived to ahve unlimited proliferative capacity and extensive differentation capability into a wide range of cell types. (Itoh, “Generation of 3D skin equivalents fully reconstituted form human induced pluripotent stem cells (iPSCs) PLOS ONE, October 2013, 8(10))
Commercially Available Disease Models:
Bio-hive Innovations (offer iPSC-derived skin organoids for R&D and say they can customize them for genetic and inflammatory disease modeling and compound efficacy analysis. Their organoids can contain epidermis, dermis, hair follicles, neurons and adipocytes.)
Protheragen (advertises custom skin-organoid development services. They describe using patient-derived or skin-specific iPSCs and developing customized 3D skin disease models for drug screening)
Richoh (offer broader iPSC disease-modeling services. Ricoh says it can develop disease-specific differentiated cell models and even develop new differentiation protocols when the desired cell type isn’t already available.)
Mount Sinaie Skin biology (offers human skin disease modeling, generation and characterization of patient-derived iPSCs, CRISPR editing, and differentiation of pluripotent stem cells into keratinocyte-like cells.)
Patient-dervied iPSC disease modeling is an in vitro disease-modeling approach in which patient-derived somatic cells are reprogrammed into induced pluripotent stem cells and differentiated into disease-relevant cell types that retain the patient’s genetic background.
Cell Sources for iPSCs:
For human iPSC work, skin fibroblasts and peripheral blood/PBMCs as two of the most established starting materials.
Adipose-derived stromal/stem cells — obtained from adipose tissue.
Dermal fibroblasts — one of the classic and most extensively used sources. Usually obtained by skin biopsy.
Keratinocytes — commonly obtained from skin or hair follicles and can reprogram quite efficiently.
Dental pulp cells — obtained from extracted teeth and used particularly in dental/regenerative research.
PBMCs are a mixture that includes T cells, B cells, NK cells, monocytes, and some progenitor populations. Particular nucleated cells within that population can be reprogrammed to pluripotency.
Typically, the cells are exposed to reprogramming factors such as OCT4, SOX2, KLF4, and c-MYC (or related combinations). After successful reprogramming and selection/expansion, colonies with iPSC characteristics are established and characterized for pluripotency.
Ali, “Keratinocytes dervied form patient-specific induced pluripotent stem cells recapitulate the genetic signature of psoriasis diease” Stem cells and Devleopment, 29(7), 2020) discloses generation of human iPSCs from PBMCs isolated form blood samples suing Ficoll-Paque Premium (Sigma-Aldrich). The PBMCs were cultured in StemPro-34 SFV Complete Medium (Bigco) for at elase 4 days before reprogramming. The cells were transduced with the CytoTune-iPS 2.0 Sendai reprogramming kit (Thermo Fisher Scietntific). At day 3 after reprogramming, the cells were seeding on Matrigel-coated plates and cultured in StemPro medium without cytokines. At day 7, half of the medium was replaced with ReproTeSR medium (Stem Cell Technologies) and the medium was compeltely changed to ReproTeSR at day 8. The iPSCs colonies generated bwteen days 15-30 were manullay picked, expanded and maintained in mTESR-1 medium.
Urinary epithelial cells — attractive because urine collection is noninvasive.
Inducement Factors:
Introduction: An iPSC is pluripotent, meaning it can potentially differentiate into cell types belonging to all three embryonic germ layers: (1) Ectoderm: neurons, keratinocytes, retinal cells, etc. (2) Mesoderm: cardiomyocytes, skeletal muscle, bone, blood-cell lineages, etc. and (3) Endoderm: hepatocytes, pancreatic cells, intestinal epithelial cells, etc.
During differentiation, signals such as WNT, BMP, FGF, TGF-β/Activin/Nodal, and SHH activate or inhibit particular transcription factors. Those transcription factors then establish lineage-specific gene-expression programs.
Neural differentiation involves transcription factors such as SOX1, PAX6 and NEUROG2 at different stages.
Cardiac differentiation involves factors including NKX2-5, GATA4 and TBX5.
Hepatic differentiation involves factors such as HNF4α and FOXA2.
There is also an epigenetic component—DNA methylation, chromatin accessibility, histone modifications, etc.—that stabilizes those changes in gene expression.
Several transcription factors, including Oct3/4 and Nanog function in the maintenance of pluripotency in both early embryos and ES cells. Several genes that are frequently upregulated in tumors, such as Stat3, E-Ras and beta-catenin have been shown to contribute to the long-term maintenance of the ES cell phenotype and the rapid proliferation of ES cells in culture. c-Myc and Klf4 are essential factors in maintaining plripotency (Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell 126, 663-676, August 25, 2006).
c-Myc protein: has many downstream targets that enhance prolfieration and transformation, many of which may have roles in the generation of iPS cells. Of note, it associates with histone acetyltrasnferase (HAT) complexes, including TRRAP, which is a core subunit of the TIP60 and GCN5 HAT complexes and p300. Within the mamalian genome there may be up to 25,000 c-Myc binding sites and many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. (Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell 126, 663-676, August 25, 2006).
Differentiation of iPSCs into Particular Cell Types:
Fibroblasts: Fibroblasts, the second predominant cellular componetn of the skin have been differentiation from iPSCs. (Itoh, “Generation of 3D skin equivalents fully reconstituted form human induced pluripotent stem cells (iPSCs) PLOS ONE, October 2013, 8(10))
Keratinocytes: have been differentiated form iPSCs. (Itoh, “Generation of 3D skin equivalents fully reconstituted form human induced pluripotent stem cells (iPSCs) PLOS ONE, October 2013, 8(10))
Neurons: Scientists at Genentech are using iPSC derived NGN2 induced neurons (or iNeurons) are differentiated form human iPSCs with transcription factor Neurogenin-w (NGN2) to rapidly become neurons.