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Induced Pluripotent Stem Cells (iPSC)

Pluripotent stem cells (PSC) have the differentiating capacity to serve as the starting point for wide-ranging research into tissue development, drug discovery and toxicology, disease progression, and regenerative medicine.

For cell therapies, induced pluripotent stem cell (iPSC) banks derived from “universal” donors offer the promise of less costly, more rapidly available, and more tightly controlled allogeneic therapies.

At Bio-Techne, our mission is to deliver innovative solutions that enable cell and gene therapies to reach more patients.

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stem cell research

About Pluripotent Stem Cells and Cell Therapy

Regenerative medicine and immune cell therapy offer revolutionary promise for the treatment of degenerative diseases, pathogenic genetic defects, tissue damage, and cancer. To produce these cell therapies, somatic cells are recovered from the patient (for autologous therapies) or from immune-compatible donors (for allogeneic therapies) followed by reprogramming to an undifferentiated state by using small molecules. The resulting iPSCs have the capacity to differentiate into multiple cell lineages of the endoderm, mesoderm, and ectoderm germ layers.

An effective cell therapy product requires long term viability and functionality, the ability to home to the correct target tissue, and the ability to evade host immune rejection. Cell and gene therapies that are based on live cells are classified as advanced medicine therapeutic products (ATMPs) which require extensive quality control testing because of their inherent variability.

Listen as Tenielle Ludwig, Director of the WiCell Stem Cell Bank, defines correct use of the terms stemness and pluripotency. These terms are often used interchangeably by researchers and within scientific publications. Dr. Ludwig discusses how consistent use of this nomenclature can positively impact the field of pluripotent stem cell research.

Stemness vs. Pluripotency – Definitions and Methods to Verify

iPSC Workflow

Use the icons to navigate to products and services for each step of the iPSC workflow below:

Somatic Cell Isolation and Selection

iPSC Isolation icon

Terminally differentiated somatic cells can be easily recovered from a variety of tissues (e.g. skin fibroblasts or peripheral T cells) and used for generating iPSC.

Somatic Cell Reprogramming to iPSC

iPSC Reprogram icon

iPSC Reprogramming

There are four general groups to categorize an iPSC reprogramming strategy:

  • Integrative Reprogramming techniques require the reprogramming factors to be inserted permanently into the host cell genome. This strategy can be further divided into viral and non-viral methods. The pioneering iPSC experiments by Takahashi & Yamanaka (2006) were conducted by integrating four transgenes using Retroviral Vectors.
  • Viral reprogramming  cells to iPSCs, using lentiviral or retroviral-based transduction methods are the most efficient but have distinct drawbacks for clinical and translational applications
  • Non-viral transposons technology such as TcBuster™ is a next-generation solution that avoids some of the pitfalls of virus transductions.
  • Non-integrative Reprogramming techniques are the preferred methodology for clinical and translational iPSC generation. They require no genomic integration, and therefore have significantly reduced chance of introducing harmful mutations. Small molecules are widely used in non-integrative reprogramming.
Diagram of iPSC Reprogramming Groups

After reprogramming cells to iPSC, confirm their phenotype by the detection of appropriate stemness markers. See Cell Characterization below on this page.  Cell Banking Reagents Including Cryopreservation Media, CEPT Cocktail Kit (Catalog # 7991), and ROCK Inhibitors.

graphic of somatic cell reprogramming to iPSC

Verification of stemness marker expression by multi-color flow cytometry. Human cells were stained using reagents included in the Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit and simultaneously analyzed for SSEA-1, SSEA-4, Oct-3/4, and SOX2. The strong expression of SSEA-4 and Oct-3/4 but not SSEA-1 indicate the undifferentiated status of these cells.

iPSC Expansion and Culture

images of stemness marker expression on iPSC
graphs of stemness marker expression on iPSC

Human iPSC cultured in ExCellerate™ iPSC Expansion Medium maintain the expression of stemness markers over long-term culture. These cells express undifferentiated stem cell markers Oct-3/4 (red) and TRA-1-60 (red) along with F-Actin (green) and DAPI (blue) (A). iPSC lines express high levels of Oct-3/4, SSEA-4, SOX2, and no SSEA-1 as assessed by the H/M Pluripotent Stem Cell Multicolor Flow Cytometry Kit (B-C). Undifferentiated stem cell marker expression is >97% across 4 cell lines after more than 45 passages. Graph shows average ± standard deviation.

Gene Engineering for iPSC

iPSC Gene Engineering icon

Engineer cells to mask them against host immune rejection, improve tissue homing and engraftment, and introduce new functionalities like chimeric antigen receptors (CARs).

image of TcBuster-transposed gene in iPSC by DNAscope

Direct visualization of TcBuster-transposed iPSC using the DNAscope™ Assay. Detection was based on a DNAscope probe targeting the TcBuster vector backbone. Wild type iPSC (A), mixed population of TcBuster-transduced iPSC (B), selected clone isolated from the mixed population (C).

Pluripotent Stem Cell Differentiation

iPSC Differentiation icon

Simplify batch bridging during extended cell differentiation processes with reagents strictly qualified for lot-to-lot consistency.

Neuronal Differentiation

immunocytochemistry of neuronal differentiation from iPSC

Immunocytochemistry of neurons differentiated from iPSC by using the StemXVivo Neural Progenitor Differentiation Kit. Neurons are indicated by Neuron-specific beta-III Tubulin (TUJ1) expression (A), neural progenitors by Pax6 (B), and undifferentiated iPSCs by Oct-3/4 (C). Quantification of images at day 10 and 32 of neuronal differentiation grown on Vitronectin and in ExCellerate iPSC Expansion Medium.

Cardiomyocyte Differentiation

immunocytochemistry of cardiomyocytes differentiated from iPSC

Immunocytochemistry of iPSC differentiated by using the StemXVivo Cardiomyocyte Differentiation Kit. Cells were maintained in ExCellerate iPSC Expansion Medium and differentiated to cardiomyocytes as indicated by Human Troponin T (Cardiac) expression (cTNT1).

Hepatocyte Differentiation

immunocytochemistry of hepatocytes differentiated from iPSC

Immunocytochemistry of iPSC differentiated by using the StemXVivo Hepatocyte Differentiation Kit. Cells were maintained in ExCellerate iPSC Expansion Medium and differentiated to hepatocytes as indicated by Albumin and HNF-4 alpha expression.

Serum-Free and Animal-Free Cell Culture

Increase the consistency of your cell cultures as you approach translational studies for regenerative medicine and cell therapy programs. Adopting these media will

  • Reduce variability in media composition
  • Simplify compliance with regulatory guidelines
  • Simplify comparability testing for raw material changes

Cell Characterization

iPSC Characterization icon

Cell product qualification is important from start to finish with iPSC programs. Keep a close eye on cell phenotype, secretory profile, culture heterogeneity, and the presence of contaminating particles. After reprogramming, iPSCs should express endogenous pluripotency factors, similar to ESCs. These include both transcription factors, such as Oct4, Sox2, and Nanog, as well as surface markers, like SSEA-4 and TRA-1-60. Interestingly, SSEA-1 is expressed on the surface of mouse, but not human, iPSCs. Learn more about Stem Cell Marker products.

Pluripotent Stem Cell Characterization Kits and Antibodies

Analytical Instrument Platforms and Immunoassays

Verification of pluripotency by mmunocytochemistry/ immunofluorescence. (Left) Confocal immunofluorescence analysis of Mouse Anti-Human Nanog Antibody (1E6C4) (Catalog # NBP1-47427) (green). Actin filaments have been labeled with DY-554 phalloidin (red). Nanog staining was confined to the nucleus. (Right) ADLF1 induced pluripotent stem cell line stained with Mouse Anti-Human TRA-1-60 (TRA-1-60) (Catalog # NB100-730) and Anti-Mouse IgG Secondary Antibody (red) and counterstained with DAPI (blue). TRA-1-60 staining was confined to the cell surface.

iPSC verification image showing cell markers
Single-Cell Western data showing neuron differentiation from iPSC

Single-Cell Western analysis of neuronal differentiation from iPSC. Each dot represents a single cell. iPSC were treated with GMP SB 431542 and GMP Recombinant Human Noggin, followed by terminal differentiation with GMP Recombinant Human FGF, GMP N-2 MAX Media Supplement (100X), and ascorbic acid. Cells were analyzed for Pax6 and Neuron-specific beta-III Tubulin (Tuj). In iPSC, Pax6 was undetectable and 46% of the cells expressed Tuj, while 85% of neurons were Tuj+ Pax6+. See our application note for more details.

iPSC Applications

Disease research often relies on the use of animal models or two-dimensional (2D) in vitro culture systems. Though extremely useful, animal models are limited in their ability to recapitulate complex diseases and accurately model human cellular responses to new drugs and therapies. Traditional in vitro culture systems rely on examining cellular responses in a contrived 2D environment, with cells grown either in a monolayer plastic dish or in suspension surrounded by culture media. Advancements in cell culture techniques to include organoid and 3D cultures that more closely recapitulate in vivo tissue microenvironment, exponentially expand the applications for iPSCs.

Illustration highlighting the application of iPSCs

Diverse applications of iPSCs. Somatic cells are harvested from patient and reprogrammed into iPSCs. The resulting patient specific iPSCs can then be used in disease modeling and drug screening to generate disease and patient-specific therapies. Additionally, patient-specific iPSCs can be modified to repair genetic mutations. These repaired iPSCs can then be transplanted into the patient to restore tissue functionality.

Resources

Custom Solutions

We’re committed to providing optimized solutions to optimize your iPSC workflow. Our custom services team will work with you to deliver reagents and immunoassays that fit your process. Importantly, we have experience developing certified animal-free (AF) reagents as custom projects in cases where AF grade is not otherwise available. We’re experts in the requirements for regulatory compliance as well as custom formulation, vialing, and packaging.

Translational Programs for Cell Therapy

When it’s time to advance your cell therapy product to clinical manufacturing, partner with us for reliable, quality, and/or custom services. We will work with you to provide reproducible production of reagents and assays at clinical scale, with complete documentation. We offer GMP reagents as well as 21 CFR Part 11-compliant analytical instruments for automation and high throughput. We can help you streamline the manufacture of your cell therapies.

Background Information

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