Induced Pluripotent Stem Cells (iPSCs)


ipsc reprogramming ipsc flow panel for characterization
ipsc differentiation application of ipscs

The discovery of induced pluripotent stem cells (iPSCs), by Takahashi and Yamanaka in 2006, revolutionized the field of stem cell research. iPSCs can provide an unlimited supply of undifferentiated cells from readily available somatic, or differentiated, cells. Prior to this discovery, the primary source of pluripotent stem cells were embryonic stem cells (ESCs), derived from the blastocyst of an existing embryo. iPSCs have paved the way for an expansion in research and clinical areas of regenerative medicine, cell and gene therapy, and disease pathology.


What is an iPSC?

In their initial experiments, Takahashi and Yamanaka isolated mouse fibroblasts and reprogrammed them into cartilage, neural tissue, and columnar epithelium. More recently, iPSCs have been generated from other somatic cells, including peripheral blood mononuclear cells (PBMCs), keratinocytes, and hematopoietic stem cells (HSCs). Less controversial, and equally pluripotent, somatic cells can be reprogrammed by introduction of four transcription factors Oct4, Sox2, Klf4 and c-Myc, often referred to as “Yamanaka Factors.” The addition of two transcription factors, LIN-28 and Nanog, has been shown to increase reprogramming efficiency. After reprogramming to a “de-differentiated” state, iPSCs can generate virtually any cell type.


Illustrations showing cells that can be reprogrammed to iPSCs, the transcriptions factors associated with reprogramming, and the cell types commonly differentiated into.

What is an iPSC? Somatic cells can be cultured and reprogrammed into iPSCs using canonical pluripotency transcription factors. iPSCs are differentiated into various tissues for basic research and translational studies. *Though c-Myc is an essential transcription factor for iPSC reprogramming, high oncogenic potential makes it a poor factor to integrate for translational applications.



iPSC Reprogramming

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


 Schematic showing there are general methods of reprogramming for iPSC generation: integrative-viral, integrative-non-viral, non-integrative-viral, non-integrative-non-viral.

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 Yamanaka et al., were conducted by integrating four transgenes using retroviral vectors.


Viral

Though they are the most efficient method for reprogramming iPSCs, lenti- and retroviral based transduction methods have distinct drawbacks for clinical and translational applications:

  • Genome integration can cause mutagenesis
  • Disruption of endogenous gene expression
  • Inappropriate reactivation of transgenes after reprogramming is complete
  • Incomplete silencing of reprogramming transcription factors

Some of these challenges can be addressed with newer technologies. For example, instead of using four separate transgenes with four separate integration sites and, therefore, four chances for mutation, researchers have developed polycistronic lentiviruses, which express all four transgenes sequentially with 2A Peptide self-cleavage sites. Additionally, researchers can flank their transgene insert with loxP sites and use transient transfection with Cre-recombinase to excise the integrated transgenes after reprogramming is complete.


Illustration of lentiviral vector insert with polycistronic transcripts for reprogramming of mouse fibroblast cells.

Schematic of polycistronic insert with Yamanaka factors for iPSC reprogramming. (Top) 2A peptide cleavage sites are highlighted. (Bottom) One solution for removing integrated reprogramming genes is flanking loxP sites. Schematic of loxP sites flanking polycistronic insert.


Non-viral

Transposon technology is a next-generation solution to avoid traditional downfalls of virus transductions. Transposable elements, or transposons, are mobile genomic elements that can move around the genome. In the case of genetic editing and iPSC generation, it refers to a plasmid carrying a transposon with all the reprogramming elements.

TcBusterTM is a transposon-based technology developed by Bio-Techne. In addition to reducing mutagenic potential and cargo-size constraints conferred by traditional viral transduction, gene editing with TcBuster is faster and safer for clinical and translational samples. TcBuster technology can also be used to engineer additional genetic changes in iPSCs.

Image of stem cell with DNA inside illustrating the iPSC gene engineering services at Bio-Techne. Leverage our experts to engineer quality, validated iPSCs.

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. However, non-integrative methods are less efficient, and expression of reprogramming factors is more transient. If non-integrative methods are chosen, there are many small molecules available to increase reprogramming success.



Method

Advantages

Challenges

Viral
  • No chance of mutation
  • Broad cellular tropism
  • Large capacity for transgene expression (Adenovirus 8-36 kb, SeV 3.2 kb)
  • Inefficient reprogramming
  • Complete removal of virus from culture
  • Transient transgene expression
Non-Viral
  • mRNA
  • Eipsomes
  • Minicircle DNA
  • Direct protein transfer of recombinant proteins
  • miRNA
  • No chance of mutation
  • Broad cellular tropism
  • Inefficient reprogramming
  • Transient transgene expression


iPSC Characterization

The fastest and simplest way to verify successful generation of iPSCs is by morphology, or cell shape. iPSCs should form a culture of tightly packed cells with large nucleus, large nucleoli, and very little cytoplasm. More empirical verification can be done by staining for markers associated with stem cell signaling. 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. To maintain and expand iPSCs, cells are traditionally cultured and passaged with feeder cells, such as fibroblasts. For clinical and translational applications, Bio-Techne’s ExCellerate iPSC Expansion Medium is animal-free and removes the need for feeder cells and supplemental growth factors.


Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Panel (Cat # FMC001)

Target Antibody

Fluorochrome

Species

Clone

SOX2 PE (+) Human /Mouse 245610
Oct-3/4 APC (+) Human /Mouse 240408
SSEA-4 CFS (+) Human /Mouse 240408
SSEA-1 PerCP (+) Human /Mouse 240408

Isotype Control

Fluorochrome

Species

Clone

Mouse IgG2A PE Human /Mouse 20102
Rat IgG2B APC Human /Mouse 141945
Mouse IgG3 CFS Human /Mouse 133316
Mouse IgM PerCP Human /Mouse Polyclonal


Data image showing iPSC morphology and verification of pluripotency transcription factors and surface markers.

Verification of pluripotency after reprogramming by surface and intracellular flow cytometry. (Left) Morphology- Human iPSC colonies (passage 4) with smooth edges and a non-differentiated phenotype. Magnification: 10x. Scale bar: 80 µm. (Right) Using H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit to verify pluripotency of human iPSC multi-color flow cytometry. The majority of cells were positive for (a,d) SOX-2, (a, b) SSEA-4 and (c) OCT-3/4 and were negative for (b) SSEA-1. Image adapted from Harbuzariu, A., Pitts, S., Cespedes, J.C. et al. Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids. Sci Rep 9, 19162 (2019). https://doi.org/10.1038/s41598-019-55631-8, provided by CC-BY license.



R&D Systems antibody for CCR6 was used to detect the GPCR on mouse splenocytes with flow cytometry.

Verification of pluripotency by Immunocytochemistry/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.

Back to the Top


iPSC Differentiation

One of the most convincing methods to confirm successful reprogramming is to functionally verify pluripotency. To be considered “functionally pluripotent”, iPSCs must be able to differentiate into endoderm, ectoderm, and mesoderm layers. All cell and tissue types develop from one of these layers. Bio-Techne supplies kits to reliably and reproducibly differentiate pluripotent stem cells into cardiomyocytes, neural progenitors, and hepatocytes, in addition to individual growth factors and small molecules.

Immunocytochemistry images showing differentiation of iPSCs into ectoderm, mesoderm, and endoderm layers with antibody staining.

Functional Verification of iPSC Pluripotency. iPS2 human induced pluripotent stem cells were differentiated to ectoderm, mesoderm, and endoderm using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). The kit also contains antibodies targeting Otx2 (ectoderm), Brachyury (mesoderm), and SOX17 (endoderm) for the confirmation of differentiation status. To further evaluate lineage commitment, cells were stained with a Goat Anti-Human SOX1 Antigen Affinity-purified Antibody (Catalog # AF3369), a Goat Anti-Human HAND1 Antigen Affinity-purified Antibody (Catalog # AF3168), and a Goat Anti-Human HNF-3 beta/FoxA2 Antigen Affinity-purified Antibody (Catalog # AF2400). The cells were stained using the NorthernLights™557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue).


Explore Solutions for iPSC Reprogramming, Differentiation, and Characterization


Back to the Top


Applications of iPSCs

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.


Schematic diagram showing varied applications for 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.



Select applications:

Drug toxicity screening: new therapies and drugs often fail in human trials due to unforeseen toxicity. To limit this, iPSCs can efficiently be differentiated into organoids to screen target drugs for toxicity. Hepatocytes, neurons, and cardiomyocytes are the three most common tissue for drug toxicity screening.

Tissue

Select Markers of Differentiation

Illustration of three hepatocytes.
Illustration of one neuron.
Illustration of cardiomyocyte.

Disease modeling: iPSCs allow for direct studying of disease state from the patient’s own cells. Additionally, researchers can directly alter patient cells via gene editing and determine the impact for the individual patient.

Regenerative Medicine: With the ability to generate disease-free tissues using off-the-shelf or patient-derived cells, iPSCs have a bright future in regenerative medicine.


Back to the Top



Limitations of iPSCs

During somatic cell reprogramming to iPSCs, there are many genetic and epigenetic changes that occur. With current reprogramming technologies, the epigenetic landscape of the somatic cells is often incompletely or aberrantly modified. This phenomenon, known as epigenetic memory, often biases the iPSCs to differentiate toward their cell of origin.


Back to the Top


Select References

Raab S, Klingenstein M, Liebau S, Linta L. A Comparative View on Human Somatic Cell Sources for iPSC Generation. Stem Cells Int 2014;2014:. https://doi.org/10.1155/2014/768391.


S K, J B, JE C. Induced Pluripotent Stem Cells in Disease Modeling and Gene Identification. Methods Mol Biol 2018;1706:17–38. https://doi.org/10.1007/978-1-4939-7471-9_2.


Scesa G, Adami R, Bottai D. iPSC Preparation and Epigenetic Memory: Does the Tissue Origin Matter? Cells 2021;10:. https://doi.org/10.3390/CELLS10061470.


Wang L, Su Y, Huang C, Yin Y, Chu A, Knupp A, et al. NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities. Biol Open 2019;8:. https://doi.org/10.1242/BIO.047225.


Singh VK, Kalsan M, Kumar N, Saini A, Chandra R. Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol 2015;3:. https://doi.org/10.3389/FCELL.2015.00002.


Foltz LP, Clegg DO. Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells. J Vis Exp 2017;2017:56274. https://doi.org/10.3791/56274.


Sakai Y, Kiyotani K, Fukumura M, Asakawa M, Kato A, Shioda T, et al. Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication. FEBS Lett 1999;456:221–6. https://doi.org/10.1016/S0014-5793(99)00960-6.


Klingenstein S, Klingenstein M, Kleger A, Liebau S. From Hair to iPSCs—A Guide on How to Reprogram Keratinocytes and Why. Curr Protoc Stem Cell Biol 2020;55:e121. https://doi.org/10.1002/CPSC.121.

Abbar A Al, Ngai SC, Nograles N, Alhaji SY, Abdullah S. Induced Pluripotent Stem Cells: Reprogramming Platforms and Applications in Cell Replacement Therapy. Biores Open Access 2020;9:121. https://doi.org/10.1089/BIORES.2019.0046.

Goyak KMO, Laurenzana EM, Omiecinski CJ. Hepatocyte Differentiation. Methods Mol Biol 2010;640:115. https://doi.org/10.1007/978-1-60761-688-7_6.

Rajala K, Pekkanen-Mattila M, Aalto-Setälä K. Cardiac Differentiation of Pluripotent Stem Cells. Stem Cells Int 2011;2011:. https://doi.org/10.4061/2011/383709.