peyt

Induced Pluripotent Stem Cells (iPSCs)

Accurate cell counting for differentiation success in regenerative medicine

Induced pluripotent stem cells (iPSCs) are a form of pluripotent stem cell derived from somatic cells. Developed by Shinya Yamanaka’s lab in 2006 (mouse iPSCs)1 and 2007 (human iPSCs)2, this type of cell has revolutionized the work with pluripotent stem cells, as they allow investigators to experiment on stem cells of different genetic disease backgrounds. In addition, before the introduction of iPSCs, stem cell research was done on embryonic stem cells (ESCs) derived from human embryonic tissue. Using ESCs holds ethical concerns about the derivation and culture of the cells, and therefore is illegal or restricted in many countries. The introduction of iPSCs overcame most of these ethical concerns of working with embryonic cells. As a result, iPSCs have been embraced by scientists worldwide.

Generation, culture & differentiation of iPSCs

Somatic skin cells can be reprogrammed into iPSCs and differentiated into different cell types.
New lines of iPSCs are continuously generated by overexpression of the four ‘Yamanaka factors’, i.e. Oct3/4, Sox2, Klf4, c-Myc in somatic cells, which turns them into iPSCs. This allows research into the differentiation potential when presented with different genotypic backgrounds. Investigation of the relationship between the genetic origin and phenotypical presentation provides insight into disease advancement and potential drug treatment.

Traditionally, iPSCs are cultured on feeder cells with bovine serum-containing medium in a 2D setting. In an effort to create a controlled stem cell niche, culture settings have moved towards continuously restricted conditions, where all components are serum-free, or even xeno-free, and with various plastic coatings instead of cells to attach to in the cell dish. A restricted environment improves maintenance conditions for the stem cell niche and encourages a successfully directed differentiation towards the desired progenitor or mature cell type(s): There is better control of advantageous cell-to-cell signaling by the growth factors and inhibitors, which cause the type of directed differentiation you are interested in.

Recent developments have seen stem cell culturing shift from traditional 2D methods to more physiologically relevant 3D methods such as spheroid and organoid culture, allowing for proper cell-to-cell signaling and interactions during cell growth and differentiation. These 3D structures have great promise in the fields of drug discovery and regenerative medicine as they allow researchers to better simulate how their treatments affect tissues in a physiologically relevant growth setting (i.e. the stem cell niche), rather than in isolated layers of cells only.

Most predominantly are the intestinal organoids developed by Toshiro Sato and Hans Clevers3 which have been used for drug screening enabling better treatment options for cystic fibrosis patients4. Brain organoids were introduced initially by Madeline Lancaster5, where iPSCs are differentiated into different cell types and areas of the brain to allow for complex disease modeling of microcephaly and drug testing, for example.

What’s the challenge?

Apart from culture media, surface coatings and pluripotency level of the cells you work with, the cell density is the fourth major contributor to experimental success. Intercellular signaling is difficult to control and manipulate but seeding cells at the right concentration can contribute vastly to getting the directed differentiation of interest. For instance, differentiation protocols towards mesoderm and endoderm cell types e.g. cardiac cells, have shown that low cell densities positively affect the percentages of cells of interest6,7. As an added benefit, low cell densities mean lower cytokine concentrations to obtain the same effect, in the end, saving costs at the tissue culture step.

Conversely, neural differentiation protocols often require high cell densities, as cell-to-cell signaling is indispensable and cell density at protocol onset determines the ratio of, for example, central nervous system and neural crest progeny8,9.

Maintenance cultures of iPSCs are usually done by passaging dense cultures as medium-sized aggregates (clumps) of cells instead of dissociating them to single cells before re-plating. This ensures that the iPSC niche is not disrupted too much during passage.

A female scientist is passaging cells in a LAF cabinet using a serological pipette.

Solutions for iPSC work

When initiating differentiation protocols on iPSC cultures, counting cells for seeding is tedious and somewhat inaccurate: It is time-consuming and can be difficult to fully dissociate cells from the maintenance culture, and relying on an aggregate count is inaccurate, as aggregate sizes vary and therefore the total cell number is variable and an estimate at best. With the NucleoCounter® NC-202™, you can determine a direct aggregate dissociation at the time of counting using the Aggregated Cell Count Assay and obtain an accurate cell count and viability percentage in two minutes.

The NC-202™ does not rely on full dissociation to single cells, since it counts the nuclei of live and dead cells based on fluorescent stains. Thus, the instrument can better discern individual cells in small clumps, as there is less overlap between the nuclei of cells in a clump, compared to automated cell counters based on bright-field imaging.

By using the Via2-Cassette™, you eliminate user-to-user bias in counting and avoid handling cell staining reagents, as they are embedded in the cassette. Individually volume-calibrated, the Via2-Cassette™ supports the robustness of the NC-202™, ensuring low instrument variation for higher differentiation success. Furthermore, the instrument is 21 CFR Part 11-ready for integration into protocols requiring GMP compliance.

Our Field Application Scientists (FAS) resolve questions on instrument use for all our customers. The FAS also visit labs and conduct online instrument demonstrations and are available to discuss your iPSC counting methods. At installation, the FAS trains staff in using the instrument and runs performance quality tests to ensure a successful installation. With a Service Plan, you can extend the product warranty and we will carry out regular service visits to validate instrument performance and train staff members.

Documents

TitleCategoriesLink

References

  1. K Takahashi and S Yamanaka: Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006; 126, 663–676.
  2. K Takahashi, K Tanabe, M Ohnuki et al.: Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007; 131, 861–872.
  3. T Sato and H Clevers: Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science. 2013;340(6137):1190-4.
  4. SF Boj, AM Vonk, M Statia et al.: Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients. J Vis Exp. 2017;(120):55159
  5. MA Lancaster, M Renner, C-A Martin et al.: Cerebral organoids model human brain development and microcephaly. Nature. 2013;501(7467):373-9.
  6. MNT Le, M Takahi, K Maruyama et al.: Cardiac differentiation at an initial low density of human-induced pluripotent stem cells. In Vitro Cell Dev Biol Anim. 2018;54(7):513-522.
  7. H Ninomiya, K Mizuno, R Terada et al.: Improved efficiency of definitive endoderm induction from human induced pluripotent stem cells in feeder and serum-free culture system. In Vitro Cell Dev Biol Anim. 2015. 51(1):1-8.
  8. S Srimasorn, M Kirsch, S Hallmeyer-Ellgner et al.: Increased Neuronal Differentiation Efficiency in High Cell Density-Derived Induced Pluripotent Stem Cells. Stem Cells Int. 2019. Volume 2019 |Article ID 2018784.
  9. SM Chambers, CA Fasano, EP Papapetrou et al.: Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009. 27(3):275-80.
  10. M Shafa, F Yang, T Fellner et al.: Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers. Front Med (Lausanne). 2018;5:69.