Author: Kevin Bielic
This is a guest post written by Kevin Bielec, a Scientist at RheinCell Therapeutics GmbH, a Düsseldorf-based company that specializes in research-grade production and characterization of human induced pluripotent stem cells (iPSC) produced from cord blood. Previously, Kevin worked as an iPSC Scientist for Roslin Cells, a world leader in the production of iPSCs for use in research, drug discovery and cell therapy.
The Transformative Power of iPSCs
“It might walk like a duck, it might talk like duck. That doesn’t mean it is a duck!”
These are the words of a great scientist and brilliant mind which are on my mind a few weeks after starting up work within the iPSC industry. Sitting in my office with a fantastic view of Arthur’s Seat in Edinburgh, I suddenly find myself in a discussion about the fundamental pros and cons of using reprogrammed cells instead of “the golden standard” – human embryonic stem cells (ESCs).
Like most questions in life, there is no right or wrong, no black or white answer. Both iPSCs and ESCs have advantages and disadvantages.
The only fact we can say for sure is that iPSCs have undoubtedly changed the landscape of modern stem cell research and offer distinct advantages, including their relative ease of production and lack of ethical controversy.
For me, the discovery of taking fully differentiated cells and changing their plasticity is one of the greatest inventions of the 21st century.
Future Directions for iPSC Research
Based on my experience as a researcher, the following are represent future directions for iPSC research.
1. Gaining deeper insight and control over genetic and epigenetic changes to guarantee genetic and phenotype stability
With iPSC-based products moving rapidly toward clinical applications, it is important to ensure the long-term safety of the iPSC-derived therapeutics.
The use of non-integrating reprogramming methods already reduces the median number of copy number variations (CNVs) and significantly lowers the incidence of single nucleotide polymorphisms (SNPs) or genetic mosaicism, as compared to integrating methods. Replacing carcinogenesis-related reprogramming factors like c-Myc with less carcinogenic gene family members like L-Myc, also reduces the occurrence of chromosomal aberrations.
Further, initially higher numbers of genetic variations seem to be lost by selection pressure during early passaging in a pluripotent state.
In my opinion, ongoing investigation into the improvement of reprogramming, cell culture conditions, and the regular assessment of produced cell lines throughout a differentiation process are essential to ensuring the genetic stability, as well as the long-term safety and efficacy, of iPSC-based therapeutics.
2. Improving the efficiency of non-integrating reprogramming methods
Using the most common non-integrating methods (episomal, Sendai virus, mRNA), reprogramming efficiency is a rather stochastic than deterministic event, usually ranging from 0.02% to 1% and being strongly dependent on the reprogrammed cell type.
In 2010, Warren, et al., achieved a reprogramming efficiency of 4.4% employing synthetically modified mRNA using a stable BJ fibroblast cell line under feeder-dependent conditions. Several small molecules like Vitamin C, Valproic Acid, butyrate or CHIR99021 have been shown to significantly enhance reprogramming efficiencies and even make some reprogramming factors redundant. However, the overall reprogramming efficiency still remains low.
In a thrilling paper from 2013, Rais et. al. identified the Mbd3/NuRD (Nucleosome remodelling and deacetylation) repressor complex as the predominant molecular block for deterministic induction of pluripotency. The inhibition of Mbd3, and thus the Mbd3/NuRD repressor complex, should under certain conditions lead to reprogramming efficiencies nearing 100%.
Although researchers have started to question Rais’ outcomes as being not reproducible, the impact of this paper and the increasing use of various small molecules during reprogramming show demand for a significant improvement of the reprogramming efficiency.
3. Development of high-throughput systems for the production, expansion and banking of pluripotent cells
Usually iPSCs are cultured as adherent monolayers in extracellular matrix-coated tissue culture plates or flasks. Despite the robustness of this method, it is also a major drawback for scalable mass production, due to the restriction to ECM-coated growth areas.
For example, considering the need of approximately 5×108 CD34+ cells for hematopoietic stem cell (HSC) transplantation into a 70 kg adult patient, the large-scale production of pluripotent stem cells is a key process to sufficient enough cell counts for differentiation protocols.
At the same time, it needs to be performed in a streamlined and cost-effective manner.
The invention of a commercially available, affordable and scalable automated suspension-culture bioreactor would be a game-changer for induced pluripotent stem cell (iPSC) research.
Future Directions for Clinical Applications of iPSCs
Regarding future directions for iPSC-based therapeutics, the following points should be emphasized.
1. Production of allogenic ATMP on the basis of an HLA homozygous iPSC bank
I have not personally worked according to GMP conditions, but as a R&D scientist I have come in contact with a production department that did and observed first-hand the workload required. Although iPSCs could theoretically be derived from individual patients for autologous transplantation, the production of individual GMP-grade iPSC lines would impose a major logistical challenge and financial burden.
Alternatively, a number of GMP-grade iPSC lines could be produced that HLA-match with a large number of patients.
Specifically, Okita, et al., pronounced that approximately 140 HLA homozygous iPSC lines for the most common HLA haplotypes could generate immune tolerated cellular therapeutics for about 90% of the Japanese population. Similar outcomes have been found for the UK population.
Although immunogenicity can and should not be neglected, using HLA homozygous iPSC-based cellular therapeutics represents a valuable option for providing a vast part of a population with advanced therapy medicinal products (ATMPs).
2. Non-therapeutic use of iPSCs
The clinical use of iPSCs for organogenesis or tissue replacement is the most obvious area of application. However, from a regulatory and financial perspective, it is also the most difficult.
Other areas of non-clinical application are using patient-specific iPSCs for disease modelling and drug screening. Both could allow for a deeper understanding of the molecular mechanism of an underlying disease and for a more personalized treatment of various medical conditions.
Another, less prominent application to consider might be the use of iPSC-based host cells for industrial protein expression.
Why not use iPSC-based β-cells for the in vitro production of insulin, instead of using bacterial expression systems like E. coli? Why not using an iPSC-based B-cell product for the production of monoclonal antibodies? This list could go on and on.
As mentioned above, I am not a production scientist and mammalian (especially human) expression systems are more expensive and difficult to use than their bacterial counterparts. However, this approach would allow the use of streamlined intracellular machinery to produce the protein needed, including a full range of post-translational modifications.
BioInformant is the first and only market research firm to specialize in the stem cell industry. Our management team comes from a BioInformatics background – the science of collecting and analyzing complex genetic codes – and applies these techniques to the field of market research. BioInformant has been featured on news outlets including the Wall Street Journal, Nature Biotechnology, Xconomy, and Vogue Magazine.
To learn more , view the “Compete 2015-16 Induced Pluripotent Stem Cell (iPSC) Industry Report.”