Path to Better Drugs through Disease-Specific iPSCs

Induced human pluripotent stem cells

The recent finding that pluripotency, the ability to differentiate into all cell types typically associated with embryonic stem cells, can be induced in somatic cells may be the molecular equivalent of the discovery of antibiotics or vaccines in the last century [1].

iPSC-based disease modeling

Recent studies have described the generation of induced pluripotent stem cells (iPSCs) from patients with a full range of genetically inherited or sporadic diseases, and in vitro differentiation of these iPSCs to cell types relevant to the disorder with certain disease features.

Example 1 (out of ~20): Progressive motor neuron loss during differentiation of iPSCs derived from spinal muscular atrophy (SMA) patients, reflecting developmental loss seen in the disease.
Example 2: iPSCs made from RETT syndrome give rise to glutamatergic neurons with fewer synapses than controls, a better treatment was found from a panel of candidates based on this model.
Example 3: Neurons differentiated from iPSCs that have been derived from early or late onset Alzheimer’s disease were shown to display different properties and potential interference points.
The identification of novel pathways or drugs that could prevent disease is the ultimate goal of the iPSC-based disease modeling approach.

Major steps towards efficient iPSC disease modeling

The first hurdle for feasible application of patient-specific disease modeling is to achieve efficient generation of iPSCs from large cohorts of patients quickly and at a low cost while eliminating “clonal variations”. As described in a recent publication [2], the Allele Biotechnology team has shown that human fibroblasts can be converted to stem cells in just over a week, achieving bulk conversion efficiency without any chromosome modifications. The process is also xeno-free and feeder-free, enabling both fundamental scientific research and clinical applications.

The next major advancements required for disease modeling are robust lineage-specific differentiation protocols that provide a large number of desired cells for drug testing and screening. Cardiomyocytes derived from iPSCs have been the best known example of large expansion; other cell types will become available in the near future. Allele Biotechnology has commenced differentiating iPSCs along several lineages using our own iPSCs of superior quality.

With cells of disease-matching tissue types derived from patients’ iPSCs, cell-based assays can be designed and developed using various assay formats. Allele Biotech’s leading capacities in fluorescence and bioluminescence, gene silencing, delivery vehicles and single-domain targeting agents will be of unmatched value to drug discovery partners.

1. Review: Wu, SM and Hochedlinger, K. “Harnessing the potential of induced pluripotent stem cells for regenerative medicine ” 2011, Nature Cell Biology, V13-5, 497-505.
2. Allele Biotech publication: Warren, L., Ni, Y., Wang, J. and Guo, X. “Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA” 2012, Nature’s Scientific Reports, doi:10.1038/srep00657.

For business development contact:
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Related products for academic customers: Non-Integrating iPSC Generation Product Line http://www.allelebiotech.com/non-integrating-ipsc-generation/

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Expression of iPS Factors from Transfected mRNA

Differentiated cells can be reprogrammed to pluripotency by enforced expression of certain combinations of stem cell-specific protein factors in them. The power of this method was first demonstrated by Yamanaka’s group using retroviruses carrying Oct3/4, Sox2, c-Myc, and Klf4. Alternative factors such as Lin28 and Nanog, and additional factors such as the human telomerase gene hTert and shRNA against p53 were also shown to contribute to reprogramming. From the very beginning it was realized that viral integration would pose a major problem in using the induced pluripotent stem cells (iPSCs) for clinical purposes. There have been multiple attempts to circumvent this problem by using non-integrating vectors such as plasmid, minicircle DNA, adenovirus, baculovirus, removable transposons, episomal DNA, or by introducing recombinant proteins with a transmembrane domain into target cells. From reports in the field and customer feedbacks it seems that retroviral or lentiviral systems are still the most efficient in reprogramming. mRNA is about the only option left unreported, until an article by Warren et al was published in Cell Stem Cell online recently.

From that report, it is clear that the reason that it took so long for RNA-induced iPSCs (RiPSCs) to appear in the literature was because synthetic mRNAs activate interferon responses in mammalian cells, reminding us of the early days of RNAi. The authors took a number of steps to reduce interferon responses, including adding a 5’-cap (actually a fairly standard step in in vitro transcription), using a phosphatase to remove 5’ triphosphates on uncapped mRNAs, and using modified C and U bases (5-methucytidine or 5mC and pseudouridine or psi) during T7 promoter-driven in vitro transcription. The prepared mRNA was then administered everyday for 17 days at an amount not clearly defined in the paper. The main benefit of this method is of course that there is no gene integration to alter the chromosome. The efficiency of the new method was also compared to using viral vectors and it was shown that 1.4% conversion efficiency was achieved vs retroviral systems’ 0.01% (although we have experienced better results using lentivirus, at least the 4-in-1 version).

The DNA templates used for in vitro transcription of the iPS factors were created by multiple PCR reactions and bridged ligation; it could also be done by other cloning strategies. For those excited about trying this new way of making iPSCs, the major hassle would be preparing modified mRNAs good and abundant enough for 17 consecutive transfections. Allele Biotech would like to provide custom services, before offering shelf products, for creating such mRNAs as the method sounds potentially very helpful to many researchers in the iPSC field.

    New Product of the Week 100410-101010:

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