Appearance of iPSCs–Different Reprogramming Stages within the Same Well

Previously scientists at Allele Biotech have reported near uniform conversion of human fibroblasts using our proprietary mRNA mixtures. The first picture below shows a well of cells after 7 days of growing fibroblasts with the new Allele mRNA mix.

This month, by adjusting the mRNA dose while testing Allele’s own reprogramming medium formulation, we observed various stages of cells going through the transition in the same well (see pictures 2 to 5). All stages of reprogramming typically observed over a span of weeks can actually be seen within 1 well of a 6-well plate when we treated human fibroblasts at half the dose of our standard mRNA mix, on day 10, and using Allele Biotech’s new formulation of reprogramming medium.

(1) Warren, Ni, Wang, and Guo 2012 (pdf download)

Previous bulk conversion on Day 7 of reprogramming at full dose mRNA, improved upon our published efficiency (1)

iPSCs forming small colonies from single cells within a 24-hour time frame

Reprogramming en masse: post mesenchymal-to-epithelial (MET) transition cells start to become iPSCs without surrounding fibroblasts (as opposed to the above figure)

Large patches of cells that became iPSCs in what we call bulk-conversion

Large colonies become highly compact, with sharp edges, and composed of mature stem cells of small cell body and tight bundling

Tags: bulk convertion, fibroblast reprogramming, foot print free, highly efficient reprogramming, iPSC, mRNA reprogramming, stem cell clone

Picture Blog: How Do You Like Your iPSCs, Clonal or Bulk-Conversion?

Reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is commonly considered a stochastic process, i.e. with randomness, which offers an excuse for the commonly seen low efficiency and low constancy of making iPSCs. We have demonstrated time and again that by using potent mRNA cocktails, the majority of the fibroblasts seeded in a well can be converted into pluripotent stage in a nearly synchronized manner (Warren et al. 2012, Warren and Wang 2013, and this Allele Picture Blog series). mRNA molecules can function robustly yet transiently while avoiding the need of entering the nucleus, a bottle-neck for all DNA-based vehicles.

Other researchers are used to the idea of clonal expansion partly because isolating iPSCs from “clones” was a common step during reprogramming using viruses or other low efficiency methods, even though those clones were not necessarily from single precursor cells. This week, the Allele iPSC team developed a new way of managing our mRNA reprogramming that allowed us to achieve clonal iPSCs that appear to be a lot purer and more likely true clones compared to previous reports, without compromising any of the main benefits of our protocol, e.g. feeder-free, xeno-free, footprint-free, very fast and highly efficient. This work is currently supported by an NIDA/NIH grant to Dr. Jiwu Wang at Allele Biotech.

Traditional bulk-conversion by the Allele mRNA reprogramming protocol developed by Warren et al. The picture shows large patches of cells becoming stem cells almost overnight around the 9th day of adding mRNA-cocktail supplement to the media.

Clonal iPSC formation using a modified mRNA reprogramming protocol. The picture shows a typical clone of stem cells that originated from likely single cells.

Warren, Ni, Wang, and Guo, Scientific Reports, 2012
Warren and Wang, Current Protocols, 2013, in press

Tags: bulk conversion, clonal expansion, footprint free, integration-free, iPSC clone, iPSCs, mRNA reprogramming, pluripotency, single cell reprogramming

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:
iPS@allelebiotech.com
858-587-6645
Fax 858-587-6692
www.allelebiotech.com
6404 Nancy Ridge Drive
San Diego, CA 92121

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

New Product of the week: 6F mRNA Reprogramming Premix: $995 for 10 reprogramming!

Tags: cell therapy, disease model, drug screening, iPSC, Klf, Lin28, M3O, mRNA reprogramming, Nanog, Oct, Sox