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

Weekly update on an ongoing NIH project for improving mRNA reprogramming at AlleleBlog

Reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is commonly considered a stochastic process, i.e. with randomness, which could offer a convenient excuse for the historical inefficiency and inconstancy 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