Safer Pluripotent Stem Cells using Recombinant Protein Bath to Convert Cells

In Vitro and In Vivo Pluripotency of piPSCs
(A) piPSCs can effectively differentiate invitro into cells in the three germ layers, including neural progenitor cells (Pax6+), characteristic neurons (TUJ1+), mature cardiomyocytes (CT3+), definitive endoderm cells (Sox17+), pancreatic cells (Pdx1+), and hepatic cells (ALB+). Images were merged with DAPI (blue) staining.
(B) RT-PCR analysis of invitro differentiation of piPSCs.
(C) piPSCs incorporate into the ICM of the blastocytes after aggregation with eight-cell embryos (left). Chimeric fetuses (13.5 dpc, middle) were obtained after transfer of the piPSC aggregated embryos into pseudopregnant mice. piPSCs contributed to the germline cells (Oct4-GFP positive) in isolated genital ridge tissue from chimeric fetuses (found in 3 out of 17 fetuses, right).
(D) GFP genotyping confirmed piPSC contribution to multiple three germ layer tissues in chimeric fetuses, including heart, liver, brain, tail, and gonad tissues. A representative genomic PCR of GFP was shown for embryo 9 that also contains piPSC germline contribution.

A group of researchers at the Scripps Research Institute and other institutions have achieved a breakthrough in converting adult cells all the way back to the most primitive embryonic-like cells without using the dangerous genetic manipulations associated with previous methods.

The team of scientists accomplished this extraordinarily challenging feat by engineering and using recombinant proteins, that is proteins made from the recombination of fragments of DNA from different organisms. Many different recombinant proteins have been therapeutically and routinely used to treat human diseases. Instead of inserting the four genes into the cells they wanted to reprogram, the scientists added the purified engineered proteins and experimented with the chemically defined conditions without any genetic materials involved until they found the exact mix that allowed them to gradually reprogram the cells.

The scientists found that those reprogrammed embryonic-like cells (dubbed “protein-induced pluripotent stem cells” or “piPS cells”) from fibroblasts behave indistinguishably from classic embryonic stem cells in their molecular and functional features, including differentiation into various cell types, such as beating cardiac muscle cells, neurons, and pancreatic cells.

The first author of the article, “Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins” was Hongyan Zhou of Scripps Research.

8 pages of supplemental data on the experiments

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