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Generation of Induced Pluripotent Stem Cells Using Recombina

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Alan Iturbe Avila

on 1 June 2017

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Transcript of Generation of Induced Pluripotent Stem Cells Using Recombina

Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins
Groundbreaking work demonstrated that ectopic expression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc, could reprogram murine somatic cells to induced pluripotent stem cells
To address the safety issues arose from harboring integrated exogenous sequences in the target cell genome, a number of modified genetic methods have been developed and produced iPSCs with potentially reduced risks
All of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications by the exogenous sequences in the target cells.
One possible way to avoid introducing exogenous genetic modifications to target cells would be to deliver the reprogramming proteins directly into cells, rather than relying on the transcription from delivered genes.
It demonstrated that such piPSCs can long-term self-renew and are pluripotent in vitro and in vivo.
piPSCs can effectively differentiate in vitro into cells in the three germ layers, including neural progen- itor 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.
piPSCs incorporate into the ICM of the blastocytes after aggregation with eight-cell embryos (left).

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).
To generate recombinant proteins that can penetrate across the plasma membrane of somatic cells, we designed and fused a poly-argi-nine protein transduction domain to the C terminus of four reprogramming factors: Oct4, Sox2, Klf4, and c-Myc.
These proteins were expressed in E. coli in inclusion bodies, which were then solubilized, refolded, and further purified
Following protein refolding and purification,
Oct4 (lane 2), Sox2 (lane 3), Klf4 (lane 4), and cMyc (lane 5) were analyzed by 4-12% Bis-Tris
NuPage Gel and Coomassie blue staining. The protein standards were shown in lane 1.
The protein identities were confirmed by mass spectrometry and western blot analysis
Stability of the four recombinant reprogramming proteins under the cell culture condition was
examined by Western blot analysis. Proteins (8 μg/ml) were added into mESC growth media
and incubated at 37 ºC for 12 hr. Medium samples were then collected and subjected to
Western blot analysis. The specific antibodies against Oct4, Sox2, Klf4, and cMyc were used.
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