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Whitehead Develops Dox-Inducible iPS Cells For Library Screens, Reprogramming Studies

Scientists at the Whitehead Institute for Biomedical Research and the Massachusetts Institute of Technology have generated genetically homogeneous populations of secondary somatic cells that can be used for genetic or chemical screening to improve reprogramming, according to a Whitehead Institute researcher.
Their work was recently published online in Nature Biotechnology.
The ability to generate large numbers of genetically homogeneous cells with identical proviral integrations could eventually lead researchers to a better understanding of the mechanisms involved in cellular reprogramming.
“That, I think, is the real interest here,” Rudolph Jaenisch, a member of the Whitehead Institute and a professor of biology at MIT, told CBA News last week.
The method uses genetically homogeneous secondary somatic cells to carry the reprogramming factors Oct4, Sox2, Klf4, and c-Myc as defined doxycycline-inducible transgenes.
He said the approach can also be used to screen for small molecules that replace each of the four factors by genetically deleting one particular factor in the reprogrammed, pluripotent fibroblasts.
Furthermore, this technique is not limited to fibroblast cultures, but can in principle be applied to other somatic cell types that are difficult to transfect with retroviruses, such as lymphocytes or intestinal epithelium, Jaenisch said.
The cellular and genetic heterogeneity of randomly infected fibroblasts complicates the exploration of important molecular events that occur during reprogramming, and limits the scalability required for high-throughput analyses, Jaenisch said.
In addition, it is sometimes tricky to infect or culture cells such as fibroblasts, skin cells, nerve cells, bone marrow stem cells, hematopoietic cells, immune cells, and intestinal cells.
“Our work seeks to overcome these limitations,” said Jaenisch. He explained that his group tried to do this by using drug-inducible viruses.

“[Generating cells that are] appropriate for genetic or chemical screening to improve reprogramming … I think is the real interest here.”

To generate cell populations that were homogeneous with respect to the number and location of proviral integrations, the researchers constructed doxycycline-inducible lentiviral vectors encoding the four reprogramming factors.
Mouse embryonic fibroblasts comprising both a reverse tetracycline transactivator and a phosphoglycerate kinase promoter-driven puromycin resistance gene targeted to the ROSA26 locus and a green fluorescent protein gene targeted to the endogenous Nanog locus were infected with the four lentiviruses.
The investigators also infected ROSA26 MEFs comprising the Oct4 cDNA under control of the tetracycline operator targeted to the type I collagen locus, and a neomycin resistance gene in the endogenous Nanog locus with dox-inducible lentiviruses encoding Klf4, Sox2, and c-Myc.
After viral transduction, the scientists added doxycycline to the culture medium to activate the transgenes and initiate the reprogramming process. As they expected, clonal iPS cell lines were established. To generate somatic tissues comprising genetically homogeneous cells carrying identical proviral insertions, they injected several of these clonal primary iPS cell lines into blastocysts.
The resulting dox-inducible iPS cell chimeras were allowed to gestate for 13 days, at which point MEFs were isolated, and from those, iPS cell-derived cells were isolated.
These so-called ‘secondary’ MEFs were isolated from chimeric iPS cell embryos generated from three distinct, clonal primary iPS cell lines (one Nanog-neo and two Nanog-GFP lines), and were cultured in the presence of doxycycline to see if the integrated lentiviral vectors retained competence to mediate epigenetic reprogramming after differentiation in the developing embryo.
The researchers found that the addition of doxycycline to these cultures initiated dramatic morphological changes, and ‘secondary’ iPS cell lines were efficiently isolated from these cultures by neomycin selection or GFP expression and subsequently propagated in the absence of doxycycline.
They also found that cells derived from the chimeras reprogram upon exposure to doxycycline with efficiencies 25- to 50-fold greater than those derived using direct infection and drug selection for pluripotency marker reactivation.
The scientists confirmed the pluripotency of these cell lines by their ability to form cells of endodermal, ectodermal, and mesodermal lineages in teratoma formation assays, and by their ability to contribute to adult chimeric mice upon blastocyst injection.
The researchers also sought to determine the range of tissue types amenable to reprogramming by isolating secondary cells from 3- to 4-month-old iPS-cell chimeras generated from the NNeo and one of the NGFP cell lines, and examining the reprogramming ability of multiple cell types derived from these chimeras.   
They found that iPS cells could be derived from many other tissues, including brain, epidermis, intestinal epithelium, mesenchymal stem cells, tail tip fibroblasts, kidney, muscle, and adrenal gland through exposure to doxycycline, indicating that the proviruses were appropriately activated in cell types other than MEFs.
In terms of the next step in this work, Jaenisch said his team will probably make secondary cells lacking a virus encoding one of the four reprogramming factors, and then try to replace the virus with a small compound.
“I do not think it will be long before secondary iPS cells generated using this technique are used in drug discovery, because in principle, I think we are almost there,” Jaenisch concluded.   

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