Normally, it takes researchers many weeks to get the slightest .01 to 10 percent yield when making pluripotent stem cells from ordinary cells via the Nobel Prize-winning induced pluripotent stem cell (iPSC) approach.
But by adding just three compounds to the above recipe— including a shot of vitamin C— a New York group reports in Stem Cell Reports a huge 90 to 100 percent stem cell haul (in mice) in under a week.
The approach, when viewed in light of two other recent new methods, improves the chance researchers may become highly adept at routinely making stem cells from ordinary cells for study—and maybe the clinic.
“We are very pleased to see follow-up reports by alternative pathways showing that iPSC efficiency can be brought up drastically to near 100 percent efficiency,” Jacob Hanna told Bioscience via email. The Weizmann Institute’s Hanna was not involved in the recent study. But he was the first to increase the yield of the iPSC approach to near 100 percent in a Nature paper last year (in that case, in both mouse and human cells).
“In colonies undergoing reprogramming, almost no cell is left behind,” said the new paper’s senior author, New York University’s Matthias Stadtfeld, via email to Bioscience.
“An important step forward,” agreed Thomas Graf of the Center for Genomic Regulation, also by email. Uninvolved in the recent study, Graf has also devised a reprogramming approach dramatically improving efficiency in mice. “Exciting,” he said.
The Yamanaka factors
In 2007, Kyoto University stem cell researcher Shinya Yamanaka published a paper in Cell reporting that he turned ordinary differentiated human cells into extraordinary stem cells by genetically tweaking four genes (since called the “Yamanaka Factors”). While this approach was complex, it was also consistent, reliably producing stem cells from differentiated cells, in labs around the world, about one percent of the time.
In 2012, Yamanaka won the Nobel Prize for the work.
But for years, efficiency remained at .01 to ten percent. And the process took weeks. One reason was the fact that many cells only partially dedifferentiated— or did not dedifferentiate at all— to the stem cell state. Yamanaka has been studying these cells.
Another potential reason for the low reprogramming rate: the standard iPSC process creates mutations.
But last year, by transiently disabling a fifth gene (Mbd3), Hanna generated stem cells from adult blood and skin cells in mice, and adult skin cells in humans, with near 100 percent efficiency— in one week. He has said he has since been analyzing the cells to see if this extra step also improves quality.
And early this year, Graf’s team also tweaked a fifth gene— the CCAAT/enhancer binding protein-a gene (C-EBPa)— to get near-perfect reprogramming efficiency. He did this using early lymphocyte cells— also in only a few days.
“Striking” results—sans extra gene tweaking
This week, Stadtfeld’s team reported near-perfect reprogramming efficiency in a greater variety of mouse cells. Within a week, he scored near 100 percent reprogramming efficiency in mouse blood progenitors and hepatoblasts, which, during normal reprogramming, results in 30 percent efficiency. And he scored 80 percent efficiency reprogramming more recalcitrant mouse embryonic fibroblasts.
He did all this sans additional gene tweaking. Instead, his team just added three compounds (to cells genetically altered with the standard Yamanaka Factors): the anti-oxidant vitamin C; a compound to activate Wnt signaling (CHIR99021); and a compound to inhibit TGF-B signaling (ALK-5 inhibitor II).
“In addition to reprogramming efficiency and speed, a striking difference of our approach compared to basal reprogramming [genetic tweaking with the Yamanaka Factors alone] is the homogeneity of pluripotency-gene reactivation,” Stadtfeld told Bioscience.
Furthermore, the results were cell-specific
“All of the compounds we tested had been implicated in pluripotency and/or reprogramming before. We were simply curious whether they would act synergistically,” Stadtfeld said. Also, their effect hadn’t been studied extensively outside of fibroblasts, “so we wanted to fill that gap” especially for blood cells. “Because of their accessibility, they are especially interesting for IPC cell derivation.”
The mutation issue
Stadtfeld noted that, in his study, it seemed “unlikely” that functionally significant mutations were generated by his approach “based on our observation that all of the iPSC lines we tested were able to generate chimeric mice— an established assay for developmental potency…Nevertheless, it is an important question whether the compounds modulate biological properties of reprogrammed cells in a lasting manner, and we are investigating this possibility.”
He will also be analyzing the other approaches for clues to natural dedifferentiation processes the three papers may have unearthed between them. While the Graf paper “is based on genetic engineering and works only in early B lymphocytes, these cells seem to reprogram similarly…to our blood progenitor cells. It's very intriguing to figure where these approaches [including Hanna’s Mbd3 approach] molecularly intersect.”
Intriguing parallels
Agreed Graf by email: “It remains to be seen whether the new findings can be applied to human cells, where reprogramming efficiencies remain abysmally low” outside of Hanna’s work. (While there has been one reported problem reproducing Hanna's work, Hanna notes there is a related paper, and says four high-profile labs have reported to him success using his system.) Still, "there are intriguing parallels in [Stadtfeld’s] findings [where] cells that could be reprogrammed most rapidly and with the highest efficiency are granulocyte macrophage progenitors (GMPs), extending earlier findings by other labs. “
He continued: “What makes these findings so exciting is that the sequence-specific transcription factor (TF) C/EBPa, known to be strictly required for GMP formation, poises pre-B cells for high-efficiency iPSC reprogramming when activated transiently before over-expressing the OSKM [Yamanaka] factors,” as shown in Graf’s paper. “And it does not end here: over-expression in B cells of C/EBPa without OSKM induces their highly efficient transdifferentiation into macrophages, with the cells going through an intermediate stage that has some features of GMPs. And while neither B cells nor macrophages are susceptible to OSKM-induced reprogramming, the intermediates are--again, consistent with the idea the GMP stage is special.”
Finally, Graf noted, the C/EBPa effect is “both cell-type and transcription-factor (TF) specific: C/EBPa neither facilitates reprogramming of fibroblasts nor can it be replaced by other transdifferentiation-inducing TFs, including the erythroid, neuron, and muscle inducing TFs GATA-1, Ascl1 and MyoD, pointing to the regulation of a cell-type specific gene expression signature required for reprogramming.”
In short, Graf said, much points to “a key role of C/EBPa in causing the extraordinary facility with which GMPs can be reprogrammed into iPSCs.” Unveiling the secret of how the factor acts may bring “understanding of reprogramming to pluripotency—at least in blood cells.”
A fourth paper reporting dramatic improvement of the iPSC method is due out shortly.
