A team of Whitehead Institute researchers, led by Massachusetts Institute of Technology (MIT) biology professor and principal investigator Rudolph Jaenisch, has discovered a way to reduce the time it takes to create induced pluripotent stem cells (iPS cells). Jaenisch told BioTechniques that the team set out to determine why reprogramming somatic cells into iPS cells using the overexpression of the transcription factors oct4, sox2, klf4, and c-myc only resulted in a minority of adult cells achieving a state of pluripotency. Jaenisch’s team not only determined what was limiting pluripotency, but also discovered a novel way to reduce the time it takes to create an iPS cell by half.
Reprogramming somatic cells is a continuous stochastic process, said the authors, and almost all donor cells will eventually achieve pluripotent capabilities given continued expression of growth and transcription factors. The authors also stated that additional expression of the cancer-related genes p53 and p21 resulted in an increased rate of cell division that accelerated the kinetics of iPS cell formation. The overexpression of the gene nanog also sped up reprogramming in a manner independent from the rate of cell division.
Quantifying cell progress during in vitro reprogramming has previously been problematic due to the heterogeneity of de novo infected somatic cells, said Jaenisch. To reduce the heterogeneous nature of the somatic cells, a new reprogramming method was devised where all somatic cells expressed the same pattern of drug-inducible viral transgenes. “We used a large base of knowledge to do this research,” said Jaenisch. “Our previous knowledge of mouse genes was very helpful.”
The researchers grew individual mouse immune cells using this method, and allowed them to divide over a period of several weeks. The scientists recorded the rate of cell division, and monitored when the cells began emitting the chemical signals that indicate pluripotency. According to the paper, up to 20% of the created cells gained pluripotency after 3-4 weeks of transgene expression, and only 8% failed to gain pluripotency after an 18-week period.
The researchers were able to cut the time required to generate the iPS cells by increasing the expression of the cancer-linked genes p53 and p21. “Honestly, we’re building on research from the last ten years here,” said Jaenisch. “We’ve all been wondering which genes would be required to make the process more efficient.” According to Jaenisch, activating the molecular pathways involved in cancer-forming cells was found to increase the rate at which immune cells divided, which increased their rate of reprogramming simultaneously. Expression of the gene nanog was found to reduce the number of cells divisions required to achieve pluripotency, which also shortened reprogramming time.
Jaenisch said that reprogramming may involve a series of epigenetic events as cells divide. This hypothesis fits with the team’s findings, since it is known that when cells divide rapidly, they also quickly increase their pluripotency, and cancer cells are known to divide very quickly. “One idea we had is that the over-expression of these oncogenes changes the epigenetic state of the cell,. We think that these oncogenes help with the reprogramming process, essentially loosening up the genome to make it more efficient,” said Jaenisch.
The research team came up with two hypotheses to explain why the overexpression of certain genes would speed up the reprogramming process. Cell division could amplify the daughter cells of partially reprogrammed cells so that each individual cell has an independent chance of becoming pluripotent, or nuclear changes during cell division could enable the acquisition of epigenetic marks that allow the re-establishment of pluripotency. Jaenisch said that this research will continue for many years. “This discovery will be important for studying disease mechanisms in humans,” said Jaenisch.
The paper, “Direct cell reprogramming is a stochastic process amenable to acceleration” was published in the November edition of the journal Nature.