stem cells in their environment

The quest for efficient cell reprogramming – how far have we come?

03 February 2014
By Christine Weber

Hardly any science field these days is as rapidly developing as the iPS field. The ability to generate induced pluripotent stem cells (iPSC) in a dish from mature and fully specialised adult cells has revolutionised the whole field - and created its own in the process. But how efficient are today's reprogramming techniques? How stably and reproducibly can we manipulate a cell's fate?

Dr Konrad Hochedlinger from the Harvard Stem Cell Institute shed some light on these questions in his recent talk at KCL. Starting off with a short introduction to the history of stem cell research, he soon pointed out the current main challenges of transcription factor-induced pluripotency: low efficiency, low yields, and instability of the generated cells. Taking into account the time-consuming and elaborate nature of today's protocols, this is a rather discouraging outcome. Plenty of research groups have addressed the difficulties in reprogramming efficiency, with promising results only recently being published by Rais et al. As the authors claims to have found a way to convert nearly 100% of cultured skin cells into iPS cells, low efficiency rates might actually be a thing of the past. However, the molecular mechanisms of induced pluripotency are still largely unknown and will need to be unravelled before reproducible and safe reprogramming techniques can become a standard.

Konrad Hochedlinger has made notable contributions to the field since the very beginning of his career. During his PhD with Rudolf Jaenisch he reprogrammed adult cells back into embryonic stem cells using nuclear transfer cloning in mice. Later, he used the Yamanaka transcription factors (Oct4, Sox2, Klf4, c-Myc or OKSM) for the same purpose and began to think about how he could dissect the mechanisms at work when a cell regains pluripotency.

To facilitate the reprogramming of adult cells, the Hochedlinger lab at the Harvard Stem Cell Institute created a "reprogrammable mouse model”. Inserted into the genome of this animal is an inducible reprogramming cassette that stably expresses all four Yamanaka factors after the mouse has been treated with doxycycline, thereby starting reprogramming. Using these powerful tools, his lab started out to develop a molecular roadmap of cellular reprogramming which identifies specific surface markers that appear during distinct phases of reprogramming. When the OKSM factors are expressed for 8-10 days, cells reactivate their endogenous pluripotency program and become stably reprogrammed. During this time cells down-regulate certain somatic markers before they turn on telomerase and the pluripotency genes. This means that the reprogramming factors regulate genes in defined and timed patterns. Depending on where the cell is on its journey back to pluripotency certain markers will manifest themselves. Hochedlinger hopes that being able to trace this distinct sequence of molecular events with specific markers will enable scientists to define reprogramming stages more reliably.

As he mentioned during his talk, the outcome of reprogramming also depends very highly on the cell you start out with. Cells in various stages of differentiation respond differently to reprogramming factors, with some being more amenable than others. The addition of those factors not only changes transcription of genes but also chromatin and DNA-methylation. This is a well-known problem by now, undoubtedly also in light of future treatment options in a clinical setting. A challenge for personalised medicine is the fact that it is necessary to standardise patient-specific cells. Technically, every cell line you get is different.

But we don't have to look that far ahead. Another consequence of the high degree of variation among cells has recently become an emerging area of interest in the iPS field. Until fairly recently current methods only allowed reprogramming in a stochastic manner. Whenever a population of cells has been treated with the reprogramming factors some will turn on their pluripotency program sooner than others; and some cells might actually get stuck in a partially reprogrammed state. This limits efficiency and it also denotes one of the field's bottlenecks. Nobody knows why cell behaviour differs so widely and why some of them encounter a roadblock on the way. What are these roadblocks and can they perhaps be removed? What are optimal culture conditions that facilitate rapid reprogramming under deterministic conditions where - after a short time - all cells jump to an iPS state? Hochedlinger and others spearheading the field are working on these issues and we can certainly look forward to exciting years ahead in iPS research.

References

•  Deterministic direct reprogramming of somatic cells to pluripotency

•  Removing Reprogramming Roadblocks: Mbd3 Depletion Allows Deterministic iPSC Generation

•  A molecular roadmap of reprogramming somatic cells into iPS cells

•  Konrad Hochedlinger: The new kid of nuclear reprogramming

•  A reprogrammable mouse strain from gene-targeted embryonic stem cells

Konrad Hochedlinger
Image © 2009 Leah Fasten
Source: The Scientist

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