Scientists develop method to define, standardise stages of stem cell reprogramming.


In a study that provides scientists with a critical new understanding of stem cell development and its role in disease, UCLA researchers have established a first-of-its-kind methodology that defines the stages by which specialized cells are reprogrammed into stem cells resembling those found in embryos.  The opensource study is published in the journal Cell.

Induced pluripotent stem cells, known as iPSCs, are cells that can be generated from adult cells and then, like embryonic stem cells, be directed to become any cell in the human body. Adult cells can also be reprogrammed in the lab to change from a specialized cell back to an iPSC (and thus becoming a cell similar to that of an embryonic stem cell).

Reprogramming takes one to two weeks and is a largely inefficient process, with typically less than one percent of the starting cells successfully becoming an iPSC. The exact stages a cell goes through during the reprogramming process are not well understood. This knowledge is important, as iPSCs hold great promise in the field of regenerative medicine, as they can reproduce indefinitely and provide a single source of patient-specific cells to replace those lost to injury or disease. They can also be used to create novel disease models from which new drugs and therapies can be developed.

The team developed a roadmap of the reprogramming process using detailed time-course analyses. They induced the reprogramming of specialized cells (that could only make more of themselves, and not other cell types), then observed and analyzed on a daily basis or every other day the process of transformation at the single-cell level. The data was collected and recorded during a period of up to two weeks.

The team found that the changes that happen in cells during reprogramming occur in sequentially, and that importantly, the stages of the sequence were the same across the different reprogramming systems and different cell types analyzed.

The team state that the exact stage of reprogramming of any cell can now be determined with the current study providing simple and efficient tools for scientists to study stem cell creation in a stage-by-stage manner. Most studies to date ignore the stages of reprogramming, however, the medical community can now seek to better understand the entire process on both a macro and micro level.

The team further discovered that the stages of reprogramming to iPSC are different from what was expected. They found that it is not simply the reversed sequence of stages of embryo development. Some steps are reversed in the expected order; others do not actually happen in the exact reverse order and resist a change until late during reprogramming to iPSCs.  This reflects how cells do not like to change from one specialized cell type to another and resist a change in cell identity.  Resistance to reprogramming also helps to explain why reprogramming takes place only in a very small proportion of the starting cells.

The lab plans future studies to actively isolate specific cell types during specific stages of reprogramming. They also hope the research will encourage further investigation into the characteristics of iPSC development.

The team summise that the research has a broad impact, because by understanding cell reprogramming better the medical community has the potential to improve disease modeling and the generation of better sources of patient-specific specialized cells suitable for replacement therapy.  This can ultimately benefit patients with new and better treatments for a wide range of diseases.

Source:  UCLA Health

 

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.  X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency.  Plath et al 2014.

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming. X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency. Plath et al 2014.

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