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Heck Lab Leads Proteomic Characterization of Stem Cell Reprogramming Process

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NEW YORK (GenomeWeb) – A team led by researchers at Utrecht University has characterized the proteomic changes that occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs).

Detailed in a paper published today in Nature Communications, the effort offers insights into the proteins and pathways involved in cell reprogramming that could provide scientists with avenues for improving the speed and efficiency of the reprogramming process, Albert Heck, chair of the Biomolecular Mass Spectrometry and Proteomics group at Utrecht University and senior author on the paper, told GenomeWeb.

In addition, the researchers characterized a new class of pluripotent cells – dubbed f-class cells – that, Heck said, could in some instances serve as an alternative to iPSCs. 

The study is part of a larger project led by Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Toronto researcher Andras Nagy, a co-author on the Nature Communications paper. Named Project Grandiose, the effort aims to perform deep molecular characterization of the cellular reprogramming process, including genomic, epigenomics, transcriptomic, and proteomic analyses.

The effort involved roughly 50 researchers around the world, resulting in the publication today of five papers – two in Nature and three, including the Heck-led proteomic characterization, in Nature Communications.

To generate the proteomic profiles, the researchers used mass spec analysis on a Thermo Fisher Scientific LTQ Orbitrap instrument. They looked at the cells at 13 different time points across the three weeks of the reprogramming process identifying 7,250 proteins in total, 4,454 of which they quantified in all of the samples analyzed. Of that, 2,452 had changed more than two-fold at at least one of the time points compared to the original secondary mouse fibroblast cell.

Mass spec-based proteomic analysis of stem cells was once a challenge due to the difficulty of producing the relatively large amounts of material required for such analysis, Heck noted. However, he said, with current methods, this is no longer a problem.

"In general, here we had about a million or so cells, so it's not a real difficulty," he said.

In the traditional method of generating iPSCs, developed by Kyoto University researcher Shinya Yamanaka, ectopic expression of the transcription factors Oct4, Klf4, c-Myc, and Sox2 enables reprogramming of somatic cells into iPSCs. The Project Grandiose team used a modified version of this approach in which they tweaked the system so that these transcription factors could be switched on by the addition of doxycycline. This allowed the researchers to more finely control the reprogramming process such that the large populations of cells needed for their analyses could be generated in a more uniform manner, enabling better comparison of the molecular characterizations.

Comparing the cell proteomes across the reprogramming, a picture emerged of a stepwise process with significant remodeling occurring at discrete time points followed by lower levels of activity.

"The data shows that the reprogramming process actually takes place in steps," Heck said. "At day one when you induced these [transcription] factors something dramatic happens in the cell, but then for a couple of days nothing changes. Then, after five days a dramatic change happens again. Then, until 11 days nothing changes much, and then at 11 days it changes significantly again." 

"So [we] wanted to know which genes, which proteins, which RNAs, which microRNAs are really involved in these steps in the reprogramming process," he said. 

Heck noted that while many previous studies have done molecular characterizations of iPSCs, these studies have typically looked only at the starting somatic cells and the end product iPSCs. Less common, he said, are studies looking at the process across many time points as did the Project Grandiose researchers.

One result of this approach was the discovery by project researchers of the previously unidentified f-class cells.

"People looked at the cells that became [iPSCs], but they never looked at the cells that didn't become [iPSCs]," Heck said. "We started to analyze all the steps in the reprogramming, and we found this alternative [f-class] endpoint."

The cells, which he said represent a stable reprogramming endpoint, are like iPSCs in that they can differentiate into other cells, for instance, neurons. However, Heck said, they are quite distinct from iPSCs at a molecular level. 

From a practical perspective, the f-class cells are interesting in that they are easier to produce and more stable to culture than traditional iPSCs, he said. At the same time, they are, like iPSCs, pluripotent stem cells with the genetic background of the original somatic cell.

"So we believe that for drug testing and other kinds of testing experiments, these "f-class" cells might be a good alternative [to iPSCs]," Heck said.

Indeed, tackling the difficulty of generating IPSCs – a process that takes three weeks and provides very low yields – is one of the main goals behind the Project Grandiose characterizations, he noted.

With the data provided by the group's various analyses, the researchers now have a sense "for every protein, every microRNA, every RNA, [etc.] — at what stage in the reprogramming they are important," Heck said.

"People have tried over the years to make this reprogramming of cells more efficient," he said. "And I think our resource and data gives many clues to which [molecules] we should manipulate to make this programming more efficient."

Heck said that he and his colleagues are now looking into how they might manipulate the process to make it either faster or more efficient by, for instance, inducing expression of a particular gene or protein shown to be important at a particular stage of the process.

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