Stem Cells Forward and Back

By Matthew Busse

A pair of papers from Associate Professor Sheng Ding's lab at The Scripps Research Institute describes research into one type of adult stem cell, shedding light on the detailed mechanisms that control both how these stem cells choose their specialized fate, and how specialized cells can unwind that choice.

The papers, published recently in the Proceedings of the National Academies of Sciences (PNAS), focus on a type of adult stem cell known as mesenchymal stem cells, which are cells that normally reside in bone marrow and other tissues, giving rise to a variety of cell types, including bone, muscle, fat, cartilage, and neurons. In contrast to embryonic stem cells, adult stem cells can be found in the body at any age.

"Tissue-specific (or adult) stem cells are regarded as the source for normal tissue maintenance and repair," says Ding. "They also provide tremendous promise for regenerative medicine, because of their capacity to proliferate and differentiate into a variety of mature cell types."

The Potential of Large-Scale Screens

The first paper, published online in an advance edition PNAS on May 29, investigates differentiation, the process by which stem cells develop into specialized cell types, such as bone or skin cells.

In the paper, the researchers describe the first large-scale RNAi screen ever performed on human stem cells. RNAi, short for RNA-interference, can be used as a tool to specifically inhibit the action of a single protein. In a large-scale screen, many different substances are tested simultaneously.

"This is a proof-of-concept study," says Ding, "showing what is feasible in stem cell biology using the most cutting-edge technology."

Ding and colleagues used a cell-based assay to sift through an RNAi library targeting 5,000 genes, identifying the proteins that block the development of mesenchymal cells into bone-forming cells known as osteoblasts. The screen identified 53 candidate genes coding for proteins inhibiting osteoblast development.

Of these, Ding chose 12 to investigate in depth. Interestingly, the team found several candidates that function at different levels of one well-known signaling pathway, called the cyclic adenosine mono-phosphate (cAMP) pathway. Ding notes that finding multiple proteins in the same pathway confirms the essential role of such pathway in the studied biological process, and potentially enables researchers to fine tune the pathway's activity, as each protein represents a possible new drug target.

While studying the bone cell fate of mesenchymal stem cells, Ding's group made an important additional discovery—bone cell fate and fat cell fate are two sides of the same coin for mesenchymal stem cells, linked together by cAMP. If the cAMP pathway is active, the stem cells develop into fat cells. If the cAMP pathway is deactivated, the stem cells instead develop into bone-forming cells.

These findings are examples of the underlying genetic networks controlling differentiation that Ding aims to uncover using genome-wide techniques such as large-scale screens. Understanding the fundamental biology of mesenchymal stem cells, says Ding, is the key to learning how to control the differentiation of stem cells for therapeutic purposes. While stem cell based therapies may still be a long way off, Ding's work could be used in the near-term to identify targets for drugs to treat diseases such as osteoporosis and diabetes.

In addition to Ding, the paper, "A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells," was authored by Yuanxiang Zhao. The research was supported by Scripps Research and the Genomics Institute of the Novartis Research Foundation (GNF).

Shedding Light on Reversine

The second paper, published in PNAS June 12, focuses on dedifferentiation, the process of restoring specialized skin, muscle, or bone cells to a stem cell state.

This paper follows up on work published three years ago, in which the Ding lab, in collaboration with the laboratory of Scripps Research Professor and GNF Director Peter Schultz, discovered a small molecule—which the scientists dubbed "reversine"—that could turn muscle cells back into stem cells. The current study sheds light on reversine's mechanism of action.

Using a variety of biochemical techniques, the team found that reversine binds to and inhibits two proteins—mitogen activated extra-cellular signal regulated kinase (MEK1) and nonmuscle myosin II heavy chain (NMMII). MEK1 is a well known protein that regulates many processes, including stem cell differentiation, metabolism, and proliferation. The recent study suggests that, by inhibiting MEK1, reversine suppresses the activity of several cell fate-determining genes, leading cells to lose their specialized cell fate.

NMMII plays a role in the cell's cytoskeleton, the internal scaffolding that gives each cell support and shape; and in the regulation of the cell cycle, the process through which cells replicate themselves. The team found that reversine causes cells to spend extra time preparing to replicate. Although the exact mechanism is still unknown, this effect likely contributes to the dedifferentiation of the cells.

Adding to their work with muscle cells, the team demonstrated that fibroblast, fat and bone cells can also be dedifferentiated into mesenchymal-like stem cells using reversine, and then redifferentiated into different specialized cell types.

Ding believes reversine offers several advantages over other approaches that have been shown to dedifferentiate cells, such as genetically engineering specialized cells to express stem cell genes, which Ding believes carries a significant risk of causing cancer. A small molecule such as reversine acts in a chemically defined manner, allowing for more control over its action. Additionally, reversine acts transiently and is easily reversible.

Currently Ding is working on discovering and/or designing additional small molecules that can dedifferentiate specialized cells all the way back to embryonic-like stem cells, capable of developing into any type of specialized cell found in the body. He also imagines a day, years down the road, when a chemical cocktail can revert any specialized cell to any developmental stage, and a second chemical cocktail can differentiate the cell into any type of cell needed for therapeutic applications. His two most recent papers along with his earlier small molecule discoveries, such as pluripotin and QS11, are steps in this direction.

In addition to Ding and Schultz, the June PNAS paper, "Reversine increases the plasticity of lineage-committed mammalian cells," was authored by Shuibing Chen, Shinichi Takanashi, Qisheng Zhang, Wen Xiong, Shoutian Zhu, and Eric C. Peters. The research was supported by the National Institutes of Health, GNF, and Scripps Research.

Send comments to: mikaono[at]scripps.edu

"Tissue-specific (or adult) stem cells... provide tremendous promise for regenerative medicine, because of their capacity to proliferate and differentiate into a variety of mature cell types."

-Sheng Ding