RIKEN Center for Developmental Biology

2003 Annual Report

Stem cell biology

Stem cells of various types, characterized by their abilities to self-renew and to generate more highly specialized cells indefinitely, are the key to replenishing the body's cells. The study of these cells stands at the heart of some of the central questions in development, but many aspects of stem cell biology, such as the limits (if any) of the ability to reprogram them to produce progeny cells of diverse lineages, and the identities of the intrinsic and extrinsic molecular factors that regulate a cell's decisions whether to maintain stemness or to commit to differentiation have yet to be resolved.

A stem cell's location and immediate environment can be important factors in defining its role. Recent findings that stem cells may arise from one tissue type and then later be directed to give rise to another strongly substantiate the model in which a stem cell's behavior and properties are influenced by external signals. By studying the microenvironments, known as 'niches,' in which stem cells originate, abide and proliferate, the Nishikawa research group seeks to gain insight into the identity and function of molecules involved in stem cell activity, and the potential of stem cell-based therapeutic applications.

Characterizing the stem cell niche

Working to isolate the defining characteristics of specific stem cell niches, the Nishikawa group has chosen melanocyte stem cells as one of the main model systems for their research, as melanocytes are readily identified by the presence of a melanocyte-specific promoter, Dct, and are amenable to experimental manipulations that allow specific cell types to be isolated from mixed populations. As the existence of niche cells and the identification of their characteristics can only be determined by first isolating the stem cells they support, Nishikawa's lab has developed an approach in which stem cells are dissected from their surroundings, dissociated and used to construct single cell libraries, a process involving the technically challenging initial step of isolating single intact hair follicles from the skin. Targeted GFP expression is next used to label the follicular melanocytes, making it possible to distinguish them from other cells. These cells are used to construct cDNA libraries of (GFP-positive) melanocytes from the bulge and matrix regions of the follicle, as well as all of the GFP-negative cells from the bulge and the subset of GFP-negative cells found specifically to adhere to bulge melanocytes.

These follicular regions were selected based on the results of a previous study that showed that the bulge and matrix are the two main sites of localization of melanocyte stem cells, making them excellent sites for studies of niche properties. Each of these areas hosts a specific sub-population of stem cells. The matrix niche, the site of active hair growth and pigmentation, provides an environment in which amplifying progeny become committed to a stem cell fate, while the bulge serves as a reservoir for stem cells in their resting, or quiescent, state. Subtractive comparisons of cDNAs from stem cell, differentiated cell and 'niche-like' (keratinocyte) libraries revealed approximately 200 non-redundant genes, providing the lab with targets for further experiments in which the individual genes will be conditionally knocked out and the resulting phenotypes examined and analyzed. Other related studies, for which single cell libraries of other resting stem cells are now being constructed, will entail profiling gene expression in melanocytes and resting stem cells, which promises to provide a new resource for scientists investigating stem cells' genetic signatures.  

Differentiation of mesoderm and endoderm

In another ongoing project, members of the Nishikawa group are developing software to allow for improved DNA chip analyses and using it to build a database of the progressive intermediate states that endothelial cells pass through in the journey from ES (embryonic stem) to terminally differentiated cell. A number of mesoderm and endoderm cell lineages and their intermediates are being used, as these are available in pure cultures and their in vitro differentiation can be steered by the addition of the appropriate factors.

The mesendodermal lineage, which has been clearly defined at cell level by the Nishikawa group, includes a number of important cell types, including endothelial cells, the primary constituent cells in many important organs including the lungs, liver and pancreas. Mesendodermal differentiation is characterized by the activity of specific marker genes whose expression is switched on or off, depending on the pathway a cell has followed and the stage it has reached. Analysis of the stages in this stepwise process has also made it possible to develop a system for guiding differentiation from ES cell to endodermal, mesenchymal or mesodermal (paraxial and lateral) fates in vitro using combinations of extrinsic factors. By identifying the characteristic markers of cells at each step on each branch of the lineage tree and collecting samples to produce DNA arrays, the Nishikawa lab is working to build a database of cDNA profiles of the intermediate stages in ES cell differentiation in culture, which can be queried and visualized using software developed in the lab. Analyses of this cDNA chip database promise to lead to a better understanding of the process of endothelial differentiation, and of stem cell specification in general. This powerful new DNA analysis software application, which promises to make a great contribute to the harmonization of multiple DNA array databases, is being developed in collaboration with the Institute of Biomedical Research and Innovation, located adjacent to the CDB in the Kobe Biomedical Industry Project park.

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