RIKEN Center for Developmental Biology

2003 Annual Report

Self-renewal and pluripotency

Their ability to self-renew indefinitely and to differentiate into cells of all three germ layer types (a differentiative capacity termed 'pluripotency'), makes embryonic stem (ES) cells one of the most promising subjects of study in regenerative medicine, as well as an attractive model system for research into a spectrum of developmental processes. However, the mechanisms by which ES cells are able to maintain these capabilities are incompletely understood, and a better understanding of the stemness of these cells will be necessary in order to be able to take best advantage of their remarkable properties.

Two of the biggest challenges that now face stem cell research are the determination of the factors that allow ES cells to generate limitlessly self-renewable progeny, and the identification of molecules that direct the dividing ES cell to produce daughter cells of specific types. Hitoshi Niwa's research addresses both of these challenges, with the aims of developing solid scientific foundations and reliable technologies to support this exciting field of biomedicine.

Inducing differentiation

The development of methods by which undifferentiated ES cells can be prompted to commit to a specific cell lineage is a field of central importance to the stem cell research community. Studying factors identified in work on knockout mice, the Niwa research team has been engaged in the analysis of transcription factors with potential roles in the development of extraembryonic cell lineages, extraembryonic endoderm and trophectoderm.  Extraembryonic endoderm derivatives include the parietal and visceral endoderm, which give rise to the yolk sac covering embryos in utero during development, and trophectoderm derivatives, the source of the placenta that sustains the developing mammalian embryos.

In previous research, the Niwa lab showed that the overexpression of GATA transcription factors such as Gata-4 or Gata-6 resulted in the specific conversion of undifferentiated ES cells into extraembryonic endodermal cells, and that the exogenous expression of either of these factors simultaneously induced the expression of the other from the endogenous gene. New work suggests that the homeobox gene Cdx-2 can serve as a similar trigger for the induction of trophectoderm from ES cells expressing low levels of the pluripotency maintaining gene, Oct 3/4. Using regulable activation of Cdx-2 in vitro, Niwa successfully induced ES cells to differentiate into trophoblast stem cells. These results, in combination with the GATA study, have led to the development of a model in which Oct 3/4, when maintained at an appropriate mid-range level, inhibits the differentiation of ES cells into either the trophectodermal or primitive endodermal lineages by suppressing Cdx-2 and Gata-6, respectively.

Maintaining pluripotency

The POU-family transcriptional regulator Oct 3/4 was the first factor found to elicit multiple differentiative outcomes dependent on its expression level. ES cells expressing median levels of Oct 3/4 maintain their pluripotency, while its overexpression results in differentiation into primitive endoderm and mesoderm and its inhibition causes the cells to take up a trophectodermal fate. These findings established Oct 3/4 as a primary regulator of pluripotency in ES cells, but the function of this regulator in the commitment of more specific lineages remains an open issue.

In 2003, the Niwa lab contributed to a study that demonstrated a role for Oct 3/4 in neurogenesis promoted by stromal cell derived inducing activity. The researchers found that SDIA acts to maintain Oct 3/4 expression in ES cells, which appears to be important as a promoter of the differentiation of ES cells into neural lineages. ES cells from which Oct 3/4 had been deleted lost their ability to differentiate into neurons, while heightened levels of Oct 3/4 intensified the neurogenic effects of SDIA. This finding raises the possibility that, in addition to its function as a maintainer of pluripotency, Oct 3/4 also plays a second key role by regulating neuronal differentiation in a concentration-dependent manner.

ES cell growth in culture

This goal of controlling culture conditions to achieve specific outcomes is important to the growth of ES cells in vitro as well. The Niwa lab is working to develop a serum- and feeder-cell–free system for culturing mouse ES cells, which necessitates developing a detailed picture of both the extrinsic factors and the intrinsic networks that function in these cells in culture. It is known that a single ES cell in isolation will fail to proliferate, while colonies of such cells grow normally. It is also known that even isolated single cells can be induced to proliferate if the culture medium from a larger ES cell colony is transferred to the single cell's plate, suggesting that ES cells produce a growth-stimulating factor that acts by community effect. Niwa has developed an assay system to isolate candidate molecules for this putative 'stem cell autocrine factor' (SAF), and is actively pursuing the characterization of the most promising candidates. The results of these analyses should improve the ability of researchers to grow ES cells in culture, facilitating the study of these fascinating and potentially revolutionarily important cells.