UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

Studies of Germ Cell Development from hESCs

Human embryonic stem cells (hESCs) were first derived in 1998. Since that time, there has been much excitement regarding their potential for novel therapies. Yet, few studies have explored the basic properties of hESCs. We have characterized three major properties of independently-derived hESC lines, on the NIH registry, that we grow in my laboratory [HSF1 and HSF6 (from UCSF) and H1, H7, H9 and H14 (from WiCell)], in detail: (1) differentiation potential, (2) genetic imprinting status, and (3) gene expression and generation of hESCs.

These studies have led to several surprising results. First, we demonstrated that hESCs can differentiate not only to somatic cell lineages but also to the germ cell lineage. This is a fundamental property of mouse ESCs that has been used to distinguish “good from bad” lines. It indicates that hESCs possess potential beyond that which we initially expected and clearly indicates that hESCs are not restricted in their potential to sub-branches of somatic development. Furthermore, the ability to differentiate hESCs to the germline will revolutionize human genetic studies of infertility and germ cell development. For the first time, we can dissect the function of genes, such as the DAZ ( Deleted in AZoospermia) genes (which we previously identified), during germ cell development on the background of the human genome. Second, we observed that genetic imprinting, at different loci, is variable in different hESC lines. The human genome contains at least 77 imprinted loci; imprinting at each may ultimately bear upon the utility of hESCs in therapeutics as abnormal imprinting can lead to tumorigenesis. Our studies allowed us to probe this important property in hESCs and in human germ cells differentiated from hESCs. Third, we observed that gene expression in hESCs and in cells isolated from the inner cell mass is distinctly different indicating that these two cells types are not equivalent. Further, we noted that expression of germ cell specific proteins in hESCs is noted throughout the cell colonies but is absent in cells of the inner cell mass. These observations suggest to us that hESCs appear to be an epiblast derivative, most closely related to embryonic germ cells, that have developed beyond the initial state of inner cell mass cells. These basic studies form the foundation for our research and for the formation of a program in human embryonic stem cell biology as described below.

The Program in Human Embryonic Stem Cell Biology

The newly developed tools of human genetics and human embryonic stem cell biology have strengthened our resolve to pursue human biology. Yet, human genetics has long suffered from the lack of a rigorous system to experimentally manipulate human genes of interest and likewise, human stem cell biology suffers for lack of genetic rigor in its early stages. An overall goal of the Program in Human Embryonic Stem Cell Biology is to establish a combined program of human genetics and stem cell biology that allows the use of stem cells to address human genetic questions and vice versa. In addition, the program will train physicians in rigorous genetics and stem cell biology and train basic scientists in rigorous analysis of clinical problems.

Experiments in my own laboratory will focus on characterization of fundamental properties of hESCs, in particular, the analysis of cell types differentiated, imprinting and gene expression. These experiments will require the same reagents that are already, or soon will be, required by other UCSF researchers: (1) multiple NIH-approved lines, (2) newly-derived lines cultured on human feeder cells, (3) ability to silence, disrupt and overexpress genes specifically in human ES cells, and (4) ability to transplant labelled cells to an appropriate model in vivo (primates or rodents). Thus, the Program in Human Embryonic Stem Cell Biology fuses the activities of generation of hESC lines and genetic modification and transplantation of undifferentiated and differentiated cell types. The fusion of hESC biology with human genetics will increase the power of both at UCSF. The program is co-directed by me (UCSF School of Medicine) and Dr. Susan J Fisher (Tissue and Cell Biology; UCSF School of Dentistry).

Selected References

Moore FL, Jaruzelska J, Fox M, Urano J, Firpo MT, Turek PJ, Dorfman DM, Reijo Pera RA. (2003) Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-Like proteins. Proc. Natl. Acad. Sci. USA 100, 538-43.

Clark AT, Bodnar MS, Fox MS, Rodriquez RT, Abeyta MJ, Firpo MT, Reijo Pera RA (2004) Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum. Mol. Genet.13, 727-39

Clark AT, Rodriguez R, Abeyta MJ, Firpo MT, Reijo Pera RA (2004) Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hot-spot for teratocarcinoma. Stem Cells 22, 169-79.

Abeyta MJ, Clark AT, Rodriguez R, Reijo Pera RA and Firpo MT (2004) Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum. Mol. Genet. 13, 601-8.

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