Hans R Scholer
School of Veterinary Medicine
University of Pennsylvania

Stem cells are crucial ingredients in the progressive restriction of cellular developmental capacity during mammalian embryogenesis. Until recently, only embryonic stem cells (ESC) had been shown to exhibit the potential to differentiate into cells of all three germ layers. However, adult stem cells (ASC) can regain pluripotency (potential to differentiate into various lineages [1]) if transferred from one stem-cell microenvironment into another, but it is not known whether they pass through an ESC-like state. It is also unknown whether ASCs can de-differentiate into ESCs and be kept in culture as such. The POU transcription factor Oct4, expressed in ESCs and germ cells, is strongly implicated in the process of maintaining as well as regaining stem-cell pluripotency. The scientific hypothesis is that ESCs, ASCs, and germ cells can be converted into each other and that the function of Oct4 is essential for some of the underlying processes.

Schematic overview of the focus of our laboratory.
Currently, mice are used as a model organism to assess these processes in vivo.

One major focus of research is to generate autologous somatic cells of mouse by in vitro procedures and to understand the underlying processes. Our laboratory attempts to elucidate, at the molecular level, how a somatic cell nucleus is reprogrammed to ensure that the genetic program is maintained and to develop procedures that may be useful for stem cell therapies.

Part of the previous and current work is to define the function and regulation of Oct4 as a means to elucidate the regulatory network in stem and germ cells [2-5]. In those studies, knowledge and tools developed by our laboratory were applied. Previous research findings of our laboratory and relevant to this research focus are:

• The Oct4 gene is not active in any somatic cell, including adult stem cells [2, 6]
• The Oct4 gene is expressed in embryonic stem and germ cells [2, 6]
• Distinct genomic fragments of Oct4 are ESC- and germ-cell-specific. These fragments can be used both in mice and cultured cells to specifically express genes in either cell types, for example, the viable marker green fluorescent protein (GFP) was used in several studies in our laboratory [7-10]
• Expression of Oct4 protein is essential but not sufficient to maintain pluripotency of embryonic stem cells and survival of germ cells [5]

This research is comprised of several overlapping areas, of which two are discussed below.

Derivation of ESCs after nuclear transfer into oocytes.

The majority of mammalian clones die early in development, presumably as a consequence of nuclei failing to effectively reestablish a normal embryonic gene expression program. The role of Oct4 expression is crucial in this process. Abnormal Oct4 expression in the embryo was visualized in tandem with observed reprogramming failure in blastocyst-stage mouse clones and accounts for more than 90% of the embryos that fail to develop [11]. Whereas correct spatial and temporal expression, together with correct protein levels, appear to be prerequisites for embryonal development, the requirements to derive ESC lines are less stringent ([11] and unpublished).. In those studies, which were done in collaboration with the laboratory of Prof. McLaughlin at the Center for Animal Transgenesis and Germ Cell Research, transgenic mice were used that reproduce the endogenous expression pattern of Oct4 by GFP, as a marker for cellular pluripotency. Few cells within the early embryo expressing Oct4/GFP at the right level appear to be able to derive ESC lines. Without sufficient expression, none of the blastocyst outgrowths resulted in ESC lines, but with adequate Oct4 levels, about 30% of the outgrowths successfully gave lines, which is in the range of rates obtained with normal blastocysts.

A number of genetic and epigenetic problems that may be associated with reproductive cloning were discussed. Mutations are more likely to happen in genes that are not used in a certain cell-type of an organism. For example, muscle-specific genes are packed away in brain cells, because in the normal life of an organism they will be never used again. When mutated, the muscle-specific genes in brain cells are less likely to be repaired than brain-specific genes. Such phenomena are well known and were described several years ago [termed: transcription-coupled DNA repair]. Since the human body has more than 200 different cell types, it is not difficult to imagine which problems such mutations - that were silent in the one cell used for cloning - might cause in all the other cell types in the newly generated organism. Moreover, problems, like the poor quality of somatic DNA that becomes worse and worse with age, cannot be solved. It was discussed in detail why the birth of an organism generated by cloning will always be accompanied by organisms that die as fetuses, at birth or later in life. And one cannot prevent that organisms are born with malformations or sick based on the fact they were cloned organisms. Importantly, those that first look normal might become sick later. Even if the cloned organism is viable, there will be no guarantee that the next generation will not suffer or die due to genetic problems.

In contrast, cell lines obtained by nuclear transfer therapies promise much more potential. Several reasons were given. One is that cell lines can be tested and screened in the culture dish before they are used for therapy. Another is that one can use the nuclei of cells that are from the same organ as the one that should be repaired or at least from a closely related cell type, thus minimizing the risk of mutations in genes that need to be expressed after therapy.

The importance of research on cloning problems described in a recent publication [11] was shown when that publication was included in the Top 100 List of 2002 Science Stories (See January 2003 issue of Discover Magazine, also online http://www.discover.com/ under Recent Issues / No 1. “The Year of Cloning”).

Nuclear transfer to generate stem cells may turn out to be a powerful system, but it is considered problematic because so far it has required the usage of oocytes. As a way around this, our laboratory is trying to develop several alternative approaches that are presented in the first figure. One promising approach is described below.

Derivation of oocytes from ESCs

Thus far, it has not been possible to generate germ cells from ESCs. They are being studied in an in vitro system that allows them to obtain germ cells from ESCs. This system has important implications for development of targeting sites for male contraception, as well as providing insight into the multi-factorial causes of infertility. In particular, this system is used (1) to analyze genetic reprogramming after nuclear transfer of somatic cells and (2) to establish conditions by which autologous ESC lines can be derived. This discovery holds tremendous implications, as it would render the generation of embryonic stem cells from human embryos obsolete; an issue surrounded by much ethical debate and controversy. This system also holds great potential for the generation of new tissues and organs to replace those damaged by diseases, external conditions, and disabilities, such as Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.

References
[1] Boiani, M., Schöler, H.R., Determinants of pluripotency in mammals, in In: Principles of cloning, R. Lanza, Cibelli, J., and West, M., Editors. 2002. p. 109-152.
[2] Pesce, M., Schöler, H.R.: Oct4: Control of totipotency and germline determination. Mol. Reprod. Dev., 55, 452-457 (2000).
[3] Pesce, M., Gross, M.K., Schöler, H.R.: In line with our ancestors: Oct4 and the mammalian germ. Bioessays, 20, 722-732. (1998).
[4] Fuhrmann, G., Chung, A.C., Jackson, K.J., Hummelke, G., Baniahmad, A., Sutter, J., Sylvester, I., Schöler, H.R., Cooney, A.J.: Mouse germline restriction of oct4 expression by germ cell nuclear factor. Dev Cell, 1, 377-387 (2001).
[5] Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Schöler, H.R., Smith, A.: Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95, 379-391 (1998).
[6] Pesce, M., Schöler, H.R.: Oct4: Gatekeeper in the beginnings of mammalian development. Stem Cells, 19, 271-278 (2001).
[7] Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., Schöler, H.R.: Germline regulatory element of Oct4 specific for the totipotent cycle of embryonal cells. Development, 122, 881-894 (1996).
[8] Anderson, R., Copeland, T.K., Schöler, H.R., Heasman, J., Wylie, C.: The onset of germ cell migration in the mouse embryo. Mech Dev, 91, 61-68 (2000).
[9] Yoshimizu, T., Sugiyama, N., De Felice, M., Yeom, Y.I., Ohbo, K., Masuko, K., Obinata, M., Abe, K., Schöler, H.R., Matsui, Y.: Germline-specific expression of the Oct4/green fluorescent protein (gfp) transgene in mice. Dev. Growth Differ., 41, 675-684 (1999).
[10] Szabo, P.E., Hubner, K., Schöler, H.R., Mann, J.R.: Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev, 115, 157-160 (2002).
[11] Boiani, M., Eckardt, S., Schöler, H.R., McLaughlin, K.J.: Oct4 distribution and level in mouse clones: Consequences for pluripotency. Genes Dev., 16, 1209-1219 (2002).

Biographical Sketch

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