Rutgers New Jersey Medical School

Back

References: Honda A et al, Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells. Proc Natl Acad Sci USA 2007;104:12389.

Summarized by: Mary Dovlatyan, Fall 2007

LAY REVIEW

Thecal cells are specialized cells that make up a layer of the ovarian follicles. Their primary purpose is the production of a hormone that is the precursor to estrogen and structural support which are performed by the theca interna and theca externa cells, respectively. Originally the primary goal of the authors was to isolate female germ line stem cells. Germ cells are those cells that contain half the DNA necessary for reproduction. They come from either a male or female and produce the egg and sperm of an organism. The discovery is that the apparent stem cells were instead, somatic cells rather than germ cells. Thus, the authors proceeded to characterize the isolated somatic cells as well as functional studies.

The thecal stem cells were isolated from ovaries of newborn mice, 2-4 days old ICR and green fluorescent mice. Granulosa cells that were not contaminated with thecal cells were also studied. Characterization was addressed by electron microscopy and gene expression analysis, which led to the supposition of theca stem cells.

Functional studies were determined in the ability of the cells to differentiate by using culture conditions that induce differentiation. For example when serum was added, the round colonies spread and appeared to begin hormone production. Additional growth factors led to the observation of specific organelles. The isolated granulosa cells, stated above, were added to the culture. This induced further differentiation of the theca stem cells. The newly formed granulosa cells were identified from the added granulosa cells by added granulosa cells from the “green” mice. This would be different from granulosa cells that are not expected to be green. The level of androstenedione, which is the hormone necessary for estrogen production, in the media also increased as the thecal cells differentiate.

The final stage in the authors’ study was to perform intraovarian transplantation of green thecal stem cells into non-green mice. The green stem cells were identified in the ovaries by fluorescence microscopy. Finally the host mice were given equine chorionic gonadotropin to produce follicular development. When the ovaries were removed and observed the green fluorescent cells proliferated and surrounded the fully developed follicles.

I found that this article informative and well written. More importantly, the authors clearly outlined their thought processes. They began with the studies with the intent to do something completely different and when they hit the obstacle of the cells being somatic cells rather than germ cells. The authors instead, used this obstacle as an opportunity to study something new. Also they raised questions throughout the article and then performed experiments to address their questions. For example if these were not germ cells what kind of stem cells were they? They used microscopy and gene expression profiling. One question is when they were applying the cells to different growth factors did true differentiation occur or was it an artifact of the media? I believe that these cells were actually stem cells because they were able to culture multiple passages and showed success in their transplantation. I believe that the addition of different growth factors created a microenvironment that facilitated differentiation. For example in the last stage they showed that granulosa cells were needed for final differentiation. In ovaries, granulosa cells, which are located next to the thecal cells may therefore be cellular component of the microenvironment for the differentiation of natural thecal cells. It would be wonderful to see future studies in animal models for translational studies with thecal stem cells. Also they could use these experiments to further study the effect of the microenvironment on stem cells and perhaps apply the findings to other types of adult stem cells.

SCIENTIFIC REVIEW

Thecal cells are specialized cells that make up a layer of the ovarian follicles. Their primary purpose is hormone production and structural support which are performed by the theca interna and theca externa cells respectively. Originally the primary goal of the authors was to isolate female germ line stem cells. However, instead of germ stem cells, the cells were somatic. The authors proceeded to characterize the isolated somatic cells and also performed functional in vitro and in vivo studies.

To obtain the thecal stem cells the authors initially collected ovaries from newborn mice that were 2-4 days old for cell culture. The in vivo studies were done with ICR and “green” fluorescent mice. When culturing these cells they were able to perform four to five passages before the cells stopped proliferating. They also cultured granulosa cells and eliminated contamination with thecal cells by molecular analyses. During the initial isolation of the thecal cells, a serum-free medium was used containing growth factors. The floating cells were then passaged to a second culture. The second culture resulted in small round colonies and stained similar to other stem cells. The cultured cells were also treated with BrdU. The small cell colonies that stained positive for BrdU, which indicated mitotic activity did not stain for MVH (a germ cell marker). The results indicated that the cells within the colonies were somatic cells rather than germ cells. In the next step, the authors identified the somatic cells as interstitial, based on electron microscopy. These cells were also distinguishable from granulosa cells by alkaline phosphatase reactions. Finally, gene expression analysis determined the expressions of theca cell markers, Ptch1 and Gli3, but not markers of granulosa cells. In total, the results suggested the isolation of thecal stem cells.

The next step performed in vitro differentiation of the theca stem cells from serum-free media. This was done by replating the cultured cells in five different conditions. Each condition contained serum and different growth factors. Each condition resulted in more matured cells, indicating variations of cell maturity. For example, when serum was added the original round colonies spread and appeared to begin steroidogenesis. As more growth factors were added, the cells showed higher levels of differentiation, which was apparent by the observation of organelles specific for steroidogenesis. As stated earlier granulosa cells were also isolated. When the media was supplemented with these cells, the thecal cells showed further differentiation. The theca cell-derived granulosa cells were identified from the added granulosa cells since the latter were obtained from the “green” mice. The level of androstenedione in the media also increased as the cells progressed in their level of differentiation.

The final stage in the authors’ study was to perform intraovarian transplantation of the thecal stem cells. The thecal cells that were used were from “green mice” and were transplanted into the ovaries of nontransgenic mice. Therefore the authors were able to determine if the thecal stem cells had proliferated in the ovaries by using fluorescence microscopy. They performed the transplantation by first removing the host ovary, injecting it with the donor thecal stem cells and then inserting the ovaries into the empty bursa of another host nontransgenic mouse. This procedure was replicated twice. The reason the donor cells were not directly injected into the ovaries while still in the animal is because this caused heavy bleeding and the donor cells did not survive. Finally the host mice were given equine chorionic gonadotropin to produce follicular development. When the ovaries were removed and observed the green fluorescent cells had proliferated and surrounded the fully developed follicles.

Back