Given that stem cell populations are generally associated with conditions of cellular hyperproliferation and tissue remodeling, we have examined the possibility of stem cells isolated from menstrual blood. While previously it was suggested that mesenchymal stem cells are found in endometrial tissue , numerous other tissues also have been found to possess endogenous mesenchymal stem cell populations which do not necessarily correlate with angiogenesis. For example cells with mesenchymal stem cell properties have been found in liver [16, 17], lung , skin , pancreatic  and kidney tissues . It was our hypothesis that the extreme angiogenesis occurring during the build-up of the endometrium would allow for specialized populations of stem cells to accumulate which could be extracted by culture of menstrual blood. We observed that the adherent fraction of menstrual blood cells could be expanded up to 68 doublings without losing karyotypic normality or developing tumorigenic potential. The cells appeared to possess some markers of mesenchymal stem cells such as CD9, CD29, CD41a, CD44, CD59, CD73, CD90, and CD105 while lacking hematopoietic stem cell markers such as CD14, CD31, CD33, CD34, CD133, and the pan-leukocyte marker CD45. Additional characteristics however make this a unique population from endometrial mesenchymal stem cells based on: 1) higher rate of proliferation compared to control cord blood derived mesenchymal stem cells; 2) lack of STRO-1 expression; 3) expression of the embryonic stem cell marker Oct-4; and 4) high expression of matrix metalloproteases. Given the ability of these cells to differentiate into tissues representative of all three germ layer components, we have named these cells "endometrial regenerative cells" (ERC).
ERC appear to have high rate of proliferation in comparison to other control mesenchymal stem cells. The positive expression of Oct-4, but negative expression of Nanog and SSEA-4 on these cells may be similar in some ways to amniotic fluid derived stem cells in that they express some but not all embryonic stem cell markers as well as telomerase reverse transcriptase . One drawback of our experiments is that we did not perform functional assessment of telomerase activity using TRAP assays. These experiments are currently underway.
The possibility of ERC to be shed endometrial tissue-resident mesenchymal stem cells seems unlikely in light of several findings. Specifically, tissue stem cells of the endometrium have previously described to be bone marrow derived and to express CD34 and CD45 , markers which are not found on ERC. Tissue mesenchymal-like stem cells from the endometrium express STRO-1 (13a), a marker not found on ERC. Additionally, the proliferative rate of ERC (1 doubling every 19.4 hours) appears to be faster than that described for cells derived from putative uterine derived stem cells . Finally, it is interesting that culture of ERC with specific "differentiation media" was able to generate cells of all three germ lines, something which has not been reported for endometrial tissue stem cells. One possible explanation for the pluripotency of ERC may be that these cells have some relationship to the "circulating oocyte progenitors" described by Tilly's group. Specifically, it was reported that bone marrow derived cells have the potential to transdifferentiate into oocyte precursors and that the presence of these cells in peripheral blood and bone marrow fluctuated with menstrual phase . We are in the process of assessing ERC for expression marker's reported to be found on circulating oocyte precursors such as Vasa, Dazl and Stella. Another possibility is that ERC are involved in the angiogenesis phase of the menstrual cycle and contribute to the high level of tissue remodeling. In agreement with this hypothesis is the high level of matrix metalloprotease and growth factor production in comparison to control mesenchymal stem cell lines.
Regardless of biological significance, ERC appear to possess numerous advantages compared to other stem cell sources that make them attractive of future investigation. Firstly, the ease of collection of ERC allows for the creation of patient-specific banking. Given that the cells are expandable, as well as possessing ability to differentiate into various tissues, the cells can not only be banked until future use, but can also be expanded and pre-differentiated into various tissues so that patient-specific tissues are "on standby' and ready for use when needed. Other stem cell sources such as bone marrow and adipose tissue do not allow for such wide-spread expansion and ease of collection. Secondly, the finding that the cells can be expanded for 68 doublings without evidence of karyotypic or functional abnormalities implies that from one starting cell enough cells theoretically can be produced to treat every human being in the world. This relatively unlimited potential allows for generation of unique cell lines that can be transfected with different genes to induce specific effects. For example, cell lines can be engineered with angiogenic agents , neurotrophic factors , or to express insulin . Lastly, ERC appear to have several-fold higher expression of matrix metalloproteases as compared to stem cells of other lineages. Physiologically, it is known that major remodeling of tissue is associated with the process of menstruation. Given the potential role of these cells in remodeling the endometrium, it may be reasonable to suggest that these cells are useful for treatment of fibrotic conditions such as cirrhosis in which regenerative cells with tissue degradation activities are desired. These possibilities are currently under investigation by our laboratory.
In conclusion, we have discovered a novel stem cell source from the menstrual blood that is easily accessible, highly expandable in vitro, and possesses pluripotency. This cell population may become a practical solution of choice for autologous stem cell therapy.