Stem cell-like properties of the endometrial side population: implication in endometrial regeneration.

BACKGROUND: The human endometrium undergoes cyclical regeneration throughout a woman's reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium. METHODOLOGY/PRINCIPAL FINDINGS: We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo. CONCLUSIONS/SIGNIFICANCE: These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis.

Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRalpha+Sca-1+CD45-TER119-) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

Two distinct stem cell lineages in murine bone marrow.

Mesenchymal stem cells (MSC), a distinct type of adult stem cell, are easy to isolate, culture, and manipulate in ex vivo culture. These cells have great plasticity and potential for therapeutic application, but their properties are poorly understood because of their low frequency and the lack of knowledge on cell surface markers and their location of origin. The present study was designed to address the undefined lineage relationship of hematopoietic and mesenchymal stem cells. Genetically marked, highly purified hematopoietic stem cells (HSCs) were transplanted into wild-type animals and, after bone marrow repopulation, the progeny were rigorously investigated for differentiation potential into mesenchymal tissues by analyzing in vitro differentiation into mesenchymal tissues. None/very little of the hematopoietic cells contributed to colony-forming units fibroblast activity and mesenchymal cell differentiation; however, unfractionated bone marrow cells resulted in extensive replacement of not only hematopoietic cells but also mesenchymal cells, including MSCs. As a result, we concluded that purified HSCs have no significant potency to differentiate into mesenchymal lineage. The data strongly suggest that hematopoietic cells and mesenchymal lineage cells are derived from individual lineage-specific stem cells. In addition, we succeeded in visualizing mesenchymal lineage cells using in vivo microimaging and immunohistochemistry. Flow cytometric analysis revealed CD140b (PDGFRbeta) could be a specific marker for mesenchymal lineage cells. The results may reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics. Disclosure of potential conflicts of interest is found at the end of this article.

Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/gamma c(null) immunodeficient mice.

Human uterine endometrium exhibits unique properties of cyclical regeneration and remodeling throughout reproductive life and also is subject to endometriosis through ectopic implantation of retrogradely shed endometrial fragments during menstruation. Here we show that functional endometrium can be regenerated from singly dispersed human endometrial cells transplanted beneath the kidney capsule of NOD/SCID/gamma(c)(null) immunodeficient mice. In addition to the endometrium-like structure, hormone-dependent changes, including proliferation, differentiation, and tissue breakdown and shedding (menstruation), can be reproduced in the reconstructed endometrium, the blood to which is supplied predominantly by human vessels invading into the mouse kidney parenchyma. Furthermore, the hormone-dependent behavior of the endometrium regenerated from lentivirally engineered endometrial cells expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescence imaging. These results indicate that singly dispersed endometrial cells have potential applications for tissue reconstitution, angiogenesis, and human-mouse chimeric vessel formation, providing implications for mechanisms underlying the physiological endometrial regeneration during the menstrual cycle and the establishment of endometriotic lesions. This animal system can be applied as the unique model of endometriosis or for other various types of neoplastic diseases with the capacity of noninvasive and real-time evaluation of the effect of therapeutic agents and gene targeting when the relevant cells are transplanted beneath the kidney capsule.