Endometrial stem/progenitor cells: the first 10 years

Bone marrow has been proposed as a source of cells that cross lineage barriers to differentiate into specialized cell types of several organs including the endometrium ( Krause et al. , 2001 ; Du and Taylor, 2009 ). Bone marrow comprises small populations of haematopoietic, mesenchymal and endothelial stem/progenitor cells and large numbers of myeloid cells at various stages of differentiation. Several reports suggest that bone marrow gives rise to endometrial stromal, epithelial and endothelial cells. These studies used donor-specific markers to identify cells from transplanted bone marrow in the endometrium of recipient patients, mice and baboons. As the endometrium is an immunologically active tissue that recruits bone marrow-derived immune (myeloid and lymphoid) cells, the identity of bone marrow-derived endometrial cells needs to be carefully verified by the absence of immune cell markers and/or the presence of endometrial cell-specific markers to distinguish between a stem cell and immune cell origin. The first study to report bone marrow-derived endometrial cells examined endometrium from patients who had received HLA-mismatched bone marrow transplants ( Taylor, 2004 ). Donor-derived cells were detected by reverse transcriptase–polymerase chain reaction and immunostaining for their HLA type and their phenotype determined by location and morphology. Bone marrow-derived cells accounted for up to 48% of the epithelial cells and 52% stromal cells. Subsequent studies using the Y-chromosome as a marker of bone marrow-derived cells in the endometrium of patients receiving sex-mismatched bone marrow transplants also reported contributions to epithelia and stroma, albeit at more modest levels of <10% ( Ikoma et al. , 2009 ; Cervelló et al. , 2012 ). In keeping with reports of bone marrow-derived endothelial progenitors, bone marrow-derived endometrial endothelial cells have also been reported ( Mints et al. , 2008 ). However, bone marrow-derived cells did not contribute to the endometrial SP of putative stem/progenitor cells in the human endometrium ( Cervelló et al. , 2012 ). Thus, the question still remains whether the bone marrow-derived cells incorporating into the human endometrium are stem cells or immune cells. Mouse models have also been used to examine the role of bone marrow-derived cells in endometrial regeneration. Bone marrow transplantation studies, using conditioning to ablate host marrow stem cells and allow haematopoietic engraftment, reported donor bone marrow-derived stromal, epithelial and endothelial cells in the mouse endometrium ( Bratincsák et al. , 2007 ; Du and Taylor, 2007 ; Mints et al. , 2008 ; Du et al. , 2012 ; Morelli et al. , 2013 ). Factors driving the formation of bone marrow-derived endometrial cells include estrogen, which increased the incorporation of bone marrow-derived endothelial progenitors into uterine vasculature ( Masuda et al. , 2007a ). Hormone-driven endometrial cycling showed no detectable effect on rates of bone marrow cell integration into endometrial stroma and epithelium ( Du et al. , 2012 ), arguing against a proposed role for bone marrow-derived cells in cyclic regeneration. However, uterine ischaemia or trauma approximately doubled the rates of stromal engraftment, without increasing epithelial engraftment ( Du et al. , 2012 ; Alawadhi et al. , 2014 ). These observations suggest a role for bone marrow-derived stromal cells in repairing endometrial injury, rather than normal cyclic regeneration. Exposure to cigarette smoke decreases the recruitment of both stromal and epithelial bone marrow-derived cells, a finding linked to both infertility and reduced rates of endometriosis in smokers ( Zhou et al. , 2011 ). An important aspect of this type of study is the ability to clearly distinguish endometrial cells from bone marrow derived-leukocytes through co-immunolocalization and confocal microscopy. Several micrographs of uterine CD45 immunostaining in some studies of bone marrow-derived endometrial cells ( Du et al. , 2012 ; Alawadhi et al. , 2014 ) are unusually devoid of CD45 + cells, suggesting a low detection rate for leukocytes, possibly leading to misclassification of bone marrow-derived leukocytes as stromal and epithelial cells. Most studies of bone marrow-derived endometrial cells have not addressed the type of bone marrow cells contributing to the endometrium. Protocols used have been typically designed to facilitate engraftment of HSCs. HSCs have been widely reported to give rise to non-haematopoietic lineages in other tissues ( Krause et al. , 2001 ), but this has been disputed ( Wagers and Weissman, 2004 ). In support of HSCs as the source of bone marrow-derived endometrial cells, transgenic mouse models tracing the CD45 + bone marrow haematopoietic lineage demonstrated the existence of bone marrow-derived endometrial epithelium in a small number of animals ( Bratincsák et al. , 2007 ). In contrast, human patients and baboons transplanted with mobilized and purified HSCs failed to exhibit evidence of HSC-derived endometrial stroma ( Wolff et al. , 2013 ). Also arguing against HSC-derived contributions to the endometrium is the finding that granulocyte-colony stimulating factor, which mobilizes bone marrow HSCs, reduced the engraftment of bone marrow-derived stromal cells in the endometrium ( Du et al. , 2012 ). It was proposed that bmMSC, a population not mobilized by granulocyte-colony stimulating factor, rather than HSC, generated endometrial stroma. Interpretation of these studies is further complicated by the finding of a resident population of uterine haemangioblasts that generate HSCs and vascular cells in the murine endometrium ( Sun et al. , 2010 ). The concept of bone marrow as a source of transdifferentiating cells responsible for cellular replacement has been investigated in many organs including lung, liver, kidney, intestine and skin ( Krause et al. , 2001 ). Studies of these organs tell a cautionary tale relevant to ongoing work on bone marrow-derived cells in endometrial repair and regeneration. Initial enthusiasm for the concept of bone marrow transdifferentiation ( Petersen et al. , 1999 ; Krause et al. , 2001 ; Gupta et al. , 2002 ) was tempered by subsequent reports that genuine transdifferentiation of bone marrow-derived cells is either very rare or cannot be detected ( Wagers et al. , 2002 ; Duffield et al. , 2005 ; Kotton et al. , 2005 ). Cell fusion, artefactual marker staining and overlaying leukocytes are commonly cited as sources of cells that may be misinterpreted as transdifferentiated cells ( Wang et al. , 2003 ; Duffield and Bonventre, 2005 ). During the ongoing debate on bone marrow cell transdifferentiation, a common emerging theme is that rigorous technical analysis and multiple markers must be used to definitively identify and determine the phenotype of candidate bone marrow-derived cells in any organ ( Duffield and Bonventre, 2005 ; Kassmer and Krause, 2010 ). The existence of bone marrow-derived endometrial cells has yet to receive the level of scrutiny applied to many other organs.

It is 10 years since the first evidence for the existence of adult stem cell populations in the endometrium was published. In this study, rare clonogenic cells or colony-forming units (CFUs) were identified in purified populations of human endometrial epithelial and stromal cells isolated from hysterectomy tissue ( Chan et al. , 2004 ). Concurrently, it was reported that some epithelial and stromal cells in human endometrium of HLA-antigen-mismatched bone marrow transplant recipients were of donor origin ( Taylor, 2004 ). Subsequently, label-retaining cells (LRCs) were identified in mouse endometrium ( Chan and Gargett, 2006 ). This early direct evidence of stem/progenitor cells in the endometrium was then summarized in the first comprehensive review on endometrial stem/progenitor cells published in Human Reproduction Update ( Gargett, 2007 ). Later in 2007, a second publication on murine endometrial LRCs confirmed and extended the original findings ( Cervelló et al. , 2007 ). The 2007 Human Reproduction Update review also provided a blueprint on how to identify stem/progenitor populations in tissues and organs not previously characterized for adult stem cell activity, focusing on functional assays used in other organs. These included CFU activity, self-renewal, differentiation, proliferative potential, label retention and tissue reconstitution assays. It pointed out the importance of linking stem cell markers to functional stem cell activity. It also summarized the indirect evidence for stem/progenitor cells in the highly regenerative human endometrium gleaned from the literature. In this comprehensive review, we summarize the progress that has been made on the identification and characterization of endometrial stem/progenitor cells in both human and mouse models since this last review. We will focus on the identity and in vivo location of the stem/progenitor cells as specific markers and approaches that have now been identified for their purification, particularly for the mesenchymal stem/stromal cell (MSC) population. Specific markers also allow ‘omics' characterization of endometrial stem/progenitor cell populations. The role of bone marrow-derived and endogenous stem/progenitor cells in endometrial proliferative disorders, including endometriosis, adenomyosis, thin dysfunctional endometrium and Asherman's syndrome, will also be covered. The review will also describe the use of the endometrial MSCs (eMSCs) as potential cell-based therapies for several women's health and other diseases. Finally, we will raise unresolved issues facing the field, particularly the similarities and differences between eMSCs and endometrial stromal fibroblasts and the identity of bone marrow-derived cells involved in endometrial function.

Identification of specific markers for eMSCs enables profiling of these stem/progenitor cells and their differentiated progeny to identify their signatures. Comparison with bmMSCs, adipose MSCs and placental MSCs also becomes possible. This work has commenced and already shows differences between the perivascular-derived MSCs and the stromal fibroblast and their decidualized derivatives ( Spitzer et al. , 2012 ; Murakami et al. , 2014 ). RNA sequencing will uncover regulatory pathways involved in the endometrial stem/progenitor cell function and their differentiation pathways allowing comparisons between cells from normal and dysfunctional endometrium and ectopic endometrial lesions of adenomyosis and endometriosis. Epigenetics governs cellular phenotype, particularly during cellular differentiation. Interrogation of the epigenetic profiles of endometrial stem/progenitor cells and stromal fibroblasts will uncover regulatory pathways, governing the function of these cell types. Similarly, once markers for endometrial epithelial progenitors are determined, their chromatin state can be determined and compared between well-characterized cell phenotypes, shedding light on the regulation of the adult stem cell state and its differentiation. These advances in sequencing technologies are matched by the availability of single-cell analysis using Fluidigm ® technologies, enabling investigations into cell heterogeneity. For example, SUSD2 has a wide range of expressions on individual cells, but the function and phenotype of low versus high expression are currently unknown (unpublished), but could be determined using single-cell analysis and deep sequencing or epigenetic profiling. eMSCs and menstrual blood ERCs are showing promise for cell-based therapies for gynaecological disease and non-homologous use. The properties of eMSCs are favourable for further development in regenerative medicine applications as they are relatively easy to obtain compared with the current commonly used sources. Regenerative medicine frequently requires biomaterials and scaffolds to deliver cells to the injured site. These can be fabricated ( Edwards et al. , 2013 ) or can be derived from decellularized uterine matrix for endometrial applications ( Miyazaki and Maruyama, 2014 ), the latter with potential application for Asherman's syndrome or IUA. Once epithelial progenitor markers are identified, these and the MSCs could be used to seed these decellularized matrices.

This study was funded by National Health and Medical Research Council of Australia grants 1085435 (C.E.G. and J.A.D.) and 1081944 (C.E.G.) and Senior Research Fellowship ( 1042298 , C.E.G.), Royal Australian and New Zealand College of Obstetricians and Gynaecologists (K.E.S.) and Victorian Infrastructure Support Program. Funding to pay the open access publication charges for this article was provided by the National Health and Medical Research Council of Australia grants ID1085435 and ID1042298.

The published literature was searched using the PubMed database with the search terms ‘endometrial stem cells and menstrual blood stem cells' until December 2014. Only original articles in English were included. The review includes human, mouse and domestic animal studies.

C.E.G. was involved in conception and design, acquisition of data, analysis and interpretation of data, writing the article and revising it critically for important intellectual content plus gave final approval of the version to be published. K.E.S. provided data acquisition, analysis and interpretation of data, drafting the article and gave final approval of the version to be published. J.A.D. was involved in acquisition of data, analysis and interpretation of data, writing the article and revising it critically for important intellectual content plus gave final approval of the version to be published.

The ease with which endometrial tissue can be obtained by Pipelle biopsy without anaesthetic in comparison to the collection of bone marrow aspirates or adipose tissue liposuctions makes it an attractive source of MSCs for regenerative medicine ( Ulrich et al. , 2013 ). Menstrual blood is an even easier source for collection of MSC-like cells, although greater attention is required to ensure sterility of the cell product and methods for the purification of eMSCs from this source have not been refined. The potential of cultured menstrual blood ERCs as a regenerative medicine therapy for a range of allogeneic non-homologous applications has been examined in several preclinical animal models. In a rat model, cell sheet technology was used to patch an infarcted heart, resulting in ERC incorporation and differentiation into cardiomyocytes, and improved cardiac function ( Hida et al. , 2008 ). Similarly, ERC transdifferentiated into skeletal muscle myocytes by fusion in a Duchenne muscular dystrophy mouse model ( Cui et al. , 2007 ). In a rat stroke model, cultured ERCs sorted for CD117 expression showed neuroprotection by differentiating into neuronal cells, improving motor and neurologic impairments ( Borlongan et al. , 2010 ). Cultured ERCs have reversed ovarian damage in a cyclophosphamide-induced mouse model of premature ovarian failure ( Liu et al. , 2014 ). In this model, locally injected ERC labelled with a fluorescent DiO dye showed greater retention than DiO-labelled human fibroblasts (source unknown) in the ovaries after 14 days. The DiO-labelled cells also expressed ovarian markers, AMH, FSH receptor, inhibin-α and -β. cDNA profiling revealed greater similarity in gene expression with human ovaries for mice treated with ERCs compared with human fibroblasts. Ovarian weight increased and function improved as judged by normalized circulating estradiol and FSH levels in ERC-treated mice. There were also fewer atretic follicles and more normal follicles. These studies indicate that ERCs have superior retention and reparative action than fibroblasts (presumably dermal) when delivered locally to injured ovaries. Tissue engineering principles are being developed using ERC for eventual clinical translation. ERCs cultured on polycaprolactone nanofibres showed greater proliferative capacity and ability to differentiate into chondrocytes than bmMSCs ( Kazemnejad et al. , 2012 ). In evaluating ERCs for a potential cardiac tissue engineering application, a greater ability was observed to penetrate silk fibroin scaffolds and proliferate than bmMSCs ( Rahimi et al. , 2014 ). Further development of these tissue engineering constructs and their evaluation in vivo are warranted for ERCs, given their greater proliferation rates and easier acquisition than other sources of MSCs. Summarized in a recent review ( Ulrich et al. , 2013 ) are several case reports on the treatment of patients with various disorders using systemically delivered, cultured ERCs. In addition, a phase 2 clinical trial, the RECOVER-ERC launched by Medistem for treating congestive cardiac failure ( Bockeria et al. , 2013 ), is also detailed. Although there have been no side effects reported, the clinical data have not yet been published. The potential of menstrual blood stromal fibroblasts for regenerative medicine purposes looks promising for non-homologous use. However, more research is required to compare local versus systemic delivery and to determine the mechanism of action by using genetic labelling to track the cells more accurately in appropriate preclinical animal models to generate sufficient confidence for translating this potential into the clinic. Pelvic organ prolapse (POP) is a major hidden disease burden for women, a legacy of vaginal birth for 50% of postmenopausal parous women ( Nygaard et al. , 2008 ). POP is the herniation of the pelvic organs into the vagina causing urinary and faecal incontinence, voiding and sexual dysfunction. Many women (19%) have a lifetime risk of undergoing reconstructive surgery for POP ( Smith et al. , 2010 ; Wu et al. , 2014a ) and ∼30% require additional operations due to failure of native tissue surgery or from significant adverse events associated with the use of vaginal mesh ( Olsen et al. , 1997 ; FDA, 2011 ). It has been proposed that autologous eMSCs used together with new mesh designs matching the biomechanical properties of vaginal tissue ( Ulrich et al. , 2012 ; Edwards et al. , 2013 ) in a tissue engineering construct may improve surgical outcomes of vaginal mesh surgery for POP ( Gargett, 2006 ; Ulrich et al. , 2013 ). In a proof-of-principle experiment, culture-expanded SUSD2 + DiO-labelled eMSC seeded on new mesh (polyamide/gelatin composite, 2.5 × 10 5 cells) improved tissue integration of the mesh and the biomechanical outcomes of the tissue/mesh complex after 90 days of implantation in a fascial defect wound of immunocompromised nude rats, despite surviving at the site for only 14 days ( Ulrich et al. , 2014a ). The eMSC promoted early neovascularization around the implanted mesh (7 days). An early inflammatory response was also observed around the mesh with eMSCs, characterized by increased numbers of M1 macrophages, but by 30 days these had differentiated into the M2 wound-healing phenotype and at 90 days the chronic macrophage response was significantly reduced. Similar quantities of new tissue collagen were deposited whether or not eMSCs were present, but more physiological, crimped collagen was observed in the mesh with eMSCs ( Edwards et al. , 2015 ). This led to a more compliant, less stiff mesh/tissue complex than mesh alone, addressing one problem associated with the use of clinical polypropylene mesh ( FDA, 2011 ). This promising result in a heterologous small animal model of POP repair surgery has led to the development of an autologous large animal, ovine preclinical model of POP using ovine eMSCs ( Ulrich et al. , 2014b ). This animal model is required to determine whether the mechanism of action of locally delivered eMSCs is due to paracrine release of factors promoting wound repair or whether they differentiate and incorporate into the dermis and smooth muscles of the vaginal wall ( Atala, 2009 ; von Bahr et al. , 2012 ). In vitro , TGFβ1 and platelet-derived growth factor BB (PDGF-BB) induced differentiation of SUSD2 + eMSCs seeded on the polyamide/gelatin meshes into SUSD2 − smooth muscle cells expressing SM22α and smooth muscle myosin heavy chain, intermediate and late smooth muscle cell differentiation markers ( Su et al. , 2014 ). Connective tissue growth factor also differentiated eMSCs into collagen-producing fibroblasts. It remains to be determined whether autologous eMSCs delivered on the new mesh will undergo differentiation to these tissue-forming cells in vivo . To prepare eMSCs or ERCs for cell-based therapies, it is necessary to incorporate the principles of Good Manufacturing Practice guidelines into the isolation and culture expansion process to ensure that the cells produced are of desired quality, safety and efficacy for each batch ( Eaker et al. , 2013 ; Hunsberger et al. , 2015 ). This requires the removal of all animal products (xeno-free, XF) used in the dissociation of tissue, cell purification, cryopreservation and in culture expansion protocols. In particular, bovine foetal calf serum needs to be replaced in the development of optimized SF culture medium, an area of active development for MSCs from other sources. Initial steps towards the optimization and scale-up or scale-out culture of CD146 + PDGFRβ + eMSCs examining commercial and in-house XF and SF media formulations found that eMSC attachment and proliferation were optimal on a fibronectin matrix in an in-house Dulbecco's modified Eagle's medium/F-12 SF medium containing FGF2 and EGF in physiological hypoxia (5% oxygen) ( Rajaraman et al. , 2013 ). eMSCs cultured under these optimized conditions retained their MSC properties. The specific markers used to enrich eMSCs (PDGFRβ, CD146 and SUSD2) were more informative in discriminating between the various media and matrix combinations tested than the classic MSC markers (CD29, CD44, CD73 and CD105), pointing to the value of more specific perivascular markers. Further research is needed to determine the optimal XF conditions for eMSC and ERC culture expansion. In addition to their progenitor properties, MSCs have anti-inflammatory and immunomodulatory properties, regulating both the innate and adaptive immune systems, that have been exploited clinically ( Le Blanc and Mougiakakos, 2012 ; Bianco et al. , 2013 ). Tissue fibroblasts also have these anti-inflammatory and immunomodulatory properties ( Haniffa et al. , 2009 ; Bianco et al. , 2013 ), and this is why cultures of unfractionated adherent bone marrow cells containing predominantly stromal fibroblasts have been effective in many studies and clinical trials. In a proinflammatory environment, resident MSCs exposed to the inflammatory cytokine tumour necrosis factor-α and/or interferon-γ are activated to secrete anti-inflammatory mediators ( Krampera, 2011 ). These mediators include IL-10, prostaglandin E2, TGFβ, HLA-G, indoleamine oxidase and nitric oxide, which together with cell–cell interaction suppress dendritic, T, B and NK cell activation ( Le Blanc and Mougiakakos, 2012 ). The low level of expression of major histocompatibility complex II and co-stimulatory molecules confers MSCs with low alloreactivity, enabling them to be used for allogeneic transplantation. MSCs can also switch macrophages from an inflammatory M1 to a reparative M2 phenotype ( Krampera, 2011 ). Although no in vitro studies have examined the immunoregulatory function of human eMSCs or ERCs, transcriptional profiling of fresh CD146 + PDGFRβ + and cultured SUSD2 + and SUSD2 − cells revealed that eMSCs expressed inflammatory and immunomodulatory genes ( Spitzer et al. , 2012 ; Murakami et al. , 2014 ). The SUSD2 − stromal fibroblasts secreted more chemokines and inflammatory mediators than SUSD2 + eMSCs, but both cell types secreted similar levels of many cytokines and chemokines including the Th-2-associated IL-4 and anti-inflammatory IL-10 ( Murakami et al. , 2014 ). In contrast, in vivo studies showed that eMSCs and ERCs have anti-inflammatory and immunomodulatory properties. Locally delivered eMSCs reduced the chronic inflammatory response to implanted synthetic mesh in a nude rat fascial wound repair model and promoted the switch of M1 to M2 macrophages, resulting in improved biocompatibility compared with controls ( Ulrich et al. , 2014a ). Cultured endometrial stromal cells and ERCs suppressed neuroinflammation in an autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis ( Peron et al. , 2012 ) and bowel inflammation in a colitis model ( Lv et al. , 2014b ), respectively. In the multiple sclerosis model, endometrial stromal fibroblasts were delivered intraperitoneally 24 h prior to EAE induction. In the colitis model, ERCs were systemically delivered several times after disease induction. Both models showed improved clinical symptoms and histological scores, reduced leukocyte infiltrates, lower inflammatory cytokine transcription in the damaged organs and upregulated anti-inflammatory cytokine transcripts in the spleens. There were also fewer active T cells, cytotoxic T cells and dendritic cells and more regulatory T cells in the spleens of animals receiving cells. In these models, the eMSCs, endometrial stromal fibroblasts and ERCs acted in a paracrine manner to suppress inflammation, with the latter two cell types also downregulating immune responses. These findings concur with many observations reported for MSCs derived from other sources, suggesting that eMSC-like cells have similar anti-inflammatory and immunoregulatory properties.

The authors have nothing to declare.

In the absence of specific markers for identifying mouse endometrial stem/progenitor cells, label retention was initially used to characterize their phenotype and in vivo location. The quiescent or slow-cycling phenotype of many quiescent adult stem cells allows their identification by label retention assays in mice (Table  I ). The thymidine analogue bromodeoxyuridine (BrdU) is typically delivered as a pulse during development or remodelling and incorporates into the DNA of actively dividing cells. A chase period follows, when actively dividing cells dilute the label below detectable levels while quiescent and slow-cycling cells retain detectable label. The timing of the initial pulse and the length of the chase are critical variables in determining which cells incorporate and retain detectable label ( Gargett et al. , 2007 ). Several labelling and chase regimes have been used to identify LRC in the stroma and epithelium of mouse endometrium ( Chan and Gargett, 2006 ; Cervelló et al. , 2007 ; Chan et al. , 2012 ; Patterson and Pru, 2013 ; Cao et al. , 2014 ) (Table  VI ). A transgene-based label retention system was also used in the murine female reproductive tract, with labelling initiated by antibiotic-inducible expression of green fluorescent protein (GFP)-labelled histones (H2B-GFP) ( Wang et al. , 2012c ; Patterson and Pru, 2013 ). Table VI Summary of label retention papers published 2006–2014. Label Pulse Chase LRC present after chase Comments References BrdU PND 3–5P PND 19–21 Up to 12 weeks Up to 10 weeks L, G, S L, S Epithelial LRCs are Esr1 − Stromal LRCs are Esr1 +/− , αSMA + , Sca-1 −a Chan and Gargett (2006) BrdU PND 3–5 8–10 weeks S Some LRCs express Oct-4 and c-Kit Cervelló et al. (2007) BrdU Adult, model of menstrual breakdown and repair 4.5–8.5 days L, G Glandular epithelial LRCs proliferate following epithelial repair Kaitu'u-Lino et al. (2010) BrdU PND 3–5 4 and 8 weeks L, S Epithelial LRCs proliferate after estrogen LRCs initiate epithelial proliferation in prepubertal endometrium 12% of stromal LRCs proliferate after estrogen Chan et al. (2012) BrdU PND 19–22 Up to 11 weeks L, S Luminal epithelial LRCs at 5 weeks chase Stromal LRCs persist through pregnancy and proliferate postpartum Stromal LRCs express CD140b (46%), CD146 (2%), CD44 (24%), CD90 (45%), Sall4 (34%), Sca-1 (72%); ABCG2 − Cao et al. (2014) H2B-GFP Adult cycling Up to 47 weeks G, S Endometrial epithelial LRCs lost within 4 weeks Long-term epithelial LRCs in distal oviduct Long-term epithelial LRCs are Esr1 − , CD44 − , Sca-1 − , Lgr5 − , c-Kit − Wang et al. (2012c) H2B-GFP ED 13.5–PND 21 PND 21–40 Up to 47 weeks 8 months L, G, S G Endometrial LRCs are short-lived (<5 weeks) Long-term epithelial LRCs in distal oviduct and endocervical transition zone Epithelial LRCs persist Patterson and Pru (2013) ED, embryonic day; PND, postnatal day; L, luminal epithelial; G, glandular epithelial; S, stromal. a About16% stromal LRCs are Esr1 + and 84% Esr1 − . Summary of label retention papers published 2006–2014. ED, embryonic day; PND, postnatal day; L, luminal epithelial; G, glandular epithelial; S, stromal. a About16% stromal LRCs are Esr1 + and 84% Esr1 − . Postnatal (days 3–5) or prepubertal (days 19–21) administration of BrdU provides a window to label developmentally active stem/progenitor cells expected to reside in the endometrium. Stromal LRCs produced by this protocol were detectable after a chase in excess of 9 weeks ( Chan and Gargett, 2006 ; Cervelló et al. , 2007 ) (Table  VI ). Label retention studies using the H2B-GFP system during embryonic, early postnatal development and adulthood also produced stromal LRCs after a 3–8-week chase ( Wang et al. , 2012c ; Patterson and Pru, 2013 ). Stromal LRCs from postnatal or prepubertal BrdU labelling were detected at the endometrial–myometrial junction, beneath the luminal epithelium, or in a perivascular location near CD31 + endothelial cells ( Chan and Gargett, 2006 ). LRCs did not express CD45, demonstrating that they were not infiltrating leukocytes ( Chan and Gargett, 2006 ). Stromal LRCs expressed the stem cell markers Oct-4, c-Kit ( Cervelló et al. , 2007 ), CD140b, CD146, CD44, CD90 and Sall4 ( Chan and Gargett, 2006 ; Cao et al. , 2014 ). Sca1 was absent from postnatal-derived LRCs but expressed in prepubertal-derived LRCs ( Chan and Gargett, 2006 ; Cao et al. , 2014 ). Postnatal-derived LRCs in the perivascular zone expressed α-smooth muscle actin (αSMA), suggesting that they are perivascular cells or pericytes ( Chan and Gargett, 2006 ). A small proportion (16%) of stromal postnatal or prepubertal-derived LRCs expressed Esr1, the predominant ESR involved in estrogen-mediated endometrial regeneration. In summary, stromal LRCs in postnatal and prepubertal models are heterogeneous populations, and further investigation is required to determine whether subpopulations of LRCs are the MSCs of the mouse endometrium. Examining LRCs in mouse models of endometrial regeneration may identify which subpopulation of stromal LRCs functions in generating new stromal vascular tissue (see later section). Epithelial LRCs in postnatal and prepubertal models are absent or very rare after a 3–4-week chase (Table  VI ) ( Chan and Gargett, 2006 ; Cervelló et al. , 2007 ; Patterson and Pru, 2013 ). The shorter persistence of epithelial LRCs is due to higher rates of epithelial cell proliferation, particularly under the influence of estrogen once estrous cycling begins at ∼4 weeks of age ( Chan and Gargett, 2006 ). Epithelial LRCs were predominantly in the luminal rather than glandular epithelium, reflecting the higher turnover of luminal epithelium during development that facilitates labelling and subsequent dilution ( Chan and Gargett, 2006 ). These luminal epithelial LRCs did not express Esr1, unlike most non-labelled epithelial cells. Epithelial LRCs do, however, proliferate in response to estrogen, pointing to an indirect effect mediated via neighbouring Esr1 + cells ( Chan and Gargett, 2006 ; Chan et al. , 2012 ). Glandular epithelial LRCs were rare in postnatal or prepubertal models and have not been characterized in detail. H2B-GFP labelling spanning embryonic development to postnatal day 21 yielded highly persistent epithelial LRCs (9–13-week chase) in the distal oviduct and endocervical transition zone, but not endometrium ( Wang et al. , 2012c ). Peripubertal H2B-GFP labelling (postnatal days 21–40) gave rise to glandular LRCs that persisted for 8 months and through several pregnancies, further emphasizing differences between glandular and luminal LRCs ( Patterson and Pru, 2013 ). In contrast, H2B-GFP labelling in adult cycling mice did not produce long-term glandular LRC in the endometrium, suggesting that the peripubertal phase is a unique developmental window when some glandular epithelial development is permanently completed. Long-term epithelial H2B-GFP LRCs were reported in the distal oviduct after labelling of adult cycling mice ( Wang et al. , 2012c ). The LRC approach does not definitively identify stem/progenitor populations. It does, however, provide insight into patterns of development, rates of cell turnover and reactivation during endometrial regeneration and repair. LRC experiments highlight the higher turnover of luminal epithelium, relative to glandular epithelium and the stromal compartment. These observations suggest that luminal epithelium may be replenished from glandular epithelial or a stromal stem/progenitor population, but this is currently unclear ( Kaitu'u-Lino et al. , 2010 ; Huang et al. , 2012 ; Patterson and Pru, 2013 ). The location of many stromal LRCs directly under the luminal epithelium ( Chan and Gargett, 2006 ) may represent a snapshot of the ‘mesenchymal-to-epithelial transition’ (MET) believed to occur in the endometrium ( Huang et al. , 2012 ; Patterson and Pru, 2013 ). The perivascular location of other stromal LRCs ( Kaitu'u-Lino et al. , 2012 ) suggests a link to the perivascularly located human eMSCs. Unfortunately, the functional properties of BrdU-LRC have been impossible to assess directly because BrdU detection assays require fixation and treatment that kills the tissue being examined. This limitation is circumvented by the use of the transgenic H2B-GFP system, which allows the isolation of living LRCs. This transgenic system has only recently been used in the study of LRCs in the female reproductive tract ( Wang et al. , 2012c ; Patterson and Pru, 2013 ) and is compatible with in vitro and in vivo assays that could clarify the identity and potential of quiescent putative mouse endometrial stem/progenitor populations. SP cells have been identified in murine postpartum but not in the normal cycling endometrium ( Hu et al. , 2010 ). The postpartum endometrial SP was enriched in clonogenic cells, which expressed Esr1 and tended to differentiate on exposure to estrogen in culture ( Hu et al. , 2010 ). However, unlike human endometrial SP, the mouse endometrial SP was not enriched for endothelial cells and its exact identity remains unclear. Compared with the human, cell surface markers for stem/progenitor cells are less well characterized in the mouse endometrium. CD44 is a transmembrane protein expressed on many cell types, including HSCs, MSCs and cancer stem cells ( Zöller, 2011 ). In the mouse endometrium, CD44-expressing epithelial cells constituted an epithelial progenitor population, which lacked Esr1 or PR ( Janzen et al. , 2013 ). This epithelial population survived hormonal deprivation, possibly due to Wnt pathway activation. CD44 + epithelial cells generated more gland-like structures than CD44 − cells in a tissue reconstitution assay in immunocompromised mice. CD44 + cells were also proliferative, suggesting that they are distinct from slow-cycling epithelial LRCs.

In the 10-year history of endometrial stem/progenitor cells, substantial progress has been made in developing assays for their evaluation and for identifying specific markers and characterizing these populations. The in vivo identity of eMSC and SP cells has been determined, and examination of their role in endometriosis and adenomyosis has commenced. The ease with which eMSCs and ERCs can be obtained makes them attractive candidates for cell-based therapies for gynaecological disease and other regenerative medicine applications.

In contrast to adult stem/progenitor cells, pluripotent hES cells are available in large numbers, and many protocols have been developed for their differentiation into clinically relevant cell types ( Trounson, 2006 ), including endometrial cells ( Song et al. , 2015 ). Induced pluripotent stem (iPS) cells (Table  I ) offer a more attractive source of pluripotent cells to generate cells for tissue repair as they can be derived from the patient's own cells, overcoming immunological barriers associated with hES cell derivatives ( Takahashi et al. , 2007 ). Endometrial cells are an attractive source of cells for reprogramming into iPS cells as they express elevated levels of pluripotent factors and more efficiently generate iPS cells when compared with conventional cells ( Park et al. , 2011 ). Human endometrial epithelial-like tissue has been derived from hES cells by recapitulating the stages of reproductive tract development using a two-stage strategy ( Ye et al. , 2011 ). GFP-tagged hES cells were initially partially differentiated to intermediate and lateral plate mesoderm in vitro using bone morphogenetic protein 4 and activin A during embryoid body formation. This was followed by further differentiation in vivo by induction with neonatal mouse uterine mesenchyme as a tissue recombinant and transplanted into immune-compromised NSG mice. Differentiation during this 9-week process was monitored by assessing transcripts and protein for specific mesodermal differentiation markers in the differentiating embryoid bodies, Müllerian duct markers (LIM homeobox protein 1, LIM1; paired box 2, PAX2; homeobox A10, HOXA10) in early xenografts and endometrial epithelial markers (ovarian cancer-related tumour marker, CA125; ESR1, β-tubulin on ciliated cells) in late harvest xenografts ( Ye et al. , 2011 , 2012 ). These endometrial gland-like profiles were functional and proliferated in response to exogenous estrogen and upregulated glycodelin A. The ability of uterine stroma to direct the differentiation of hES cell-derived mesodermal derivatives could be used to differentiate endometrial stromal cell-derived iPS cells into endometrial epithelial cells for potential use in tissue engineering applications to regenerate endometrial tissue in Asherman's syndrome ( Gargett and Ye, 2012 ).

A key issue facing the endometrial stem/progenitor cell field is the use of numerous terms to describe the various stem/progenitor cell types (Tables  I , II and IV ). A common nomenclature recognizing the species, type of cell and its source is needed. It will be important that any nomenclature fits with that used in the broader stem cell discipline. This particularly applies to the universal definition of MSC ( Dominici et al. , 2006 ), which is increasingly recognized as inadequate for defining MSC from tissues other than bone marrow ( Bianco et al. , 2013 ). The nomenclature will need to reflect the different cell types studied, perivascular cells versus the endometrial stromal fibroblast, clonogenic versus non-clonogenic stromal fibroblasts, SP versus MP and those identified with specific markers enriching for clonogenic, self-renewing and multipotent progenitors versus unfractionated multipotent stromal fibroblasts. Although it is apparent that bone marrow-derived cells incorporate in low numbers into the endometrium, the nature of these cells is currently unknown. Careful studies using transgenic tools to track cells are needed. Together with known markers of endometrial stem/progenitor cells and those to be identified in future will enable mechanistic studies to more precisely determine the role of bone marrow-derived cells in normal endometrial physiology and in endometrial diseases. The hierarchical relationship among the clonogenic epithelial progenitor cells, endometrial and decidual SP cells, CD146 + PDGFR-β + and SUSD2 + cells and menstrual blood ERCs is not fully known. This requires more defining markers or gene signatures and determination of the in vivo activity for the various cell types. As these data are generated, it may be possible to determine whether MET links the various stem/progenitor cell types and whether this process occurs during endometrial regeneration.

As adult stem cells have a key role in maintaining tissue homeostasis, it is likely that their function is aberrant in benign gynaecological disease associated with altered endometrial proliferation. Stem/progenitor cells are regulated by the stem cell niche, which may also have roles in the development and progression of endometriosis, adenomyosis, thin dysfunctional endometrium and Asherman's syndrome (Table  VII ). Table VII Endometrial diseases in which endometrial stem/progenitor cells may play a role. Endometrial disease Description Adenomyosis A benign disease involving extensive growth and invasion of basalis endometrial tissue into the uterine myometrium with associated smooth muscle hyperplasia, resulting in an enlarged uterus and painful, heavy or prolonged periods Asherman's syndrome and intrauterine adhesions (IUAs) An acquired uterine condition characterized by complete obliteration of the endometrium with fibrotic intrauterine adhesions (IUAs) causing amenorrhea and infertility. IUA is a less severe condition involving partial replacement of the endometrium with fibrous tissue, causing hypomenorrhea, infertility and pregnancy loss. It results from trauma to the basalis endometrium following dilation and curettage (D&C) due to miscarriage, abortion or retained placenta in a setting of low estrogen and/or infection Endometriosis A benign disease affecting reproductive aged women in whom endometrial tissue grows outside the uterine cavity, most often in the pelvic cavity, around/on the ovaries and in the rectovaginal septum, resulting in inflammation, infertility and severe pelvic pain Thin dysfunctional endometrium Endometrial tissue that does not respond to estrogen stimulation and fails to reach at least 7 mm in thickness necessary for embryo implantation and maintenance of an ongoing pregnancy Endometrial diseases in which endometrial stem/progenitor cells may play a role. Endometriosis is characterized by the growth of endometrial tissue outside the uterine cavity ( Giudice and Kao, 2004 ; Viganó et al. , 2004 ). The most widely accepted theory for the pathogenesis of endometriosis is that retrograde menstruation deposits viable endometrial fragments into the pelvic cavity which attach to and invade the peritoneal mesothelium to establish ectopic growth of endometrial tissue ( Sampson, 1927 ). Despite most women experiencing retrograde menstruation, it is not known why only 6–10% of the reproductive age women develop endometriosis ( Halme et al. , 1984 ). Following the discovery of endometrial stem/progenitor cells, Sampson's hypothesis was extended to address this disparity. It is proposed that endometrial stem/progenitor cells with associated niche cells are abnormally shed during menses, where they gain access to the peritoneal cavity by retrograde menstruation and establish ectopic implants, causing endometriosis ( Starzinski-Powitz et al. , 2001 ; Leyendecker et al. , 2002 ; Gargett, 2007 ; Sasson and Taylor, 2008 ; Gargett and Masuda, 2010 ). It was also proposed that endometriosis lesions initiated by endometrial stem/progenitor cells would be more severe and invasive than lesions initiated by more differentiated transit-amplifying cells, explaining the different grades of endometriosis ( Gargett, 2007 ). Alternatively, endometrial stem/progenitor cells, with yet to be identified intrinsic abnormalities, such as carrying one of the endometriosis susceptibility alleles ( Nyholt et al. , 2012 ), may have increased propensity to implant and establish an ectopic colony. Normal endometrial stem/progenitor cells may also implant more readily on an abnormal peritoneal mesothelium ( Gargett and Chan, 2006 ; Gargett, 2007 ). It was also hypothesized that endometrial stem/progenitor cells may be involved in the pathogenesis of premenarcheal and adolescent endometriosis through retrograde neonatal uterine bleeding due to maternal progesterone withdrawal at birth ( Brosens and Benagiano, 2013 ; Brosens et al. , 2013 ; Gargett et al. , 2014 ). Endometrial stem/progenitor cells, together with niche cells, would remain dormant beneath the peritoneum until rising estrogen levels triggered at menarche activate them to initiate clonal growths of ectopic endometrium and establish early onset endometriosis prior to menstruation (Fig.  2 ). Figure 2 Schematic describing the hypothesis that endometrial stem/progenitor cells shed in neonatal uterine bleeding may play a role in early onset endometriosis. Neonatal uterine bleeding occurs in 5% of neonates. It is hypothesized that retrograde neonatal bleeding occurs because thick mucus obstructs the long neonatal cervix. Fragments of shed endometrial tissue are postulated to contain an endometrial epithelial progenitor cell (pink) and a perivascular MSC (pink) together with niche cells. These rapidly adhere to the neonatal mesothelium, invade and/or become contiguous with the mesothelial lining where they remain quiescent for ∼10 years. Rising estrogen (E2) levels associated with thelarche and menarche reactivate the stem/progenitor cells to initiate growth of endometriosis lesions on the surface of or below the peritoneal mesothelium, resulting in early onset endometriosis. Reprinted with permissions from Gargett et al. (2014) . Schematic describing the hypothesis that endometrial stem/progenitor cells shed in neonatal uterine bleeding may play a role in early onset endometriosis. Neonatal uterine bleeding occurs in 5% of neonates. It is hypothesized that retrograde neonatal bleeding occurs because thick mucus obstructs the long neonatal cervix. Fragments of shed endometrial tissue are postulated to contain an endometrial epithelial progenitor cell (pink) and a perivascular MSC (pink) together with niche cells. These rapidly adhere to the neonatal mesothelium, invade and/or become contiguous with the mesothelial lining where they remain quiescent for ∼10 years. Rising estrogen (E2) levels associated with thelarche and menarche reactivate the stem/progenitor cells to initiate growth of endometriosis lesions on the surface of or below the peritoneal mesothelium, resulting in early onset endometriosis. Reprinted with permissions from Gargett et al. (2014) . No direct evidence for the role of endometrial stem/progenitor cells in the pathogenesis of endometriosis has yet been reported. Several studies support a role for shedding of endometrial epithelial progenitor cells from the basalis in women with endometriosis. Fragments of basalis endometrium were identified more often in menstrual blood of women with than without endometriosis ( Leyendecker et al. , 2002 ). SSEA-1, a marker of endometrial basalis epithelial cells, was found in endometriotic lesions ( Valentijn et al. , 2013 ). The monoclonality of ectopic endometrial epithelial cells ( Jimbo et al. , 1997 ; Tamura et al. , 1998 ; Wu et al. , 2003 ) and identification of DNA losses/genomic imbalances demonstrating clonal proliferation ( Silveira et al. , 2012 ) in endometriotic lesions suggest the involvement of progenitor cells in the pathogenesis of endometriosis. Stem cell genes and proteins OCT4, SOX2, NANOG, Musashi and C-KIT have been observed in endometriotic lesions ( Götte et al. , 2008 ; Forte et al. , 2009 ; Song et al. , 2014 ). Cultured ectopic, clonally derived eMSCs expressed OCT-4 and demonstrated MSC phenotypic surface markers ( Kao et al. , 2011 ). Together, these studies suggest that women with endometriosis shed basalis endometrium at menstruation, increasing the likelihood of endometrial epithelial progenitor cells gaining access to the peritoneal cavity to initiate and develop into endometriotic lesions. However, these findings do not explain a role for eMSCs, which are present in both functionalis and basalis. Recent functional human studies identified stem/progenitor cells in ectopic endometriotic lesions, suggesting their potential role. Similar to eutopic endometrial stem/progenitor cells, endometriotic epithelial and stromal CFUs were observed, and these serially cloned two to three times ( Chan et al. , 2011 ). Ectopic stromal CFUs were multipotent ( Chan et al. , 2011 ; Kao et al. , 2011 ) and underwent more than 25 population doublings before senescence ( Kao et al. , 2011 ; Moggio et al. , 2012 ), similar to eutopic stromal CFUs ( Gargett et al. , 2009 ). Ectopic endometriotic stromal cell lines differentiated into cytokeratin- and E-cadherin-expressing epithelial cells in culture ( Moggio et al. , 2012 ). Ectopic MSCs demonstrated greater migration and invasion than eutopic MSCs with increased angiogenesis and invasion into surrounding tissue in a scaffold transplantation mouse model ( Kao et al. , 2011 ). Together, these studies support the presence of endometrial stem/progenitor cells in ectopic endometriotic lesions. The peritoneal fluid of menstruating women with and without endometriosis contained similar numbers of endometrial cells ( Bokor et al. , 2009 ), although markers used to identify them were not specific and also immunostained resident peritoneal mesothelial cells. As endometrial stromal cells rapidly attach to peritoneal mesothelial cells in vitro ( Lucidi et al. , 2005 ), their concentration in peritoneal fluid may not accurately represent those retrogradely shed during menstruation. The presence of endometrial stem/progenitor cells in peritoneal fluid has not yet been reported, which is crucial for identifying their role in the pathogenesis of endometriosis. The identification of the specific eMSC marker SUSD2 will enable their identification in shedding endometrium and peritoneal fluid. However, markers are currently lacking for endometrial epithelial progenitor cells. Endometriosis models in mice, rats, baboons and marmosets ( Grümmer, 2006 ) exist, but these have focused on pathophysiological mechanisms involved in the development of disease, rather than the involvement of stem/progenitor cells, although bone marrow-derived cells may have contributed to the progression of established endometriosis lesions ( Taylor, 2004 ). However, it can be inferred that endometrial stem/progenitor cells are likely responsible for lesion formation in animal models, in which menstrual debris was transplanted into the peritoneal cavity ( Greaves et al. , 2014 ) or endometrial cells/tissues were injected subcutaneously ( Wang et al. , 2014 ). Genetic labelling of endometrial stem/progenitor cells with fluorescent tags is needed to exploit these animal models for cell tracking to determine their role in ectopic lesion formation. This approach was used to label all endometrial cells with adenovirus encoding red fluorescent protein ( Wang et al. , 2014 ) or recombinant lentivirus conferring yellow fluorescent protein or click beetle red-emitting luciferase (Masuda et al. , 2007b ) for non-invasive monitoring of cell xenografts over several menstrual-like cycles. These animal models need further adapting to monitor labelled endometrial stem/progenitor cells, transplanted into the peritoneal cavity and subjected to a similar hormonal regime as the mouse model of menstruation ( Brasted et al. , 2003 ) for several cycles to examine the role of endometrial stem/progenitor cells in the initiation and progression of endometriosis. Adenomyosis is considered a dichotomous disease characterized by thickening and disruption of the endo-myometrial JZ structure ( Benagiano et al. , 2014 ). Little is known about the pathophysiology of adenomyosis, which mainly affects parous women. Theories suggest that chronic microtrauma to the JZ from chronic peristaltic myometrial contractions causes repeated cycles of tissue injury and repair ( Leyendecker et al. , 2009 ). A vicious cycle is established in which local estrogen production promotes uterine hyperperistalsis and further auto-traumatization, allowing basal endometrial glands and stroma to penetrate the myometrium and proliferate to form pockets of adenomyosis within the uterine muscle. Tissue injury typically activates adult stem cells, which may establish ectopic endometrial lesions through disruption of endometrial stem/progenitor cell niches ( Gargett, 2007 ). Abnormal differentiation of eMSCs may be responsible for the smooth muscle hyperplasia ( Gargett, 2007 ). In support of this concept, stromal cells cultured from adenomyotic tissue differentiated into typical mesodermal lineages and expressed MSC surface markers ( Chen et al. , 2010 ). Whether adenomyotic stromal cells exhibit MSC properties of clonogenicity and self-renewal or contain a subpopulation of perivascular CD146 + PDGFRβ + or SUSD2 + or SP cells is unknown. Gene expression profiling identified differences between adenomyotic and normal endometrial stromal cells ( Chen et al. , 2010 ), particularly COX-2 overexpression. COX-2 has a role in the tissue injury repair cycle and local estrogen production ( Leyendecker et al. , 2009 ). In an attempt to identify endometrial stem cells in adenomyotic tissue, immunostaining with an adult stem cell marker, Musashi-1, was undertaken ( Chen et al. , 2014 ). However, most epithelial cells and a proportion of stromal cells were Musashi-1 + , in contrast to observations of rare immunostained cells in the normal endometrium ( Götte et al. , 2008 ). Given this discrepancy and that Musashi-1 + endometrial stromal cells in eutopic and ectopic adenomyosis do not appear in a perivascular location, it seems unlikely that Musashi-1-expressing cells are endometrial stem/progenitor cells in adenomyosis. It also indicates that functional stem cell assays should accompany studies examining stem cell marker expression. More research is required to establish a role for endometrial stem/progenitor cells in the initiation and progression of adenomyosis. Dysfunctional endometrial stem/progenitor cells may be responsible for the inability of some women to generate a sufficiently thick endometrium (>7–8 mm) to support embryo implantation ( Yu et al. , 2008 ). Thin dysfunctional endometrium unresponsive to estrogen stimulation is particularly challenging in IVF clinics. In Asherman's syndrome, defined as the complete obliteration of the uterine cavity with adhesions, we hypothesize a complete loss of endometrial stem/progenitor cells. Intrauterine adhesions (IUAs) represent a continuum between Asherman's syndrome and thin dysfunctional endometrium, where we postulate that there are insufficient endometrial stem/progenitor cells, which may or may not be dysfunctional, residing in the pockets of remaining endometrial tissue ( Gargett and Ye, 2012 ). Between 2 and 22% of infertile women have Asherman's syndrome or IUA ( Yu et al. , 2008 ; Panayotidis et al. , 2009 ). Trauma to the endometrial basalis and JZ from termination of pregnancy, spontaneous miscarriage and postpartum curettage in a setting of low circulating estrogen levels hinder endometrial regeneration. Any remaining endometrial stem/progenitor cells survive a low estrogen environment, but their niche cells require an estrogen-rich milieu to activate them. When infection and inflammation also occur, the inflammatory products may damage endometrial stem/progenitor cells, critically reducing their numbers and limiting their capacity to regenerate sufficient endometrium ( Gargett and Ye, 2012 ). A mouse model of Asherman's syndrome has been developed using a needle to traumatize the lumen of both uterine horns ( Alawadhi et al. , 2014 ). Immediately following the damage, unfractionated male mouse bone marrow cells or saline was administered intravenously and the mice were examined 3 months later. Histological analysis showed reduced fibrosis and pregnancies with normal litter sizes in 90% of the bone marrow-transplanted animals, compared with 30% of saline-treated mice. A small number (0.1%) of non-leukocyte (CD45 − ) Y chromosome + cells were detected in the endometrium of the transplanted mice, but the original identity of the incorporated cells is unknown. The effective repair of damaged endometrium by this small number of cells suggests either an immediate systemic cytokine effect or an indirect activation of endogenous endometrial stem/progenitor cells or their niches. In a rat model of Asherman's syndrome in which trichloroacetic acid was instilled into the uterine lumen, cultured male rat adipose-derived MSCs were injected into one uterine horn and then intraperitoneally at 5 day intervals. Histological assessment of the endometrium revealed that 4–6% of the stroma was BrdU-labelled cells ( Kilic et al. , 2014 ). MSCs alone increased vascular and cellular proliferation and VEGF immunostaining, but had no effect on inflammation or fibrosis. When co-administered with oral estrogen, the MSCs also reduced fibrosis. In contrast to systemic delivery, local injection of allogeneic MSCs to the injured endometrium enabled a proportion to incorporate into the tissue with apparent promotion of tissue regeneration, although neither tissue architecture nor endometrial function in supporting implantation or pregnancy was assessed. Several case studies have reported the instillation of fresh CD34 + or cultured autologous bone marrow cells into the uterine cavity or subendometrial region of women with Asherman's syndrome or IUA ( Nagori et al. , 2011 ; Singh et al. , 2014 ). Very moderate increases in endometrial thickness and evidence of menstruation or pregnancy were reported. However, these must be viewed with caution as there were no control subjects; a lack of detail on bone marrow cell preparation and the increase in endometrial thickness was generally below that required for successful embryo implantation ( Gargett and Healy, 2011 ). Other sources of cells should also be considered in further development of a stem cell approach to treating thin dysfunctional endometrium, for example, allogeneic endometrial stem/progenitor cells or autologous endometrial cells derived from iPS cells. Endometrial-like tissue has been generated from hESCs ( Ye et al. , 2011 ), and iPS cells are easily created from human endometrial stromal cells ( Park et al. , 2011 ) or shed menstrual blood cells due to their plasticity and regenerative capacity ( de Carvalho Rodrigues et al. , 2012 ; Li et al. , 2013 ). Further development of the animal model is required to determine the mechanism of action before cell-based therapies for Asherman's syndrome or thin dysfunctional endometrium are trialled in humans.

Human endometrial stroma terminally differentiates into the decidua during the mid-late secretory stage of the menstrual cycle. Decidualization commences in the perivascular cells of the spiral arterioles and spreads to the subepithelial stroma. The decidua of pregnancy may therefore harbour a subpopulation of undifferentiated MSCs related to eMSCs ( Kyurkchiev et al. , 2010 ). Indeed, clonogenic SP cells were identified in the first trimester decidua ( Tsuji et al. , 2008 ; Guo et al. , 2010 ; Wang et al. , 2013 ) comprising 0.03–1.4% of cells, a lower abundance than their endometrial counterparts. SP cells sorted from short-term cultured human decidual cells expressed neither CD31 (endothelial marker) nor CD146 (MSC marker) ( Wang et al. , 2013 ), but differentiated into endothelial cells in vitro and induced neovascularization following intramascular injection in a mouse ischaemic hind limb injury model, rescuing the limb ( Wang et al. , 2013 ). This CD31 − CD146 − SP proliferated more rapidly than MP cells when cultured in 0.2% serum-containing media supplemented with either epidermal growth factor (EGF) or fibroblast growth factor 2 (FGF2), similar to clonogenic eMSCs ( Chan et al. , 2004 ). They also proliferated in IGF-1- and VEGF-containing media ( Wang et al. , 2013 ). In contrast, short-term cultured decidual SP cells required IL-6, stem cell factor and thrombopoietin for growth in serum-free (SF) medium ( Guo et al. , 2010 ). The clonogenic cells appeared more heterogeneous than endometrial clones and differentiated into prolactin-staining cells following treatment with cAMP. Confocal analysis of the decidua parietalis using specific markers for purifying bmMSC or eMSC demonstrated a vascular niche for decidua MSCs ( Castrechini et al. , 2012 ). STRO-1 co-localized with vWF (endothelial marker), in agreement with recent reports that STRO-1 was an endothelial marker in adipose tissue arterioles and capillaries ( Lin et al. , 2011 ). In contrast, CD146 was perivascular with partial overlap with vWF. Cultured stromal fibroblasts from first trimester and term decidua demonstrated characteristic MSC properties: clonogenicity (2–18%), mesodermal lineage differentiation and surface marker phenotype ( Dimitrov et al. , 2010 ; Castrechini et al. , 2012 ). Cultured placental decidua basalis stromal fibroblasts differentiated into pancreatic cells in vitro when transfected with a microRNA involved in pancreas development ( Shaer et al. , 2014 ), indicating that decidua basalis stromal fibroblasts are equally as capable of differentiating across germ lineage boundaries as endometrial stromal fibroblasts. Decidual SP cells and stromal fibroblasts respond to sex steroid hormones. Both estrogen and progesterone dose dependently stimulated greater proliferation and migration of the decidual CD31 − CD146 − cells in vitro than MP cells ( Wang et al. , 2013 ). High concentrations of progesterone (7–30 μM) upregulated HLA-G on a small proportion (5.3%) of decidual stromal fibroblasts, suggesting that they may function in immunomodulation of the implanting embryo ( Ivanova-Todorova et al. , 2009 ). It is unknown whether this HLA-G-expressing subpopulation are decidual perivascular MSCs. Nor is it known if the perivascular decidual stromal cells or HLA-G-expressing cells are SUSD2 + . More studies are required using specific markers and profiling technologies to determine the relationship among eMSCs, decidual MSCs, endometrial stromal fibroblasts and decidual stromal fibroblasts.