Kjiana E. Schwab

Clonogenicity of Human Endometrial Epithelial and Stromal Cells

Abstract The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300–500/cm2) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-α (TGFα), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGFα, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except α6-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.

Isolation and Culture of Epithelial Progenitors and Mesenchymal Stem Cells from Human Endometrium

Abstract Human endometrium is a highly regenerative tissue undergoing more than 400 cycles of growth, differentiation, and shedding during a womans reproductive years. Endometrial regeneration is likely mediated by adult stem/progenitor cells. This study investigated key stem cell properties of individual clonogenic epithelial and stromal cells obtained from human endometrium. Single-cell suspensions of endometrial epithelial or stromal cells were obtained from hysterectomy tissues from 15 women experiencing normal menstrual cycles, and were cultured at clonal density (10 cells/cm2) or limiting dilution. The adult stem cell properties—self-renewal, high proliferative potential, and differentiation of single epithelial and stromal cells—were assessed by harvesting individual colonies and undertaking serial clonal culture, serial passaging, and culture in differentiation-induction media, respectively. Lineage differentiation markers were examined by RT-PCR, immunocytochemistry, and flow cytometry. Rare single human endometrial EpCAM+ epithelial cells and EpCAM− stromal cells demonstrated self-renewal by serially cloning >3 times and underwent >30 population doublings over 4 mo in culture. Clonally derived epithelial cells differentiated into cytokeratin+ gland-like structures in three dimensional culture. Single stromal cells were multipotent, as their progeny differentiated into smooth muscle cells, adipocytes, chondrocytes, and osteoblasts. Stromal clones expressed mesenchymal stem cell (MSC) markers ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146) but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45). Adult human endometrium contains rare epithelial progenitors and MSCs, likely responsible for its immense regenerative capacity, which may also have critical roles in the development of endometriosis and endometrial cancer. Human endometrium may provide a readily available source of MSCs for cell-based therapies.

Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

BACKGROUND Human endometrium is a highly regenerative tissue. We hypothesized that the source of endometrial stromal and vascular regeneration is a resident stromal stem/progenitor cell population. Putative human endometrial stromal stem/progenitor cells have been identified using clonal assays, a retrospective functional stem cell assay. Therefore, the aim of this study was to screen potential stem cell markers for the prospective isolation of human endometrial stromal stem/progenitor cells and to determine their capacity to identify colony-forming stromal cells. METHODS Single-cell suspensions of human endometrial stromal cells were sorted using fluorescence-activated cell sorting into positive and negative populations based on STRO-1, CD133, CD90 or CD146 expression for clonal assays. All markers were immunolocalized in human endometrium. RESULTS Small populations (2-9%) of human endometrial stromal cells expressed each of the markers. Only CD146(+) cells were enriched for colony-forming cells, and CD90(hi) cells showed a trend for greater enrichment compared with CD90(lo) cells. STRO-1 and CD146 were localized to perivascular cells of the endometrium. CD90 was strongly expressed by functionalis stroma and perivascular cells, but only weakly expressed in the basalis stroma. CD133 was expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. CONCLUSIONS This study identified CD146 as a marker of colony-forming human endometrial stromal cells supporting the concept that human endometrium contains a population of candidate stromal stem/progenitor cells.

Hormone and growth factor signaling in endometrial renewal: Role of stem/progenitor cells

The human endometrium is a dynamic remodeling tissue undergoing more than 400 cycles of regeneration, differentiation and shedding during a womans reproductive years. The co-ordinated and sequential actions of estrogen and progesterone direct these major remodeling events preparing a receptive endometrium for blastocyst implantation on a monthly basis. Adult stem/progenitor cells are likely responsible for endometrial regeneration. Functional approaches have been used to identify candidate endometrial stem/progenitor cells, as there are no specific stem cell markers. Rare populations of human endometrial epithelial and stromal colony-forming cells/units (CFU) and side population (SP) cells have been identified. Several growth factors are required for CFU activity: epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and platelet-derived growth factor BB (PDGF-BB) for both epithelial and stromal CFU, and basic fibroblast growth factor (bFGF) for stromal, but not epithelial CFU. A sub-population of human endometrial stromal cells with mesenchymal stem cell properties of CFU activity and multilineage (fat, muscle, cartilage and bone) differentiation have been isolated by their co-expression of CD146 and PDGF-receptor beta. Candidate epithelial and stromal stem/progenitor cells have been identified in mouse endometrium as rare label retaining cells (LRCs) in the luminal epithelium and as perivascular cells at the endometrial-myometrial junction, respectively. While epithelial and most stromal LRC do not express estrogen receptor alpha (Esr1), they rapidly proliferate on estrogen stimulation, most likely mediated by neighbouring Esr1-expressing niche cells. It is likely that these newly identified endometrial stem/progenitor cells may play key roles in the development of gynecological diseases associated with abnormal endometrial proliferation such as endometriosis and endometrial cancer.

Endometrial stem cells

Purpose of review The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in postmenopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. This review discusses the first published evidence for the existence of endometrial stem/progenitor cells in human and mouse endometrium. Recent findings Functional approaches have been used to identify candidate endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers. Rare clonogenic cells and side population cells have been identified in human endometrial cell populations. In mouse endometrium, rare label-retaining cells have also been identified. The ability of transplanted human endometrial cells to grow endometrial tissue in animal hosts also suggests the presence of stem/progenitor cells. Summary These initial studies providing the first functional evidence for epithelial and stromal stem/progenitor cells in human and mouse endometrium lay the groundwork for further studies to characterize their stem cell properties. They also provide the impetus to discover specific markers that will enable their prospective isolation and allow their location in normal and pathological endometrium to be determined. Abbreviation LRC: label-retaining cell.

Potential role of endometrial stem/progenitor cells in the pathogenesis of early-onset endometriosis

The pathogenesis of early-onset endometriosis has recently been revisited, sparked by the discovery of endometrial stem/progenitor cells and their possible role in endometriosis, and because maternal pregnancy hormone withdrawal following delivery induces uterine bleeding in the neonate. The neonatal uterus has a large cervix to corpus ratio which is functionally blocked with mucous, supporting the concept of retrograde shedding of neonatal endometrium. Only 5% show overt bleeding. Furthermore, the presence of endometriosis in pre-menarcheal girls and even in severe stage in adolescents supports the theory that early-onset endometriosis may originate from retrograde uterine bleeding soon after birth. Endometrial stem/progenitor cells have been identified in menstrual blood suggesting that they may also be shed during neonatal uterine bleeding. Thus, we hypothesized that stem/progenitor cells present in shedding endometrium may have a role in the pathogenesis of early-onset endometriosis through retrograde neonatal uterine bleeding. During the neonatal and pre-pubertal period, shed endometrial stem/progenitor cells are postulated to survive in the pelvic cavity in the absence of circulating estrogens supported by niche cells also shed during neonatal uterine bleeding. According to this hypothesis, during thelarche, under the influence of rising estrogen levels, endometrial stem/progenitor cells proliferate and establish ectopic endometrial lesions characteristic of endometriosis. This New Research Horizon review builds on recent discussions on the pathogenesis of early-onset endometriosis and raises new avenues for research into this costly condition.

Mesenchymal stem/stromal cells in post-menopausal endometrium

STUDY QUESTION Does post-menopausal endometrium contain mesenchymal stem/stromal cells (MSC) that have adult stem cell properties and can be prospectively isolated from a biopsy? SUMMARY ANSWER Perivascular W5C5(+) cells isolated from post-menopausal endometrial biopsies displayed characteristic MSC properties of clonogenicity, multipotency and surface phenotype irrespective of whether the women were or were not pre-treated with estrogen to regenerate the endometrium. WHAT IS KNOWN ALREADY Recently MSCs have been identified in human premenopausal endometrium, and can be prospectively isolated using a single marker, W5C5/SUSD2. STUDY DESIGN, SIZE, DURATION Endometrial tissue of both the functional and basal layers, from 17 premenopausal (pre-MP) women, 19 post-menopausal (post-MP) women without hormonal treatment and 15 post-menopausal women on estrogen replacement therapy (post-MP+ E2), was collected through a prospective phase IV clinical trial over 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Post-menopausal women <65 years of age were treated with or without E2 for 6-8 weeks prior to tissue collection. Serum E2 levels were determined by estradiol immunoenzymatic assay. Endometrial tissue was obtained from women by biopsy (curettage) just prior to the hysterectomy. The effect of E2 on endometrial thickness and glandular and luminal epithelial height was determined using image analysis. Endometrial tissue was dissociated into single cell suspensions and MSC properties were examined in freshly isolated and short-term cultured, magnetic bead-purified W5C5(+) cells. MSC properties were assessed using clonogenicity, serial cloning, mesodermal differentiation in adipogenic, chondrogenic, osteogenic and myogenic induction culture media, and surface phenotype analysis by flow cytometry. Estrogen receptor α expression in W5C5(+) cells was examined using dual colour immunofluorescence. Vascularity was analysed using CD34 and alpha smooth muscle actin immunostaining and subsequent image analysis. MAIN RESULTS AND THE ROLE OF CHANCE A small population of stromal cells with MSC properties was purified with the W5C5 antibody from post-menopausal endometrium, whether atrophic from low circulating estrogen or regenerated from systemic estrogen treatment, similar to premenopausal endometrium. The MSC derived from post-menopausal endometrium treated with or without E2 fulfilled the minimum MSC criteria: clonogenicity, surface phenotype (CD29(+), CD44(+), CD73(+), CD105(+), CD140b(+), CD146(+)) and multipotency. The post-menopausal endometrial MSCs also showed comparable properties to premenopausal eMSC with respect to self-renewal in vitro and W5C5 expression. The W5C5(+) cells were located perivascularly as expected and did not express estrogen receptor α. LIMITATIONS, REASONS FOR CAUTION The properties of the MSC derived from post-menopausal endometrium were evaluated in vitro and their in vivo tissue reconstitution capacity has not been established as it has for premenopausal endometrial MSC. WIDER IMPLICATIONS OF THE FINDINGS The endometrium is an accessible source of MSC obtainable with minimum morbidity that could be used for future clinical applications as a cell-based therapy. This study shows that menopausal women can access their endometrial MSC by a simple biopsy for use in autologous therapies, particularly if their endometrium has been regenerated by short-term E2 treatment, provided they have an intact uterus and are not contraindicated for short-term E2 treatment. Endometrial MSC in post-menopausal women possess key MSC properties and are a promising source of MSC independent of a womans age. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Health and Medical Research Council (NHMRC) of Australia grant (1021126) (C.E.G., A.R.) and Senior Research Fellowship (1042298) (C.E.G.), Australian Gynaecological Endoscopic Society grant (A.R.) , Monash International Postgraduate Research Scholarship (DU), Australian Stem Cell Centre, South East Melbourne Alliance for Regenerative Therapies and Australian Stem Cell Centre top up scholarships (DU) and Victorian Governments Operational Infrastructure Support Program. Competing interests: AR receives Preceptorship fees from AMS, advisory board fees and sponsored study from Astellas, and conducts investigator led studies sponsored by AMS and Boston Scientific for other projects. TRIAL REGISTRATION NUMBER CTNRN12610000563066.

The Endometrium: A Novel Source of Adult Stem/Progenitor Cells

The human endometrium (lining of the uterus) is a dynamic remodeling tissue undergoing more than 400 cycles of regeneration, differentiation, and shedding during a woman’s reproductive years. Endometrial regeneration also follows childbirth, almost complete resection and in postmenopausal women taking estrogen replacement therapy. In nonmenstruating species (e.g. rodents) there are cycles of endometrial growth and apoptosis, rather than physical shedding. The endometrium comprises epithelial-lined glands extending from the surface epithelium to the myometrium supported by extensive stroma. Remodeling of the endometrium is regulated by the co-ordinated and sequential actions of estrogen and progesterone in preparation for blastocyst implantation on a monthly basis in women and every 4–5 days in mice. Adult stem/progenitor cells are likely responsible for endometrial regeneration. Since there are no specific stem cell markers, initial studies using functional approaches identified candidate epithelial and stromal endometrial stem/progenitor cells as colony-forming cells/units (CFU) and side population (SP) cells. Recently, a subpopulation of human endometrial stromal cells with mesenchymal stem cell–like properties of CFU activity and multilineage differentiation have been isolated by their co-expression of CD146 and PDGF-receptor \(\beta\). Candidate epithelial and stromal stem/progenitor cells have also been identified in mouse endometrium as rare label-retaining cells (LRC) in the luminal epithelium and as perivascular cells at the endometrial myometrial junction, respectively. While epithelial and most stromal LRC do not express estrogen receptor α (ERα), they rapidly proliferate on estrogen stimulation, most likely mediated by neighboring ERα-expressing niche cells. It is likely that these newly identified endometrial stem/progenitor cells may play key roles in the development of gynecological diseases associated with abnormal endometrial proliferation such as endometriosis and endometrial cancer. The endometrium may also provide a readily available source of mesenchymal stem-like cells for tissue engineering purposes with possible applications not only to urogynecology but also to heart disease, and soft tissue and bone repair.