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Perivascular Human Endometrial Mesenchymal Stem Cells Express Pathways Relevant to Self-Renewal, Lineage Specification, and Functional Phenotype

ABSTRACT: A population of endometrial cells displaying key properties of mesenchymal stem cells (eMSC) has been identified in human endometrium. eMSC co-express CD146 and PDGFRB surface markers, have a perivascular location, and likely represent the reservoir of progenitors giving rise to the endometrial stromal fibroblast lineage. Endometrial stromal cells isolated from 16 oocyte donors and 3 benign gynecologic surgery subjects were FACS sorted into four populations: CD146+/PDGFRB+ (eMSC); CD146+/PDGFRB- (endothelial cells); CD146-/PDGFRB+ (stromal fibroblasts); CD146-/PDGFRB- (mixed population) then subjected to gene expression analysis on Affymetrix Human Gene 1.0 ST arrays, and differentially expressed genes compared between eMSC, stromal fibroblast, and endothelial cell populations. Ninety-two genes were validated by multiplex quantitative RT-PCR on seventy of these sorted cell populations. Immunohistochemistry was used to verify the perivascular location of eMSCs.Principal component analysis and hierarchical clustering showed eMSC clustering discretely near stromal fibroblasts and separately from endothelial cells. eMSC expressed pericyte markers and genes involved hypoxia response, inflammation, proteolysis, and angiogenesis/vasculogenesis – all relevant to endometrial tissue breakdown and regeneration. Additionally, eMSC displayed distinct gene profiles for cell-cell communication and regulation of gene expression. Overall, the phenotype of the eMSC is that of a multipotent pericyte responsive to hypoxic, proteolytic, and inflammatory stimuli, able to induce angiogenesis, migrate and differentiate into lineage cells, and potentially respond to estradiol and progesterone. Identifying the pathways and gene families described herein in the context of the endometrial niche, will be valuable in understanding normal and abnormal endometrial development in utero and differentiation in adult uterus. The multipotent, perivascular endometrial mesenchymal stem cell has a “niche phenotype” of high Notch, TGFB, IGF, and Hedgehog and low canonical/non-canonical Wnt and EGF signaling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE31152 | GEO | 2011/11/14

SECONDARY ACCESSION(S): PRJNA144817

REPOSITORIES: GEO

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Project description:A population of endometrial cells displaying key properties of mesenchymal stem cells (eMSC) has been identified in human endometrium. eMSC co-express CD146 and PDGFRB surface markers, have a perivascular location, and likely represent the reservoir of progenitors giving rise to the endometrial stromal fibroblast lineage. Endometrial stromal cells isolated from 16 oocyte donors and 3 benign gynecologic surgery subjects were FACS sorted into four populations: CD146+/PDGFRB+ (eMSC); CD146+/PDGFRB- (endothelial cells); CD146-/PDGFRB+ (stromal fibroblasts); CD146-/PDGFRB- (mixed population) then subjected to gene expression analysis on Affymetrix Human Gene 1.0 ST arrays, and differentially expressed genes compared between eMSC, stromal fibroblast, and endothelial cell populations. Ninety-two genes were validated by multiplex quantitative RT-PCR on seventy of these sorted cell populations. Immunohistochemistry was used to verify the perivascular location of eMSCs.Principal component analysis and hierarchical clustering showed eMSC clustering discretely near stromal fibroblasts and separately from endothelial cells. eMSC expressed pericyte markers and genes involved hypoxia response, inflammation, proteolysis, and angiogenesis/vasculogenesis – all relevant to endometrial tissue breakdown and regeneration. Additionally, eMSC displayed distinct gene profiles for cell-cell communication and regulation of gene expression. Overall, the phenotype of the eMSC is that of a multipotent pericyte responsive to hypoxic, proteolytic, and inflammatory stimuli, able to induce angiogenesis, migrate and differentiate into lineage cells, and potentially respond to estradiol and progesterone. Identifying the pathways and gene families described herein in the context of the endometrial niche, will be valuable in understanding normal and abnormal endometrial development in utero and differentiation in adult uterus. The multipotent, perivascular endometrial mesenchymal stem cell has a “niche phenotype” of high Notch, TGFB, IGF, and Hedgehog and low canonical/non-canonical Wnt and EGF signaling. Oocyte donors with no known uterine pathology underwent endometrial biopsy at the time of oocyte retrieval, following comparable GnHR agonist downregulated ovarian stimulation protocols. Tissue was digested and stromal cells isolated and sorted based on expression of CD146 and PDGFRB. RNA was extracted and hybridized on Affymetrix microarrays. Resulting data were compared between sorted isolated cell populations.

Project description:Herein, we investigated eMSC and eSF freshly isolated from endometrium from women with and without endometriosis and compared them to their respective short- and long-term cultures and subsequent decidualization response to progesterone. ABSTRACT Human endometrium undergoes cyclic tissue shedding with ensuing regeneration involving stem/progenitor cells, but the role of multipotent resident endometrial mesenchymal stem cells (eMSC) as progenitors of the endometrial stromal fibroblast (eSF) has not been definitively demonstrated. Progesterone (P4)-driven differentiation (decidualization) of eSF is abnormal in endometriosis, displaying P4-resistance in vivo and in vitro. This study investigated whether eMSC are precursors of eSF and, if so, whether the endometriosis P4-resistant phenotype is inherited from eMSC progenitors or acquired by eSF lineage cells within the endometrial niche. To this end we analyzed the transcriptomes of eMSC and eSF isolated by fluorescence activated cell sorting (FACS) from eutopic endometrium of women with (eMSCFACS.endo, eSFFACS.endo) and without (eMSCFACS.control, eSFFACS.control) endometriosis, and of primary cultures derived from these sorted populations. FACS-isolated eMSC and eSF displayed distinct sets of differentially expressed lineage-associated genes (LG) with >95% of the 200 most highly expressed LG conserved in endometriosis. eSFcontrol in culture maintained in vitro expression of 45% eSF LG; whereas eSFendo displayed less in vitro LG stability, expressing 33% eSF LG. Under the same culture conditions, eMSCcontrol underwent in vitro eSF lineage differentiation (eMSC→eSFcontrol), progressively losing stemness (down-regulated 81% eMSC LG) and acquiring an in vitro eSFcontrol phenotype (up-regulated 55% eSF LG) with minimal transcriptome differences vs. eSFcontrol cultures (19 genes all <2 fold change). Likewise eMSCendo cultures underwent in vitro eSF lineage differentiation (eMSC→eSFendo), down-regulating 77% eMSC LG and up-regulating 50% eSF LG, and there were 78 differentially expressed genes (-2.73 to +2.81 fold change) in eMSC→eSFendo vs. eSFendo. The in vitro disease phenotypes of eSFendo and eMSC→eSFendo shared 49 common genes differentially expressed vs. their control counterparts. However, 78 and 89 genes, respectively, were unique to eSFendo and eMSC→eSFendo, and pathway analysis revealed a proinflammatory phenotype in eSFendo but not eMSC→eSFendo cultures, suggesting divergent niche effects for in vivo vs. in vitro lineage differentiation. Cultures of eSFcontrol and eMSC→eSFcontrol responded to P4 with IGFBP1 secretion, while eSFendo and eMSC→eSFendo cultures did not, demonstrating P4-resistance inherited from eMSCendo. These findings substantiate eMSC as progenitors of eSF and reveal novel aspects of the eSF disease phenotype in endometriosis with P4-resistance inherited from the eMSC and a proinflammatory component acquired by the eSF lineage within the endometrial niche.

Project description:Context: Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer. Objective: We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial M-bM-^@M-^\disease phenotypeM-bM-^@M-^] affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny. Design and setting: A prospective study conducted at an academic medical center. Patients and Main Outcome Measures: Proliferative phase endometrium was obtained from 6 overweight/obese PCOS (NIH criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting (FACS)-isolated endometrial epithelial cells (eEP), endothelial cells (eEN), stromal fibroblasts (eSF) and mesenchymal stem cells (eMSC). Gene expression data were validated using microfluidic Q-RT-PCR and immunohistochemistry (IHC). Results: The comparison between eEPPCOS and eEPCtrl showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSFPCOS and eSFCtrl showed upregulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSCPCOS vs. eMSCCtrl the most upregulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). IHC scoring showed increased expression of CCL2 in eEPPCOS and eSFPCOS compared to eEPCtrl and eSFCtrl and IL-6 in eEPPCOS compared to eEPCtrl. Conclusions: Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and pro-oncogenic changes, independent of BMI, especially in eEPPCOS and eMSCPCOS, compared to controls. The study reveals an endometrial M-bM-^@M-^\disease phenotypeM-bM-^@M-^] in women with PCOS with potential negative effects on endometrial function and long-term health. Proliferative phase endometrium was obtained from 6 overweight/obese PCOS (NIH criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting (FACS)-isolated endometrial epithelial cells (eEP), endothelial cells (eEN), stromal fibroblasts (eSF) and mesenchymal stem cells (eMSC). Tissue samples were obtained through the National Institute of Health (NIH)/University of California, San Francisco (UCSF), Human Endometrial Tissue and DNA Bank in accordance with the guidelines of the Declaration of Helsinki. Informed consent was obtained from all participants in the UCSF Center for Reproductive Health, and the study was approved by the UCSF Committee on Human Research. The clinical summary of the study participants is shown in Table 1. Eleven proliferative phase endometrial biopsies (Pipelle, Cooper Surgical Shelton, Connecticut) and one curettage specimen were collected from over-weight (Body Mass Index [BMI, kg/m2] M-bM-^IM-% 27 < 29.9; n=1) and obese (BMI M-bM-^IM-% 30; n=5) women with PCOS (age 30.5M-BM-1 2.1 yrs, BMI 34.13M-BM-1 2.2, NIH criteria (17) and overweight (n=2) and obese (n=4) control women (age 36.50M-BM-11.70 yrs, BMI 35.73M-BM-13.96). All PCOS subjects had normal 17-hydroxyprogesterone, prolactin and thyroid hormone levels. Control samples were obtained from healthy volunteer and women undergoing benign gynecological surgery. All controls reported menstrual cycles with regular interval (25-35 days) and no clinical evidence of having PCOS. Neither PCOS nor control subjects were exposed to hormonal medications for at least 2 months prior to tissue sampling and were confirmed not pregnant.

Project description:During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PD-L1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PD-L1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly, liver metastases were suppressed by the D-CAN / anti-PD-L1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy.

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Perivascular Human Endometrial Mesenchymal Stem Cells Express Pathways Relevant to Self-Renewal, Lineage Specification, and Functional Phenotype

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