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: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.

Project description:Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages such as adipocytes, chondrocytes and osteocytes. They can be isolated from numerous tissue sources including bone marrow, adipose tissue, skeletal muscle and others. Because of their differentiation potential and their secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression, which are considered advantageous in treating multiple disorders such as graft-versus-host disease. Since the number of MSCs derived from a tissue source is low, cellular expansion is necessary to obtain sufficient numbers for a desired cell therapy. However, after several rounds of passaging, our previous results have shown that MSCs exhibit reduced capacity for proliferation and differentiation. In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict the cell population proliferative quality. Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays. A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| > 0.70) for 8 MSC donors at multiple passages. These 24 identified genes were then confirmed using an additional set of MSCs from 8 new donors using RT-qPCR. The proliferative potential of the second set of MSCs was measured for each donor/passage by three proliferation assays for confluency fraction, fraction of EdU+ cells and population doubling time. The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only 7 of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, SPC25) demonstrated significant correlation (FDR: q < 0.05 and |r| > 0.62) with MSC proliferation regardless of passage. These 7 genes and the proliferative capacity of different non-MSC cell lines were assessed for comparison. Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the introduction of non-MSC cell lines; therefore this set of 7 genes may be more strongly associated with MSC proliferative quality. These correlative methods may be further used to identify additional markers that exhibit strong correlation with a particular MSC quality such as differentiation or immune suppression potential. Our results pave the way toward the identification of specific gene markers that could rapidly determine the quality of an MSC population for a particular cellular therapy in lieu of an extended in vitro or in vivo assay. Microarray gene expression analysis with confirmation of genes by RT-qPCR

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