Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium HumanMethylation450K Beadchip v1.2

Project description:miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (N=62) from untreated and patients exposed to hormonal therapies (GnRHa, Depo-Provera and oral contraceptives) from African Americans and Caucasians, and their regulatory functions in isolated paired (N=15) leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and leiomyosarcoma cell line (SKLM-S1). At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared to myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in microarray analysis, tissue factor (F3) and IL-8 were identified as their possible targets. At tissue level leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 which exhibited an inverse relationship with miR-93, but with limited racial or hormonal influences. Gain-of-function of miR-93/106b in LSMC, MSMC and SKLM-S1 dose-dependently repressed F3 and IL-8 through direct interactions with their respective 3M-bM-^@M-^YUTRs and indirectly through F3 repression inhibited IL8, CTGF and PAI-1 expression, confirmed by using siRNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase 3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL-8, CTGF and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms. Total RNA isolated from TF324 cells transfected with DOX-inducible lentiviral construct carrying miR-106b~25 cluster with and without Dox treatments for 6 days was subjected to gene expression profiling using Sentirx Beadchip Array HumanHT-12_v4.

Project description:Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and haematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1-tm1a/tm1a and demonstrated defects in bone marrow haematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation; and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes were due to a cell-intrinsic requirement for Mysm1 in the bone marrow. Importantly, Mysm1-tm1a/tm1a haematopoietic stem cells were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the haematopoietic progenitors. Overall these data establish a role for Mysm1 in the maintenance of bone marrow stem cell function, in the control of oxidative stress and genetic stability in haematopoietic progenitors, and in the development of lymphoid and erythroid lineages. Total RNA from different mouse tissues (liver, bone marrow, brain) and cell types (embryonic fibroblasts - MEFs, and embryonic stem cells - ESCs) from wild type, Mysm1+/tm1a (heterozygous), and Mysm1tma1/tm1a (homozygous) mice was analyzed. Tissue and MEFs comparisons are between 3-4 animals per group; ESC comparisons are between three independently passaged samples of wild type, Mysm1+/tm1a, and Mysm1tma1/tm1aES-cells, all on C57BL/6 background. Full allele name: Mysm1tm1a(KOMP)WTSI . This Series includes the data from the tissues.

Project description:Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and haematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1-tm1a/tm1a and demonstrated defects in bone marrow haematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation; and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes were due to a cell-intrinsic requirement for Mysm1 in the bone marrow. Importantly, Mysm1-tm1a/tm1a haematopoietic stem cells were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the haematopoietic progenitors. Overall these data establish a role for Mysm1 in the maintenance of bone marrow stem cell function, in the control of oxidative stress and genetic stability in haematopoietic progenitors, and in the development of lymphoid and erythroid lineages. Total RNA from different mouse tissues (liver, bone marrow, brain) and cell types (embryonic fibroblasts - MEFs, and embryonic stem cells - ESCs) from wild type, Mysm1+/tm1a (heterozygous), and Mysm1tma1/tm1a (homozygous) mice was analyzed. Tissue and MEFs comparisons are between 3-4 animals per group; ESC comparisons are between three independently passaged samples of wild type, Mysm1+/tm1a, and Mysm1tma1/tm1aES-cells, all on C57BL/6 background. Full allele name: Mysm1tm1a(KOMP)WTSI . This Series includes the data from the cells.

Dataset Information

Prostatic Stromal Cell Gene Expression

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Integrated analysis reveals critical genomic regions in prostate tumor microenvironment associated with clinicopathologic phenotypes.

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