Project description:Multipotent spermatogonial stem cells (mSSCs) derived from SSCs are a potential new source of individualized pluripotent cells in regenerate medicine such as ESCs. We hypothesized that the culture-induced reprogramming of SSCs was mediated by a mechanism different from that of iPS, and was due to up-regulation of specific pluripotency-related genes during cultivation. Through a comparative analysis of expression profile data, we try to find cell reprogramming candidate factors from mouse spermatogonial stem cells. We used microarrays to analyze the gene expression profiles of culture-induced reprogramming converting unipotent spermatogonial stem cells to pluripotent spermatogonial stem cells. Three types of spermatogonial stem cells were mechanically collected according to morphological criteria for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Multipotent spermatogonial stem cells (mSSCs) derived from SSCs are a potential new source of individualized pluripotent cells in regenerate medicine such as ESCs. We hypothesized that the culture-induced reprogramming of SSCs was mediated by a mechanism different from that of iPS, and was due to up-regulation of specific pluripotency-related genes during cultivation. Through a comparative analysis of expression profile data, we try to find cell reprogramming candidate factors from mouse spermatogonial stem cells. We used microarrays to analyze the gene expression profiles of culture-induced reprogramming converting unipotent spermatogonial stem cells to pluripotent spermatogonial stem cells.
Project description:Mammalian spermatogonial stem cells (SSCs) spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs) during in vitro expansion. Here, we examine the epigenetic signature of SSCs and MASCs, identifying bivalent histone H3-lysine4 and -lysine27 trimethylation at somatic gene promoters in SSCs and an ESC-like promoter chromatin state in MASCs.
Project description:Mammalian spermatogonial stem cells (SSCs) spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs) during in vitro expansion. Here, we examine the epigenetic signature of SSCs and MASCs, identifying bivalent histone H3-lysine4 and -lysine27 trimethylation at somatic gene promoters in SSCs and an ESC-like promoter chromatin state in MASCs.
Project description:Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. SSCs express key OSKM reprogramming factors at some levels, and do not require ectopic expression of any gene for the acquisition of pluripotency during reprogramming to mSSCs. Therefore, we reasoned that additional factors are required to regulate SSC reprogramming. In this study, we first compared the expression of reprogramming signature genes among somatic cells, iPSC, SSCs, mSSCs, and partially reprogramed cells, and found that they appear to have similar pluripotency states, whereas their transcriptional program differs. We developed a systems biology approach to prioritise genes for pluripotency regulatory factors by integrating transcriptome and interactome data on the genome-wide functional network. Then, we performed a series of systematic gene prioritisation steps and identified 53 candidates, which included some known reprogramming factors. We experimentally validated one particular candidate, Positive cofactor 4 (Pc4), which was expressed in PSCs and yielded a positive RNA interference (RNAi) response in an Oct4 reporter assay. We demonstrated that Pc4 enhanced the efficiency of OSKM-mediated reprogramming by promoting the transcriptional activity of key pluripotency factors, and by regulating the expression of many protein- and miRNA-encoding genes involved in reprogramming and somatic cell-specific genes. Pc4-overexpressing mESC lines were established by Venus (YFP)-expressing lentiviral transfection. The mESCs were split at a density of 2 ´ 104 cells onto fresh MEF feeder cells seeded into a 6 well dish (containing mESC growth medium) with virus particles, and 25 μg/ml polybrene (Sigma Aldrich) was added. After 24 h, the medium was replaced with fresh growth medium. After 4 days later, mESC colonies expressing YFP were picked and replated. Three different Pc4-overexpressing mESC lines were established.
Project description:Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. SSCs express key OSKM reprogramming factors at some levels, and do not require ectopic expression of any gene for the acquisition of pluripotency during reprogramming to mSSCs. Therefore, we reasoned that additional factors are required to regulate SSC reprogramming. In this study, we first compared the expression of reprogramming signature genes among somatic cells, iPSC, SSCs, mSSCs, and partially reprogramed cells, and found that they appear to have similar pluripotency states, whereas their transcriptional program differs. We developed a systems biology approach to prioritise genes for pluripotency regulatory factors by integrating transcriptome and interactome data on the genome-wide functional network. Then, we performed a series of systematic gene prioritisation steps and identified 53 candidates, which included some known reprogramming factors. We experimentally validated one particular candidate, Positive cofactor 4 (Pc4), which was expressed in PSCs and yielded a positive RNA interference (RNAi) response in an Oct4 reporter assay. We demonstrated that Pc4 enhanced the efficiency of OSKM-mediated reprogramming by promoting the transcriptional activity of key pluripotency factors, and by regulating the expression of many protein- and miRNA-encoding genes involved in reprogramming and somatic cell-specific genes.
Project description:Spermatogonial stem cells are the foundation of spermatogenesis and as such can serve as a tool for the treatment of infertility in prepubertal cancer survivors. Spermatogonial stem cells are unique as they develop from primordial germ cells (PGCs), which colonize the developing tubules as immature SSC precursors. It has been controversial, when SSCs are maturing to an adult-like stem cell and recent research has found that prepubertal SSCs are actually metabolically distinct from adult SSCs until puberty. Sertoli cells picture a major part of the SSC niche and undergo drastic changes with puberty and polarize to compartmentalize the seminiferous epithelium with formation of tight junctions to a tight basal part where SSCs reside and an apical part with more differentiated stages of spermatogenesis. In the study were mapping the progression of Sertoli cells maturation events to the metabolic changes SSCs undergo during prepubertal development.
Project description:Spermatogonial stem cells (SSCs) could transform into pluripotent state in long term culture without introduction of exogenous factors. And p53 deficiency rescued SSCs from extensive cell apoptosis during transformation induced by rewriting of methylation profiles in SSCs. Notably, p53 is believed as a key bottle-neck for reprogramming. Based on these studies, we compared the difference of chromatin accessibility between SSCs from wild type and p53 deficient SSCs mice using ATAC-seq, to explore the potential mechanism at chromosome level. And RNA-Seq was subsequently exerted to verify the predicted genes and related pathways in SSCs transformation. This result further reveals the role of p53 in regulating SSCs fates, which provide hints new insight for understanding the biological characteristics of germline stem cells, basic and clinic researchand molecular mechanisms of reprogramming and tumorigenesis.
Project description:Spermatogonial stem cells (SSCs) could transform into pluripotent state in long term culture without introduction of exogenous factors. And p53 deficiency rescued SSCs from extensive cell apoptosis during transformation induced by rewriting of methylation profiles in SSCs. Notably, p53 is believed as a key bottle-neck for reprogramming. Based on these studies, we compared the difference of chromatin accessibility between SSCs from wild type and p53 deficient SSCs mice using ATAC-seq, to explore the potential mechanism at chromosome level. And RNA-Seq was subsequently exerted to verify the predicted genes and related pathways in SSCs transformation. This result further reveals the role of p53 in regulating SSCs fates, which provide hints new insight for understanding the biological characteristics of germline stem cells, basic and clinic researchand molecular mechanisms of reprogramming and tumorigenesis.
Project description:Transcriptional profiling of mouse spermatogonial stem cells (SSCs) comparing control untreated SSCs with SSCs with exogenous FGF2 withdrawn and FGFR inhibitor SU5402 supplemented (-F+S). Results provide insight into the mechanisms of FGF2-supported in vitro self-renewal of SSCs. Two-condition experiment, SSCs-F+S vs. SSCs. Biological replicates: 4 control replicates, 4 -F+S replicates.