Project description:The sequential adipogenic differentiation and dedifferentiation processes resulted in novel stem cells that proliferated faster and retained multilineage potency; however, the mechanism underlying this cross-talk remained to be determined. In this context, we identified a number of differentially expressed genes during these processes. Gene expression profiling was performed to obtain a deeper molecular insight into AL cells and their subsequent dedifferentiation to primitive cell types with multilineage potency. GeneChips were generated for PDMSCs, AL cells and DePDMSCs from 3 donors. We selected numerous genes which were differentially expressed in the processes.
Project description:When PDMSCs were induced to heptocytes in vitro, cells mophology, stem cell markers, mitochondrial metabolism will change according to the differentiated status.But dedifferentiation reverses differentiated cells to a more primitive phenotype and PDMSCs will retain the multilineage potency. Furthermore, it will leads to the alteration of gene expression pattern. We used microarrays to detail the global programme of gene expression underlying dedifferentiation and hepatogenic differentiation prcocesses, we intend to identify distinct classes of differentiated genes during these processes.
Project description:When PDMSCs were induced to heptocytes in vitro, cells mophology, stem cell markers, mitochondrial metabolism will change according to the differentiated status.But dedifferentiation reverses differentiated cells to a more primitive phenotype and PDMSCs will retain the multilineage potency. Furthermore, it will leads to the alteration of gene expression pattern. We used microarrays to detail the global programme of gene expression underlying dedifferentiation and hepatogenic differentiation prcocesses, we intend to identify distinct classes of differentiated genes during these processes. Human PDMSCs at passage 5 were induced to hepatocytes for 11 days, then the inductive medium was replaced by general culture medium for 1 day. Then human PDMSCs, hepatogenic PDMSCs at 11 days, dedifferentiated PDMSCs were selected for RNA extraction and hybridization on Affymetrix microarrays. To that end, we hand-selected cells at three time-points: before hepatogenic induction (P), hepatogenic PDMSCs at 11 days (H) and dedifferentiated PDMSCs for 1 day (DH) .
Project description:Bone marrow mesenchymal stem cells (MSC) were adipogenically differentiated followed by dedifferentiation. We are interested to know the new fat markers, adipogenic signaling pathways and dedifferentiation signaling pathways.Furthermore we are also intrested to know that how differentiated cells convert into dedifferentiated progenitor cells. To address these questions, MSC were adipogenically differentiated, followed by dedifferentiation. Finally these dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated, adipogenically differentiated and dedifferentiated cells were performed. Regarding the conversion of adipogenically differentiated cells into dedifferentiated cells, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes play a curcial role in cell cycle to drive the adipogenically differentiated cells towards an arrested state to narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these cell cycle genes motivates dedifferentiation of adipogenically differentiated cells to reverse the arrested state. We also found new fat markers along with signaling pathways for adipogenically differentiated and dedifferentiated cells, and also observed the influencing role of proliferation associated genes in cell cycle arrest and progression. We have differentiated bone marrow derived mesenchymal stem cells(MSC) into adipogenically differentiated cells followed by dedifferentiation. We are intrested to know new fat markers, signaling pathways for adipogenically differentiated and dedifferentiated cells, along to observe the genes associated with cell cycle arrest and progression. Results are also provide molecular insight into the process of adipogenesis and dedifferentiation. To study the adipogenic differentiation and dedifferentiation process along with "how adipogenically differentiated cells convert into dedifferentiated cells" on the molecular level, gene expression profiling with genome-wide Affymetrix HG-U133 Plus 2.0 olgonucleotide microarrays (Affymetrix, Santa Clara, CA, USA) was applied. In total, 12 GeneChips were performed for n=3 donors and 4 times (3x4=12): 3x P4 MSC (undifferentiated state), 3x adipogenically differentiated cells at day 15 (differentiated state), 3x dedifferentiated cells at day 7 (dedifferentiated state) and 3x dedifferentiated cells at day 35 (dedifferentiated state)
Project description:Development is generally viewed as one-way traffic of cell state transition from primitive to developmentally advanced states. However, molecular mechanisms that ensure the unidirectional transition remain largely unknown. NanoCAGE sequencing identified an evolutionarily conserved zinc finger repressor, ZBTB12, as a molecular barrier for dedifferentiation of human pluripotent stem cells (hPSCs). Single cell RNA sequencing revealed that ZBTB12 is essential for three germ layer differentiation by blocking hPSC dedifferentiation. Mechanistically, ZBTB12 cooperates with NANOG to fine-tune the expression of human endogenous retroviruses (HERVs). Active HERVH loci drive the expression of overlapping long non-coding RNAs (lncRNAs) whose downregulation by ZBTB12 is necessary for dedifferentiation blockage and pluripotency exit. Overall, we have identified a ZBTB12-HERVH-lncRNA axis as molecular machinery that safeguards the unidirectional transition of stem cell fates.
Project description:Development is generally viewed as one-way traffic of cell state transition from primitive to developmentally advanced states. However, molecular mechanisms that ensure the unidirectional transition remain largely unknown. NanoCAGE sequencing identified an evolutionarily conserved zinc finger repressor, ZBTB12, as a molecular barrier for dedifferentiation of human pluripotent stem cells (hPSCs). Single cell RNA sequencing revealed that ZBTB12 is essential for three germ layer differentiation by blocking hPSC dedifferentiation. Mechanistically, ZBTB12 cooperates with NANOG to fine-tune the expression of human endogenous retroviruses (HERVs). Active HERVH loci drive the expression of overlapping long non-coding RNAs (lncRNAs) whose downregulation by ZBTB12 is necessary for dedifferentiation blockage and pluripotency exit. Overall, we have identified a ZBTB12-HERVH-lncRNA axis as molecular machinery that safeguards the unidirectional transition of stem cell fates.
Project description:Development is generally viewed as one-way traffic of cell state transition from primitive to developmentally advanced states. However, molecular mechanisms that ensure the unidirectional transition remain largely unknown. NanoCAGE sequencing identified an evolutionarily conserved zinc finger repressor, ZBTB12, as a molecular barrier for dedifferentiation of human pluripotent stem cells (hPSCs). Single cell RNA sequencing revealed that ZBTB12 is essential for three germ layer differentiation by blocking hPSC dedifferentiation. Mechanistically, ZBTB12 cooperates with NANOG to fine-tune the expression of human endogenous retroviruses (HERVs). Active HERVH loci drive the expression of overlapping long non-coding RNAs (lncRNAs) whose downregulation by ZBTB12 is necessary for dedifferentiation blockage and pluripotency exit. Overall, we have identified a ZBTB12-HERVH-lncRNA axis as molecular machinery that safeguards the unidirectional transition of stem cell fates.
Project description:During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process “transcriptome maturation”, and identified candidate “mature genes”, including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of “behavioral competence”. Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.
Project description:During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process “transcriptome maturation”, and identified candidate “mature genes”, including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of “behavioral competence”. Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.
Project description:Bone marrow mesenchymal stem cells (MSC) were adipogenically differentiated followed by dedifferentiation. We are interested to know the new fat markers, adipogenic signaling pathways and dedifferentiation signaling pathways.Furthermore we are also intrested to know that how differentiated cells convert into dedifferentiated progenitor cells. To address these questions, MSC were adipogenically differentiated, followed by dedifferentiation. Finally these dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated, adipogenically differentiated and dedifferentiated cells were performed. Regarding the conversion of adipogenically differentiated cells into dedifferentiated cells, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes play a curcial role in cell cycle to drive the adipogenically differentiated cells towards an arrested state to narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these cell cycle genes motivates dedifferentiation of adipogenically differentiated cells to reverse the arrested state. We also found new fat markers along with signaling pathways for adipogenically differentiated and dedifferentiated cells, and also observed the influencing role of proliferation associated genes in cell cycle arrest and progression. We have differentiated bone marrow derived mesenchymal stem cells(MSC) into adipogenically differentiated cells followed by dedifferentiation. We are intrested to know new fat markers, signaling pathways for adipogenically differentiated and dedifferentiated cells, along to observe the genes associated with cell cycle arrest and progression. Results are also provide molecular insight into the process of adipogenesis and dedifferentiation.