Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors).
Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors). Two histone modifications are given: H3K4me3, H3K27me3. In addition binding data of Mbd3 and Mi2B in various stages.
Project description:Ectopic expression of selected transcription factors enables reprogramming of somatic cells to pluripotency. Despite steady progress in the field the exact molecular mechanisms that coordinate this remarkable transition remain still largely elusive. To better understand the final steps during the acquisition of pluripotency, we optimized an experimental system where cells exit pluripotency and then are systematically challenged to reacquire pluripotency. Using this approach we identify a transient period of deterministic reprogramming during which cells exposed to ectopic transcription factors, but not serum/LIF alone, revert to pluripotency with near 100% efficiency before approaching somatic kinetics (1-2 weeks) and efficiencies (1-2%). Investigation of the transient phase reveals unique molecular dynamics that point to a set of cis-regulatory elements that appear central to facilitating reprogramming. Interestingly, these regions retain an epigenetic signature during in vitro and in vivo differentiation and may act as primary targets of ectopically induced factors during somatic cell reprogramming.
Project description:Ectopic expression of selected transcription factors enables reprogramming of somatic cells to pluripotency. Despite steady progress in the field the exact molecular mechanisms that coordinate this remarkable transition remain still largely elusive. To better understand the final steps during the acquisition of pluripotency, we optimized an experimental system where cells exit pluripotency and then are systematically challenged to reacquire pluripotency. Using this approach we identify a transient period of deterministic reprogramming during which cells exposed to ectopic transcription factors, but not serum/LIF alone, revert to pluripotency with near 100% efficiency before approaching somatic kinetics (1-2 weeks) and efficiencies (1-2%). Investigation of the transient phase reveals unique molecular dynamics that point to a set of cis-regulatory elements that appear central to facilitating reprogramming. Interestingly, these regions retain an epigenetic signature during in vitro and in vivo differentiation and may act as primary targets of ectopically induced factors during somatic cell reprogramming.
Project description:Ectopic expression of selected transcription factors enables reprogramming of somatic cells to pluripotency. Despite steady progress in the field the exact molecular mechanisms that coordinate this remarkable transition remain still largely elusive. To better understand the final steps during the acquisition of pluripotency, we optimized an experimental system where cells exit pluripotency and then are systematically challenged to reacquire pluripotency. Using this approach we identify a transient period of deterministic reprogramming during which cells exposed to ectopic transcription factors, but not serum/LIF alone, revert to pluripotency with near 100% efficiency before approaching somatic kinetics (1-2 weeks) and efficiencies (1-2%). Investigation of the transient phase reveals unique molecular dynamics that point to a set of cis-regulatory elements that appear central to facilitating reprogramming. Interestingly, these regions retain an epigenetic signature during in vitro and in vivo differentiation and may act as primary targets of ectopically induced factors during somatic cell reprogramming.
Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Reduced representation bisulfite sequencing (RRBS) was applied to mouse iPS cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors) from randomly selected Mbd3+/+ and Mbd3flox/- clonal cell line series. Polyclonal donor cell cultures were harvested at days 0,4 and 8 after DOX reprogramming without selection or sorting for any marker or passaging, and mapped for similarity to subcloned iPSC lines.