Project description:Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small molecule-driven approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging to develop. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine small molecules (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts towards a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study therefore provides an effective chemical approach for generating neural stem cells from mouse fibroblasts, and reveals mechanistic insights into underlying reprogramming process. Genome-wide epigenetic changes of H3K4me1, H3K4me3, H3K27me3, and H3K27ac were analyzed by CHIP-seq for tdMEFs from day 0 (ciNSLC), day 4 (D4), day 8 (D8) of M9-induced neural reprogramming, and ciNSLCs and pri-NPC.
Project description:The compound (O4I3) showed a remarkable OCT4 induction, which at least in part, is due to the inhibition of H3K4-specific lysine demethylase (KDM5, also known as JARID1). Experiments demonstrated that KDM5A, serves as a reprogramming barrier via interference with the enrichment of H3K4Me3 at the OCT4 promoter. Thus, our results introduce a new class of KDM5 chemical inhibitors and provide further insight into the pluripotency-related properties of KDM5-family members.
Project description:Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small molecule-driven approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging to develop. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine small molecules (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts towards a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study therefore provides an effective chemical approach for generating neural stem cells from mouse fibroblasts, and reveals mechanistic insights into underlying reprogramming process.
Project description:Fibroblasts can be chemically induced to pluripotent stem cells (CiPSCs) through an extraembryonic endoderm (XEN)-like state or directly converted into other differentiated cell lineages. However, the mechanisms underlying chemically-induced cell fate reprogramming remain unclear. Here, a transcriptome-based screen of biologically active compounds uncovered that CDK8 inhibition was essential to enable chemically-induced reprogramming from fibroblasts into XEN-like cells, then CiPSCs. RNA-seq analysis showed that a CDK8 inhibitor, MSC2530818, inhibited pro-inflammatory pathways that suppress chemical reprogramming, and facilitated the induction of a multi-lineage priming state, indicating the establishment of plasticity in fibroblasts. CDK8 inhibition also resulted in chromatin accessibility profile and Pol II occupation profile similar to that under initial chemical reprogramming. Moreover, CDK8 inhibition greatly promoted transgene-mediated reprogramming of mouse fibroblasts into hepatocyte-like cells, and chemical reprogramming of human fibroblasts into adipocytes. These collective findings thus define CDK8 as a general molecular barrier in multiple cell reprogramming processes, and as a common target for inducing plasticity and cell fate conversion.
Project description:Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small molecule-driven approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging to develop. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine small molecules (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts towards a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study therefore provides an effective chemical approach for generating neural stem cells from mouse fibroblasts, and reveals mechanistic insights into underlying reprogramming process. Genome-wide binding of Elk1 and Gli2 was analyzed by CHIP-seq for tdMEFs from day 0 (ciNSLC), day 4 (D4), day 8 (D8) of M9-induced neural reprogramming, and ciNSLCs and pri-NPC.