Project description:The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogenous neuronal cultures. Here we show that under permissive culturing conditions individual transcription factors can induce a desired neuronal lineage from virtually all expressing cells by a mechanism resembling developmental binary cell fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene expression validation of generated neurons and identification of novel features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into e.g. dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and promote efficient reprogramming also in more remote neural contexts, including human neural progenitor cells. We used microarray analysis to verify the identity of several mESC derived neuronal cell types. By genome-wide gene expression comparisons we gained novel insights into molecular properties of several clinical relevant neuronal cell types. Total RNA was extracted from wild-type and transcription-factor induced mESC derived post-mitotic neurons and hybridized on Affymetrix arrays. In total triplicates of 9 different samples were analyzed. Wild-type samples served as control samples.
Project description:scRNA-seq of mouse embryonic stem cells (mESC) derived from four different genetic backgrounds grown in ground state conditions and differentiated towards an epiblast stem cell like (EpiSCL) population.
Project description:We reported the allele-specific single cell RNA sequencing in highly morphogenic (C57Bl6J-maternal x CASTEiJ-paternal) male F1 mouse embryonic stem cells (mESC) and in vitro mESC derived six cardiac lineage cell types. We demonstrated distinctive gene regulation based on the parental origin of the alleles. We showed that deterministic and stochastic monoalleleic gene classes are distinctive in regulation and are involved in unique processes. In this study we highlighted the importance of parental origin-specific gene expression in development, homeostasis and disease.
Project description:We reported the allele-specific single cell RNA sequencing in highly morphogenic (C57Bl6J-maternal x CASTEiJ-paternal) male F1 mouse embryonic stem cells (mESC) and in vitro mESC derived six cardiac lineage cell types. We demonstrated distinctive gene regulation based on the parental origin of the alleles. We showed that deterministic and stochastic monoalleleic gene classes are distinctive in regulation and are involved in unique processes. In this study we highlighted the importance of parental origin-specific gene expression in development, homeostasis and disease.
Project description:We reported the allele-specific bulkl RNA sequencing in highly morphogenic (C57Bl6J-maternal x CASTEiJ-paternal) male F1 mouse embryonic stem cells (mESC) and in vitro mESC derived six cardiac lineage cell types. We demonstrated distinctive gene regulation based on the parental origin of the alleles. We showed that deterministic and stochastic monoalleleic gene classes are distinctive in regulation and are involved in unique processes. In this study we highlighted the importance of parental origin-specific gene expression in development, homeostasis and disease.
Project description:Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3094 upregulated and 6964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs. RNA profiles were generated from FACS-purified control and SMA mESC-derived motor neurons (n=3/genotype) by deep sequencing using Illumina HighSeq 2500
Project description:Although the locations of promoters and enhancers have been identified in several cell types, we have yet limited information on their connectivity. We developed HiCap, which combines Hi-C with promoter sequence capture, to enable genome-wide identification of regulatory interactions with single-enhancer resolution. HiCap analyses of mouse embryonic stem cells (mESC) identified promoter-enhancer interactions predictive of gene expression change upon perturbation, opening up for genomic analyses of long-range gene regulation. HiCap was designed by combining Hi-C with with sequence capture (for all promoters) and carried out in mouse embryonic stem cells (mESC)
Project description:Although the locations of promoters and enhancers have been identified in several cell types, we have yet limited information on their connectivity. We developed HiCap, which combines Hi-C with promoter sequence capture, to enable genome-wide identification of regulatory interactions with single-enhancer resolution. HiCap analyses of mouse embryonic stem cells (mESC) identified promoter-enhancer interactions predictive of gene expression change upon perturbation, opening up for genomic analyses of long-range gene regulation. HiCap was designed by combining Hi-C with with sequence capture (for all promoters) and carried out in mouse embryonic stem cells (mESC)