Induction of dnFGFR2bfor 3 partially overlapping intervals at the early stages of otocyst morphogenesis revealed expected and novel up and downregulated genes that were validated by in situ hybridization analysis. Cell cyle genes were enriched in the downregulated datasets and human hearingloss genes were enriched in the upregulated datasets. Overall design: Differential mRNA expression analysis of pooled Rosa26rtTA/+ (control) and pooled Rosa26rtTA/+;Tg(tetO-s(dn)Fgfr2b)/+ (experimental) embryos induced with doxycycline for the indicated intervals. N=4 biological replicates per treatment (i.e. 4 pregnant females)
Spatial and temporal inhibition of FGFR2b ligands reveals continuous requirements and novel targets in mouse inner ear morphogenesis.
Subject
View SamplesGenetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. Overall design: RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.
Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells.
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View SamplesCD34+ positively isolated from healthy donors (stimulated by G-CSF) with magnetic beads (after blood leukapheresis)
NA-Seq: a discovery tool for the analysis of chromatin structure and dynamics during differentiation.
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View SamplesThis SuperSeries is composed of the SubSeries listed below.
Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.
Specimen part
View SamplesPatient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this disease in a dish approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinsons disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the -synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells.
Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.
Specimen part
View SamplesHira has been implicated in replication-independent chromatin assembly.
Distinct factors control histone variant H3.3 localization at specific genomic regions.
Specimen part
View Samples