Multipotent and pluripotent stem cells have significant potential as sources for cell replacement therapies. However, the low yield and quality of in vitro differentiated cells produced from various stem cell sources presents a significant limitation for therapeutic applications. The most mature use of these stem cell products is in the field of transfusion medicine, where stem cell-derived red blood cells (RBCs) have clinically-proven potential as alternative transfusion products. To improve upon current approaches for RBC production, we used insight from both common and rare human genetic variation of blood counts to focus on the SH2B3 gene. By producing loss of function of SH2B3 using targeted knockdown and genome editing approaches in human hematopoietic stem and progenitor cells, as well as human pluripotent stem cells, we are able to significantly improve both the quality and yield of in vitro derived RBCs. We illustrate how insight from human genetic variation can assist in the development of broadly applicable approaches that have tremendous value for regenerative medicine.
Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells.
Specimen part
View SamplesSchimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in SWI/SNF-related matrix associated actin-dependent regulator of chromatin, subfamily A-like protein 1 (SMARCAL1). Changes in gene expression appear to underlie the immunodeficiency and arteriosclerosis of SIOD; therefore, we hypothesized that SMARCAL1 deficiency alters renal gene expression to cause the focal segmental glomerulosclerosis (FSGS) of SIOD, and that these gene expression alterations would be comparable to those observed in isolated FSGS. We tested this hypothesis by gene expression microarray analysis.
Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?
Sex
View SamplesSchimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in SWI/SNF-related matrix associated actin-dependent regulator of chromatin, subfamily A-like protein 1 (SMARCAL1). Changes in gene expression appear to underlie the immunodeficiency and arteriosclerosis of SIOD; therefore, we hypothesized that SMARCAL1 deficiency alters renal gene expression to cause the focal segmental glomerulosclerosis (FSGS) of SIOD. We tested this hypothesis by transcriptome analysis and quantitative reverse transcription PCR (qRT-PCR) of an SIOD patient kidney, a genetic screen and immunofluorescence. These showed increased expression of genes in the Wnt and Notch signaling pathways in an SIOD patient kidney, interaction of Marcal1 with genes encoding components of the Wnt and Notch signaling pathways, and increased levels of unphosphorylated b-catenin and Notch1 intracellular domain (NICD) in the glomeruli of SIOD patient kidneys. Given that increased Wnt and Notch activity are established causes of FSGS, we hypothesize that SMARCAL1 deficiency increases the activity of one or both of these pathways to cause the renal disease of most SIOD patients. Overall design: Comparison of mRNA levels between the kidney tissue of a Schimke immuno-osseous dysplasia (SIOD) patient and an unaffected control
Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?
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View SamplesWe investigated the nutritional effects on gene expression in sperm cells of F0 boars from a three generation Large White pig feeding experiment. A group of experimental (E) F0 boars were fed a standard diet supplemented with high amounts of methylating micronutrients whereas a control (C) group of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs which were investigated in a previous study. The aim of this study was to investigate if the nutrition affects gene expression in sperm cells of differentially fed boars and thus carry information in the form of RNA molecules to the next generation. Four RNA samples from sperm cells of these differentially fed boars were analyzed by RNA-Seq methodology. We found no differential RNA expression in sperm cells of the two groups based on the adjusted P-value > 0.05. Nevertheless, we performed a pathway analysis with 105 genes that differed in gene expression on the level of nominal P-value < 0.05 between the two diet groups. We found a significant number of these differentially expressed genes were enriched for the pathway maps of bacterial infections in cystic fibrosis (CF) airways, glycolysis and gluconeogenesis p.3 and cell cycle_Initiation of mitosis. The GO processes including a significant portion of differentially expressed genes were viral transcription and viral genome expression, viral infectious cycle, cellular protein localization, cellular macromolecule localization, nuclear-transcribed mRNA catabolic process and nonsense-mediated decay. In summary, the results of the pathway analysis are also inconclusive and it is concluded that RNA expression in sperm cells is not significantly affected by extensive supplementation of methylating micronutrients. Consequently, RNA molecules could not be established as epigenetic marks in this feeding experiment. Overall design: Gene expression in sperm cells from differentially fed F0 boars was measured. F0 boars received either a standard diet or a standard diet supplemented with methylating micronutrients. These boars were used to study transgenerational epigenetic inheritance in a three generation pig pedigree. Therefore it was of interest if the diet affects gene expression in sperm cells which could then be transmitted to next generations.
In search of epigenetic marks in testes and sperm cells of differentially fed boars.
Sex, Specimen part, Subject
View SamplesPowdery mildew, caused by the fungus Blumeria graminis (DC) Speer, is one of the most important foliar diseases of cereals worldwide. It is an obligate biotrophic parasite, colonising leaf epidermal cells to obtain nutrients from the plant cells without killing them. Syringolin A (sylA), a circular peptide secreted by the phytopathogenic bacterium Pseudomonas syringae pv. syringae, triggers a hypersensitive cell death reaction (HR) at infection sites when sprayed onto powdery mildew infected wheat which essentially eradicates the fungus. The rational was to identify genes whose expression was specifically regulated during HR, i.e. genes that might be involved in the switch of compatibility to incompatibility.<br></br>Powdery mildew-infected or uninfected plants were treated with syringolin two days after infection and plant material for RNA extraction was collected at 0.5, 1, 2, 4, 8, 12 hours after treatment (hat), resulting in an early (2 and 4 hat) and late pool (8 and 12 hat). Plant material that was uninfected prior to syringolin treatment was collected 8 and 12 hat (late pool of uninfected plant material), and 1 hat, respectively.
Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites.
Compound, Time
View SamplesOur data demonstrate that overexpression of the polarity protein Crb3 elicits changes in MCF-10A cells that culminate in an increase in the release of amphiregulin (AR) and the subsequent activation of EGFR signaling to drive proliferation. Microarray analysis was performed to define global changes in the transcriptional landscape induced by Crb3. Results provide insight into a FERM domain protein (EBP41L4B) required for Crb3 mediated induction of proliferation.
CRB3 and the FERM protein EPB41L4B regulate proliferation of mammary epithelial cells through the release of amphiregulin.
Cell line, Treatment
View SamplesThe tongue is a muscular organ in the vertebrate oral cavity that performs complex functions in daily life, including feeding and phonetic articulation. The tongue consists of mesenchyme cells of two distinct origins: the muscle cells are derived from occipital somites whereas the tendons and other connective tissues derived from the cranial neural crest. Cranial neural crest cells are important for the initiation of tongue swelling and proper patterning of intrinsic and extrinsic tongue muscle groups. However, little is known regarding the molecular and cellular mechanisms of tongue morphogenesis. We show that the odd-skipped related 1 (Osr1) transcription factor exhibits dynamic expression in the tongue mesenchyme during early tongue development. Tissue-specific inactivation of Osr1 in the early neural crest cells resulted in ectopic cartilage formation in the mouse tongue. We show that Sox9, the master regulator of chondrocyte differentiation, is initially widely expressed in the neural crest derived mesenchyme in the tongue and subsequently down-regulated concomitant by up-regulation of Osr1 expression. Osr1 mutant embryos exhibit persistent expression of Sox9 and chondrocyte differentiation from the neural crest derived tongue mesenchyme. Further biochemical analyses indicate that Osr1 may directly suppresses Sox9 gene expression in the tongue mesenchyme. These data reveal a novel mechanism in suppression of chondrogenic fate during tongue development. Remarkably, the ectopic cartilage in the Osr1 mutant mice resembles the entoglossal cartilage naturally develops in the avian tongue. These results suggest that modulation of expression of Osr1 may underline the evolutionary divergence in tongue cartilage formation. RNAs were isolated from microdissected E12 embryonic mouse tongue of Osr1f/-;Wnt1cre and control littermates and characterized by RNAseq Overall design: E12 mouse embryonic tongues were micro-dissceted, 3 pairs of control and mutant samples were pooled for the RNA extraction
Odd-skipped related-1 controls neural crest chondrogenesis during tongue development.
Specimen part, Cell line, Subject
View SamplesWe report the RNA profiles of both control and Kif3a f/f; Wnt1-Cre mandibular prominences of the murine face at embryonic day E11.5. We sought to determine the gene expression changes which occurr in the mandibular prominence when primary cilia are lost on neural crest cells. Overall design: The mandibular prominence from 10 control e11.5 embryos were collected and pooled, and 10 mutant e11.5 embryos were collected and pooled. RNA-seq was performed on these samples.
Cilia-dependent GLI processing in neural crest cells is required for tongue development.
Specimen part, Subject
View SamplesCombining an in vitro hNCC differentiation protocol with epigenomic profiling, we provide the first whole-genome characterization of cis-regulatory elements in this highly relevant cell type. With this data at hand, we have characterized the chromatin state and dynamics of all human gene promoters during the course of NCC in vitro differentiation. Most importantly, we have identified a large cohort of active and NCC-specific enhancers, which we showed to be functionally relevant in vivo, in the context of embryonic development. Finally, through sequence analysis of the identified NCC enhancers, we uncovered the orphan nuclear receptors NR2F1 and NR2F2 as novel hNCC transcriptional regulators both in vitro and in vivo. Overall design: RNA-seq experiments in human neural crest cells (hNCC)
Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest.
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Increased chronic lymphocytic leukemia proliferation upon IgM stimulation is sustained by the upregulation of miR-132 and miR-212.
Sex, Age, Specimen part, Disease
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