Project description:Analysis of erythroblastic cells differentiated from induced pluripotent stem cells (iPSCs) generated from peripheral blood T lymphocytes of CDA type IV patient. Type IV congenital dyserythropoietic anemia (CDA) is due to a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A). Results provide insight into molecular mechanisms underlying CDA pathogenesis.
Project description:Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is an inherited anaemia arising primarily from mutations in C15orf41 and CDAN1, however the molecular mechanisms that cause the disease remain to be fully elucidated. We use an in vitro culture system to study multiple stages of erythropoiesis from CD34+ progenitors of patients with CDA-I. Applying a number of techniques, including ATAC-seq, we show that differentiation of CDA-I patient erythroblasts closely matches that of healthy donors during the early and intermediate stages of erythroid differentiation and maturation. However, a defect in terminal erythropoiesis can be observed in the CDA-I patient derived erythroblasts, resulting in a reduction in the number of enucleated erythroid cells.
Project description:Pathogenic NOTCH1 mutations are linked to congenital heart defects. To pinpoint how NOTCH1 deficiency affects cardiac development, we generated homozygous NOTCH1 knockout (N1KO) human induced pluripotent stem cells (iPSCs). We then performed high-throughput RNA-seq to profile differential gene expression in cardiomyocytes (iPSC-CMs) and endothelial cells (iPSC-ECs) derived from wild type (WT) and N1KO iPSCs.
Project description:Red blood cell disorders can result in severe anemia. One such disease, congenital dyserythropoietic anemia IV (CDA IV) is caused by heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by paucity of suitable and adequate quantities of material from anaemic patients and rarity of the disease. We therefore took a novel approach, creating a human cellular disease model system for CDA IV, which accurately recapitulates the disease phenotype. Next, using comparative proteomics we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include down-regulated pathways governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking and global transcription, and upregulated networks governing mitochondria biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying effects of a rare mutation can reveal fundamental biology.
Project description:SMEI patient induced pluripotent stem cells (iPSCs) were derived from patient fibroblasts. In order to test the similarity between patient iPSCs and human embryonic stem (hES) cells, microarry analysis was carried out on SMEI patient iPSCs and human embryonic stem cells. SMEI patient iPSCs were derived from patient fibroblasts. Human embryonic stem cells were derived from human blastocyst.And we use the microarray method to compare the global expression of patient iPSCs and embryonic stem cells.
Project description:There are a total of four samples each for this analysis. Each sample consists of the cells grown on three 10 cm culture plates. Each plate should have 2x106 cells for a total of 6x106 cells per sample when all three plates are combined. The first sample is undifferentiated human embryonic stem cells, the second sample is human glutamatergic neurons derived from those human embryonic stem cells, the third sample is undifferentiated human induced pluripotent stem cells and the fourth sample is human glutamatergic neurons derived from those human induced pluripotent stem cells.
Project description:Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disorder marked by ineffective erythropoiesis, abnormal morphology of bone marrow erythroblasts, and iron overload. Most cases of CDA-I are caused by mutations in the CDAN1 gene, which encodes for a ubiquitous protein of unknown function, codanin-1. To study the function of codanin-1 in CDA-I erythroid pathophysiology several erythroid models were developed. Here we show that codanin-1 expression is required for erythroid progenitor development and normal erythroid cell differentiation. Erythroid cells lacking codanin-1 demonstrated morphologic changes similar to that observed in CDA-I. Global gene expression changes after codanin-1 knockdown revealed alterations in a set of key erythroid genes. In particular, the AHSP gene, which showed decreased mRNA expression after codanin-1 knockdown in CD34+ cells, also demonstrated increased codanin-1 occupancy at its gene regulatory region by chromatin immunoprecipitation coupled to high-throughput sequencing. Using cell models recapitulating many features of CDA-I, we have confirmed the importance of codanin-1 during erythroid differentiation and provide mechanistic insight into how loss of codanin-1 expression results in CDA-I.
