Project description:<p> <ol> <li>Implement an efficient, highly reproducible and 'scalable' system for the production of large numbers of sickle cell anemia-specific iPS cells (iPSC).</li> <li>Derive and characterize a novel, in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of 'clinical grade' transgene-free iPS cells; use this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes.</li> <li>Identify developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS.</li> <li>Determine the effects of the three known HbF major quantitative trait loci (QTL) on globin gene expression in disease-specific iPS cells during in vitro erythropoiesis.</li> <li>Search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment, by examining gene expression profiles and mRNA sequence of their iPSC-derived erythroid cells.</li> <li>Develop and use a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers, explore globin switching, and correct the HbS mutation in sickle iPSC lines.</li> </ol> </p>
Project description:In this study, we analyzed the genome-wide miRNAs dynamics that occur during the differentitation of HESCs into the erythroid lineage using high throughput sequencing technology. Undifferentiated HESCs as well as erythroid cells at three developmental stages-ESER (embryonic stage), FLER (fetal stage) and PBER (adult stage) were analyzed.
Project description:To explore the mechanisms controlling erythroid differentiation and development in human, we analyzed the genome-wide transcription dynamics that occurs during the differentiation of HESCs into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. Undifferentiated HESCs as well as erythroid cells at three developmental stages-ESER (embryonic stage), FLER (fetal stage) and PBER (adult stage)-were analyzed. Our findings revealed that the number of expressed genes decreased during the differentiation, while the total expression intensity increased. At the 3 transitions (HESC-ESER, ESER-FLER and FLER-PBER), differential expression of many types of genes was observed at every transition. These differentially expressed genes were involved in maintaining the pluripotency of stem cells, early erythroid specification, rapid cell growth and enucleation potential. In addition, differentially expressed genes were found to constitute networks and central nodes at each transition. Clusters of genes in some chromosomal regions switched between expression and silence. We also discovered that differentially expressed genes constituted networks and central nodes of them in each transition. Our studies provide a fundamental basis for further investigation of erythroid differentiation and development. Compare the transcriptome of embryonic stem cells and three erythroid cell types at different developmental stages
Project description:Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Knockdown of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation in a cell autonomous manner in the embryo and adult. We performed gene expression analysis to determine the expression of chd7 in adult sorted HSPC populations. Loss of Chd7 in long term hematopoietic stem cells (LT-HSCs) results in upregulation of genes that function in hematopoietic system development and function. Genes representative of each blood lineage including erythroid, myeloid, and lymphoid were upregulated, suggesting that Chd7 deficiency results in LT-HSCs that are more primed for multilineage differentiation. Together with the physical and genetic interaction, the data support a model in which CHD7 interacts with and modulates Runx1 activity to provide proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults. This represents a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation. We used microarrays to detail the expression of Chd7 in adult sorted HSPC populations.
Project description:Khajuria RK, Munschauer M, Ulirsch JC, Fiorini C, Leif S. Ludwig LS, McFarland SK, Abdulhay NJ, Specht H, Keshishian H, Mani DR, Jovanovic M, Ellis SR, Fulco CP, Engreitz JM, Schütz S, Lian J, Gripp KW,Weinberg OK, Pinkus GS, Gehrke L, Regev A, Lander ES, Gazda HT, Lee WY, Panse VG, Carr SA, Sankaran VG. Cell 2018, 173, 90–103. https://doi.org/10.1016/j.cell.2018.02.036.
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
Project description:CD34 positive hematopoietic stem cells were differentiated into erythroid lineage. Next generation sequencing (NGS) of 5hmC affinity pulldown and RNAseq were performed in four time point of different stages of erythroid differentiation. 4 RNA-Seq Samples (d0, d3, d7 and d10); 4 affinity-pulldown (d0, d3, d7 and d10), and 4 input samples (d0, d3, d7 and d10).
Project description:To explore the mechanisms controlling erythroid differentiation and development in human, we analyzed the genome-wide transcription dynamics that occurs during the differentiation of HESCs into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. Undifferentiated HESCs as well as erythroid cells at three developmental stages-ESER (embryonic stage), FLER (fetal stage) and PBER (adult stage)-were analyzed. Our findings revealed that the number of expressed genes decreased during the differentiation, while the total expression intensity increased. At the 3 transitions (HESC-ESER, ESER-FLER and FLER-PBER), differential expression of many types of genes was observed at every transition. These differentially expressed genes were involved in maintaining the pluripotency of stem cells, early erythroid specification, rapid cell growth and enucleation potential. In addition, differentially expressed genes were found to constitute networks and central nodes at each transition. Clusters of genes in some chromosomal regions switched between expression and silence. We also discovered that differentially expressed genes constituted networks and central nodes of them in each transition. Our studies provide a fundamental basis for further investigation of erythroid differentiation and development.
Project description:β-thalassemia cell lines were generated via CRISPR-Cas9 genome editing of Bristol Erythroid Line Adult (BEL-A) and differentiated to the basophilic and polychromatic erythroid cell stage. TMT comparative proteomics was then performed on stage matched WT and β-thalassemia cells isolated by FACS.
Project description:The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny, which also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter model we demonstrate here that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid specification. Accordingly the PreMegE population can be prospectively separated into âpro-erythroidâ and âpro-megakaryocyteâ populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that the level of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will provide the opportunity to further investigate and define the molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. mRNA profiles of wild type (WT), Runx1 P2-hCD4+ (P2+) and Runx1 P2-hCD4- (P2-) Bone marrow Pre-Megakryocyte/Erythroid (PreMegE) progenitors were generated from young adult (12-16 weeks) mice by deep sequencing, in triplicate, using Illumina NextSeq 500.