MicroRNAs (miRNAs) are small non-coding RNAs, which play an important role in various cellular and developmental processes. The study of miRNAs in erythropoiesis is crucial to uncover the cellular pathways that are modulated during the different stages of erythroid differentiation. Using erythroid cells derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs)and small RNA sequencing, our study unravels the various miRNAs involved in critical cellular pathways in erythroid maturation. We analyzed the occupancy of erythroid transcription factors and chromatin accessibility in the promoter and enhancer regions of the differentially expressed miRNAs to integrate miRNAs in the transcriptional circuitry of erythropoiesis. Analysis of the targets of the differentially expressed miRNAs revealed novel pathways in erythroid differentiation. Finally, we described the application of Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) based editing of miRNAs to study their function in human erythropoiesis.
Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from the patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies, without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.
Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34+ cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34+ cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34+ cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.
Enhancers are cruicial for gene regulation and demonstrate tissue, cell and developmental stage specificity. Recent studies have shown that analyzing genome wide association of CBP and p300 predicts the tissue spcific transcriptional enhancers in various tissues. Our aim was to study the transcriptional enhancers in haematopoietic stem cells and erythroid cells to identify the role of these genomic regions in determining transcriptional regulation of haematopoietic stem cell maintenance and erythroid differentiation. We obtained CD34+ cells from normal healthy donors after magnetic activated cell sorting from peripheral blood mononuclear cells. The purified CD34+ cells were differentiated by ex vivo erythropoiesis using a serum-free two-phase liquid culture system. Cells were collected at 2 different time points, before (day 5 in culture) and after differentiation (day13 in culture). ChIP assays were carried out using antibodies against CBP and p300 and two independent libraries were created using Illumina TrueSeq library kit as per the manufacturer's instruction. The libraries were sequenced in Illumina HiSeq producing 50 bp single end reads. A control sample (without antibody) was run as input as a background control. Sequenced reads were mapped to the human genome (UCSC Genome Browser hg19). To identify ChIP-Seq peaks, Model-based Alignment of ChIP-Seq (MACS) program was used with a p value of <10e-5 and enrichment factor > 5. Localization of the binding sites relative to the annotated genes and co-localization of CBP/p300 were determined using the ChIP seek analyzer. Validation of co-occupancy at selected regions was performed by quantitative-PCR analysis. Genome wide analysis of CBP and p300 enrichment showed these co-factors are highly enriched in introns and intergenic regions than the gene promoters suggesting that these co-activators efficiently mark enhancers in the haematopoietic stem cells and erythroid cells. Co-localization of CBP/p300 with erythroid transcription factors (GATA1, KLF1, NFE2 and TAL1) was performed. The results showed that CBP/p300 are highly associated with erythroid transcription factors during differentiation indicating that transcriptional activator complexes consisting of these transcription factors and CBP/p300 in enhancer mediated transcriptional regulation in erythropoiesis. The sites of CBP/p300 occupancy were correlated with the transcriptome data and it was found that most of the top regulated genes were enriched with CBP and p300 within the intronic region in erythroid cells. We then explored whether CBP and p300 are enriched in the regions associated with DNA polymorphisms relevant to erythroid cell traits and observed that CBP/p300occupy at HBD (5'UTR), BCL11A (intron 2)and HBS1L-MYB intergenic region which contains polymorphisms linked with levels of fetal haemoglobin (HbF). We also found enrichment of these co-activators in previously mapped erythroid specific enhancers such as IKZF1, ANK1 and ABO gene loci. Gene ontology (GO) was performed using GREAT for regions associated with CBP and p300 (binomial fold enrichment >4) and the results indicated that the genes associated with CBP were involved in erythrocyte development whereas the genes associated with p300 were found to regulate erythroid differentiation. These erythroid specific genes delineated in our study also showed conservation in mouse and were found to be associated with erythroid cell phenotypes. We also found significant enrichment of CBP and p300 in erythroid cells compared to haematopoietic stem cells for several genes that have not been previously characterized for erythroid differentiation. Taken together, our findings elucidated the roles of co-activators CBP and p300 in erythroid differentiation and further we identified these factors are enriched in previously known and new erythroid specific enhancers in association with cell specific transcription factors. Functional evaluation of the newly idenfied regulatory elements bound by CBP and p300 in the erythroid cells will provide insights in to erythroid cell development and differentiation. Disclosures No relevant conflicts of interest to declare.
A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms. Disclosures No relevant conflicts of interest to declare.
Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process. The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+). Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. Disclosures No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.