Red Blood Cell Differentiation Research Articles

Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia

BackgroundLong non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources).MethodsINFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo.ResultsThe INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo.ConclusionsOur approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Using Inducible Signaling Receptors to Increase Erythropoietic Output from Genome-Edited Hematopoietic Stem Cells

Introduction: Oxygen is delivered to all tissues in the body through the hemoglobin molecule uniquely present in red blood cells (RBCs). Proper production and function of RBCs is therefore critical to human health. However, genetic defects of the hematologic system such as anemias and hemoglobin disorders (broadly termed “hemoglobinopathies”) are among the most common genetic disorders in the world. Therefore, developing strategies to regulate RBC development may allow researchers and clinicians to increase therapeutic potential of hematologic cell and gene therapies. Erythropoietin (EPO) signaling is indispensable to RBC development. In its native form, two EPO receptor (EPOR) monomers dimerize in the presence of EPO cytokine to initiate a downstream signaling cascade. Prior work has shown that conformational requirements of EPOR dimerization are rather “loose” and signaling may also be initiated by agonistic diabodies or in the context of chimeric receptors. While these chimeric receptors have limited clinical applicability, inducible dimerization domains such as engineered FKBPs are currently being used in clinical trials in the form of inducible safety switches. Due to the modular nature of cytokine receptors, we hypothesized that inducible dimerization domains (such as the FKBP domain) could be paired with endogenous receptors and signaling pathways to generate chimeric receptors that place RBC development under the control of a bioavailable small molecule - and therefore in the control researchers and clinicians. This study presents a set of chimeric, synthetic receptors that support HSPC output towards the erythroid lineage via a non-immunogenic, non-toxic small molecule (AP30187, hereafter termed “BB”). Methods: In-vitro editing was performed in primary human CD34+ HSPCs by electroporating Cas9 protein complexed with sgRNA along with an AAV6 DNA repair template to facilitate gene insertion via homology-directed repair (HDR). Bulk edited CD34+ HSPCs were then differentiated into RBCs using established differentiation protocols in the presence and absence of both erythropoietin and BB. Differentiation was determined by staining for RBC surface markers (CD71 and GPA) and subsequent flow cytometry analysis. Erythropoietic output was determined by cell density counts. Allele frequency of the edited cell population was determined through droplet digital PCR. Results: We generated seven FKBP-EPOR chimeric receptors - with the basic structure of SFFV-[FKBP-EPOR]-2A-YFP-bGH - that placed the FKBP binding domain at different regions of the endogenous receptor (Figure 1) and analyzed their ability to support RBC differentiation in the absence of EPO but with BB. Out of the seven constructs we found a set of receptors - 1.4 and 1.5 - that demonstrated erythropoietin-independent, BB-mediated erythroid differentiation at 108% and 94% mock RBC differentiation respectively. Construct 1.5 - dubbed inducible EPOR (iEPOR) - was chosen over 1.4 for further optimization because it was also no longer EPO responsive. We further optimized the design of iEPOR by 1) introducing a secretory signal peptide to improve trafficking of the receptor to the cell surface; 2) inclusion of a naturally occurring EPOR truncation that results in supraphysiological RBC differentiation; and 3) modification of the genomic insertion site and promoter of the iEPOR cassette to generate a toolbox of expression profiles. Our results suggest that iEPOR mimics the native EPOR signaling cascade with high fidelity and sensitivity according to bulk RNA-Seq data. We also observe a significant increase in erythropoietic output for iEPOR version 1.4 (6.7-fold by day 11 of RBC differentiation in +BB over -BB conditions, P=0.003), which was further enhanced by the above modifications for vector iEPOR version 3.5.2 (12.9-fold by day 11 of RBC differentiation in +BB over -BB conditions, P<0.0001). Additionally, iEPOR is demonstrated to be erythropoietin-independent, dose-responsive, and tunable via expression from different loci and promoters. Conclusion:iEPORs represent a powerful new tool to alter the cellular dynamics of red blood cell development. The work demonstrated in this study has the potential to dramatically improve the efficacies of existing gene therapies, offer a route to EPO-free ex-vivo RBC production, as well as serve as a new tool by which to better understand stem cell biology and differentiation.

Dual α-Globin and Truncated EPO Receptor Knockin Restores Hemoglobin Production in α-Thalassemia-Derived Hematopoietic Stem and Progenitor Cells

Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. A traditionally lethal disease due to severe fetal hypoxia, this condition is now survivable due to the advent of in utero blood transfusions. However, patients still require life-long transfusions postnatally. While allogeneic hematopoietic stem cell transplant (HSCT) can provide a definitive cure, it is limited by the paucity of suitable donors, has a high morbidity and mortality due the prerequisite myeloablative regimens, and carries the risk of immune rejection and graft-versus-host disease. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient's red blood cells (RBCs). Methods: We identified a Cas9 gRNA that efficiently introduced indels at the HBB locus as well as an AAV6 vector that mediated efficient integration into the start codon of HBB. After designing and testing integration cassettes, we developed two candidate vectors that insert a HBA1 transgene at the HBB locus, with one bicistronic vector that also expresses a truncated form of the erythropoietin receptor (t EPOR) to increase erythropoietic output from edited HSCs. We used this approach to edit CD34 + HSCs derived from the bone marrow of three patients with ATM. Edited cells were cultured for 16 days in RBC differentiation media. Editing rates were quantified by a custom ddPCR assay. To determine the transcriptional response to these editing strategies, bulk RNA sequencing was performed comparing wild-type cells and patient cells that underwent respective editing conditions. Lastly, α-globin protein production was quantified using tetramer and single-globin high protein liquid chromatography (HPLC). Results: While effective editing frequencies in ATM HSCs were achieved by the HBA integration vector (39% edited alleles), we observed that editing rates increased over the course of RBC differentiation in the combined HBA+t EPOR edited condition, reaching 76% edited alleles by day 16. We also found that total cell counts by the end of RBC differentiation more than doubled (2.2-fold) in the HBA+t EPOR condition compared to the HBA alone condition. RNA sequencing of edited ATM-derived HSCs following RBC differentiation revealed an upregulation of HBA1, HBA2, and EPOR transcripts compared to unedited patient cells. These results were supported by HPLC, which demonstrated restoration of hemoglobin tetramers, including fetal hemoglobin (α+γ subunits), adult hemoglobin (α+β subunits), and hemoglobin A2 (α+δ subunits) compared to unedited patient cells. Complementing this with globin single chain analysis found that we had achieved 11% restoration of α-globin compared to wild-type levels. In spite of integration at the HBB locus, neither RNA sequencing nor HPLC showed disruption of β-globin production. α-globin production with a concomitant decrease in the formation of toxic β-globin dimers was confirmed by Western blot, with restoration of the normal balance of α-globin to dimeric β-globin in edited cells to wild-type levels. Conclusions: To our knowledge, these results demonstrate for the first time that gene therapy or genome editing may be used to increase α-globin production in ATM patient-derived RBCs. We found that the editing strategy that paired site-specific α-globin integration with t EPOR expression achieved the highest editing frequencies, greatest production of edited cells, and the most robust production of α-globin protein compared to α-globin integration alone. By increasing erythropoietic output using t EPOR, this approach has the potential to overcome some of the clinical challenges previously seen in HSCT for the hemoglobinopathies, including low editing and engraftment rates. Moreover, as patients with severe anemia such those with ATM have increased levels of circulating erythropoietin due to severe anemia, a t EPOR-based enrichment strategy is likely to be especially potent following transplantation into ATM bone marrow. We believe this could allow clinicians to reduce or eliminate the need for myeloablation, which stand as a major barrier to HSCT safety and efficacy. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for ATM.

Evolution of surface area and membrane shear modulus of matured human red blood cells during mechanical fatigue

Mechanical properties of red blood cells (RBCs) change during their senescence which supports numerous physiological or pathological processes in circulatory systems by providing crucial cellular mechanical environments of hemodynamics. However, quantitative studies on the aging and variations of RBC properties are largely lacking. Herein, we investigate morphological changes, softening or stiffening of single RBCs during aging using an in vitro mechanical fatigue model. Using a microfluidic system with microtubes, RBCs are repeatedly subjected to stretch and relaxation as they squeeze into and out of a sudden contraction region. Geometric parameters and mechanical properties of healthy human RBCs are characterized systematically upon each mechanical loading cycle. Our experimental results identify three typical shape transformations of RBCs during mechanical fatigue, which are all strongly associated with the loss of surface area. We constructed mathematical models for the evolution of surface area and membrane shear modulus of single RBCs during mechanical fatigue, and quantitatively developed an ensemble parameter to evaluate the aging status of RBCs. This study provides not only a novel in vitro fatigue model for investigating the mechanical behavior of RBCs, but also an index closely related to the age and inherent physical properties for a quantitative differentiation of individual RBCs.

Transcriptomic analysis reveals genetic factors regulating early steroid-induced osteonecrosis of the femoral head.

The present study aimed to explore the signaling pathways involved in development of early steroid-induced osteonecrosis of the femoral head (SONFH) and identify diagnostic biomarkers regulating peripheral blood in SONFH patients. We downloaded transcriptome data and identified differentially expressed genes (DEGs) using the R software. We used ClusterProfiler to perform enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, and analyzed protein–protein interactions using the STRING database. Network X was used to visualize the networks in Python. A total of 584 DEGs were identified, of which 294 and 290 were upregulated and downregulated, respectively. Enrichment analysis showed that the DEGs were mainly involved in red blood cell differentiation, cell protein catabolism, gas transportation, activation of myeloid leukocytes, phagocytosis, and inflammatory response. Pathway analysis revealed that these DEGs were involved in regulation of mitophagy-animal, human T-cell leukemia virus-1 infection, Forkhead box O, phagocytosis, osteoclast differentiation, and cytokine–cytokine receptor interaction. Quantitative real-time polymerase chain reaction results were consistent with findings from protein–protein interaction network analysis. Several genes, including peroxiredoxin 2, haptoglobin, matrix metallopeptidase 8, formyl peptide receptor 2, and integrin subunit alpha X, promote SONFH occurrence by regulating the redox, inflammatory response, and osteoblast and osteoclast structure and function pathways. They may be important targets for designing approaches for early diagnosis and treatment of SONFH.

HMG20A from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection

High-mobility group 20 A (HMG20A) has important biological functions, such as inhibiting the differentiation of red blood cells and nerve cells, promoting the proliferation and metastasis of cancer cells, and regulating inflammatory reaction. However, the role of HMG20A in the response to bacterial infection in the economic fish Nile tilapia (Oreochromis niloticus) remains unclear. In this study, a HMG20A homolog was successfully identified and characterized from Nile tilapia (On-HMG20A), and its expression model and biological effects on bacterial infection were analyzed. The open reading frame (ORF) of On-HMG20A was 876 bp in length, which encoded 291 amino acids and possessed a HMG domain (High mobility group domains) and coiled coil region. Results of the expression model showed that On-HMG20A was widely distributed in immune-related tissues of healthy tilapia and upregulated in a time-dependent manner after being challenged by Streptococcus agalactiae. Meanwhile, knocking down the expression of On-HMG20A can reduce the inflammatory response of tilapia and the degree of tissue damage caused by S. agalactiae. Moreover, knocking down the expression of On-HMG20A can reduce the bacterial load of tilapia tissues after being challenged by S. agalactiae and improve the survival rate. Collectively, these results showed that On-HMG20A may be related to the immune response of Nile tilapia against bacterial infection.

Human m6A-mRNA and lncRNA epitranscriptomic microarray reveal function of RNA methylation in hemoglobin H-constant spring disease

The thalassemia of Hemoglobin H-Constant Spring disease (HbH-CS) is the most common type of Thalassemia in non-transfusion thalassemia. Interestingly, the clinical manifestations of the same genotype of thalassemia can be vastly different, likely due to epigenetic regulation. Here, we used microarray technology to reveal the epigenetic regulation of m6A in modifiable diseases and demonstrated a role of BCL2A1 in disease regulation. In this study, we revealed that methylating enzyme writers including METTL16, WTAP, CBLL1, RBM15B, and ZC3H13 displayed low expression and the demethylating enzyme ALKBH5, along with reader proteins including IGF2BP2 and YTHDF3 exhibited high expression. In addition, BCL2A1 was hypo-methylated and showed low expression. We also revealed that the BCL2A1 methylation level and IGF2BP2 expression were negatively correlated. Additionally, the mRNAs expression between ALKBH5 and IGF2BP2 were positively correlated. In HbH-CS, most genes were hypo-methylated. This included BCL2A1, which may play an important role in the process of red blood cell differentiation and development of HbH-CS. Moreover, the mRNA-M6A methylation status may be regulated by the demethylating enzyme ALKBH5 via IGF2BP2.

Zmiz1a zebrafish mutants have defective erythropoiesis, altered expression of autophagy genes, and a deficient response to vitamin D

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits.To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

MO550INDOXYL SULFATE AFFECTS ERYTHROPOIESIS DURING THE COURSE OF CHRONIC KIDNEY DISEASE: A MOLECULAR STUDY

Abstract Background and Aims Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of renal function due to high serum levels of uremic toxins. Anemia is a major trouble in CKD patients that contributes to a faster deterioration of renal failure, leading to cardiovascular disease and increasing morbimortality. Erythropoietin (EPO) is known to contribute to CKD-associated anemia. Thus, accumulation of uremic toxins in blood impairs EPO synthesis, leading to a subsequent impairment of erythropoiesis in the bone marrow. Very few molecular clues explain why erythropoiesis is affected in CKD or explain why erythropoiesis-stimulating agents (ESA) are not efficient in some patients with CKD. The current study aims to characterize the impact of one of the most representative uremic toxins, Indoxyl Sulfate (IS), in CKD-related anemia. IS is a protein-bound uremic toxin derived from the tryptophan dietary metabolism which is difficult to remove by dialysis. Our study demonstrates the molecular effects of IS on the growth and the differentiation of red blood cells in an erythroid cell line and in primary cell cultures CD34+. Method Firstly, we examined in vitro the time-courses of IS under clinically relevant concentrations of IS (250 µM -1 mM) in a human leukemic cell line in which proliferation is induced by EPO, the UT7/EPO cell line. Cell apoptosis, proliferation, differentiation and cell cycle analysis were assessed by the MACSQuant flow cytometry. Erythroid gene expression analysis was assessed by RT-qPCR (Quantstudio 7 flex). The ratio A260/280 assessed the quality of nucleic acids. Western blotting experiments were performed to study protein expression. Human primary CD34+ cells were obtained from mobilized peripheral blood mononuclear cells (MNC) of healthy subjects and were isolated by magnetic microbeads separation on MACS columns. Results IS at 250 µM and 1 mM increased apoptosis of UT7/EPO cell line at 48h compared to control condition. On the other hand, we found no significant effect of IS on the phenotype of UT7/EPO, when using CD235a (Glycophorin A), as a marker for the detection of the erythroid cell lineage. Ki67 cellular levels, a cell proliferation marker, was not altered between control and IS experiments. This indicated that IS did not affect proliferation in UT7/EPO. At 48h, at the clinically relevant concentration of IS (250 µM), we observed an increase of the cell phase cycle Sub-G1. The analysis of erythropoiesis related genes shows that HIF2α was deregulated with IS (250 µM). Finally, in the Epo-EpoR signalling pathway, we studied the activation of the Jak2/Stat5 proteins. Results in human primary CD34+ cells confirmed the apoptotic effect of IS observed in UT7/EPO. Conclusion Our findings suggest that IS, a representative protein-bound uremic toxin, could affect cell viability, apoptosis and the cell cycle. This study suggests clues to develop new therapies for CKD-associated anemia.

Metabolic pathway regulates cell fate

Cell Differentiation Lineage-specific regulators direct cell fate decisions, but the precise mechanisms are not well known. Using an in vivo chemical suppressor screen of a bloodless zebrafish mutant, Rossmann et al. show that the lineage-specific chromatin factor tif1γ directly regulates mitochondrial genes to drive red blood cell differentiation. Loss of tif1γ reduces coenzyme Q synthesis and function, impeding mitochondrial respiration and leading to epigenetic alterations and repression of erythropoiesis. The loss of blood in the mutant fish can be rescued by the addition of coenzyme Q. This work establishes a mechanism by which a chromatin factor tunes a metabolic pathway in a tissue-specific manner. Science , this issue p. [716][1] [1]: /lookup/doi/10.1126/science.aaz2740

The Hsp70 chaperone system: distinct roles in erythrocyte formation and maintenance.

Erythropoiesis is a tightly regulated cell differentiation process in which specialized oxygen- and carbon dioxide-carrying red blood cells are generated in vertebrates. Extensive reorganization and depletion of the erythroblast proteome leading to the deterioration of general cellular protein quality control pathways and rapid hemoglobin biogenesis rates could generate misfolded/aggregated proteins and trigger proteotoxic stresses during erythropoiesis. Such cytotoxic conditions could prevent proper cell differentiation resulting in premature apoptosis of erythroblasts (ineffective erythropoiesis). The heat shock protein 70 (Hsp70) molecular chaperone system supports a plethora of functions that help maintain cellular protein homeostasis (proteostasis) and promote red blood cell differentiation and survival. Recent findings show that abnormalities in the expression, localization and function of the members of this chaperone system are linked to ineffective erythropoiesis in multiple hematological diseases in humans. In this review, we present latest advances in our understanding of the distinct functions of this chaperone system in differentiating erythroblasts and terminally differentiated mature erythrocytes. We present new insights into the protein repair-only function(s) of the Hsp70 system, perhaps to minimize protein degradation in mature erythrocytes to warrant their optimal function and survival in the vasculature under healthy conditions. The work also discusses the modulatory roles of this chaperone system in a wide range of hematological diseases and the therapeutic gain of targeting Hsp70.

Macrophage protease-activated receptor 2 regulates fetal liver erythropoiesis in mice

AbstractDeficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

Metabolomic Characterization of Red Blood Cell Differentiation

Background: Erythropoiesis is the highly coordinated multi-step process by which multipotent hematopoietic stem cells differentiate into mature enucleated red blood cells (RBCs). As erythroid cells become more terminally differentiated, they undergo changes in morphology and gene expression, start synthesizing hemoglobin, commit to an irreversible loss of proliferation, and eventually expulse their nuclei and other cytoplasmic organelles. Thus, RBCs must rely on their proteome and metabolome for proper function. The RBC proteome is estimated to contain 2,800 proteins, including a variety of receptors and transporters that allow RBCs to uptake xenobiotics or endogenous metabolites as they circulate for ∼120 days. Furthermore, they are metabolically active with glycolysis, nucleotide catabolism, and glutathione metabolism as the major pathways supporting cell survival and function. However, it is unclear how the metabolome is altered during erythropoiesis, what role metabolites play in normal erythropoiesis, and if dysregulation of metabolites contributes to diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Methods: Four models of erythropoiesis were used in this study. 1) Mice were treated with phenylhydrazine (Phz) to induce acute hemolysis followed by erythropoietic recovery, leading to an increase in circulating reticulocytes. 2) Mice were lethally irradiated and transplanted with wild-type or sickle cell bone marrow, leading to anemic profiles in sickle cell chimeras. 3) The mouse erythroleukemic (MEL) cell line was treated with DMSO to induce differentiation. 4) The human erythroleukemic (K562) cell line was treated with sodium butyrate to induce differentiation. For the in vivo mouse models, blood was collected from control and treated animals, and complete blood count (CBC) analysis was performed. For the in vitro cell culture models, the mRNA levels of β-globin were measured by Q-RT-PCR in control and differentiated cells, and the degree of hemoglobinization was determined visually and via staining for heme. In addition, metabolites were extracted from the collected RBCs and erythroleukemic cell lines, and a Snapshot LC/MS metabolomic platform was used to identify commonly altered metabolites. Results: We first validated our four models of erythropoiesis. Treatment with Phz decreased the number of total RBCs while increasing the RBC distribution width, indicating an increased number of reticulocytes (more immature RBCs) in circulation. Similar results were seen in the sickle cell chimeras. Treatment of MEL and K562 cells with DMSO and sodium butyrate, respectively, resulted in increased expression of β-globin, increased levels of heme, and increased red color. Then, using our Snapshot metabolomic platform, we identified global changes in RBC metabolism during erythropoiesis. Analyses of the commonly altered metabolites in the in vitro and in vivo models revealed an increase in amino acid, mitochondrial, and urea cycle metabolism during erythropoiesis. L-aspartate levels were particularly upregulated, especially in DMSO-treated MEL cells. We are now investigating the role of aspartate in the regulation of erythropoiesis. Conclusions: We defined how the metabolome was altered in multiple in vitro and in vivo models of erythropoiesis and identified global changes in RBC metabolism between the different models. Specifically, we found that L-aspartate was upregulated during RBC differentiation in all four models. Aspartate is an amino acid that plays a role in many processes in cells, including nucleotide biosynthesis, redox homeostasis, and amino acid biosynthesis. We hypothesize that aspartate metabolism is critical for RBC differentiation and that its dysregulation exacerbates disease of ineffective erythropoiesis, such as sickle cell anemia and β-thalassemia. We are currently testing its role in inducing hemoglobinization and in regulating the commitment of erythroid progenitor cells to an irreversible loss of proliferation. Overall, we believe that understanding the precise mechanisms by which cellular metabolism plays a role in proper RBC differentiation may lead to better therapies for diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Disclosures No relevant conflicts of interest to declare.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Uremic Toxins Affect Erythropoiesis during the Course of Chronic Kidney Disease: A Review.

Chronic kidney disease (CKD) is a global health problem characterized by progressive kidney failure due to uremic toxicity and the complications that arise from it. Anemia consecutive to CKD is one of its most common complications affecting nearly all patients with end-stage renal disease. Anemia is a potential cause of cardiovascular disease, faster deterioration of renal failure and mortality. Erythropoietin (produced by the kidney) and iron (provided from recycled senescent red cells) deficiencies are the main reasons that contribute to CKD-associated anemia. Indeed, accumulation of uremic toxins in blood impairs erythropoietin synthesis, compromising the growth and differentiation of red blood cells in the bone marrow, leading to a subsequent impairment of erythropoiesis. In this review, we mainly focus on the most representative uremic toxins and their effects on the molecular mechanisms underlying anemia of CKD that have been studied so far. Understanding molecular mechanisms leading to anemia due to uremic toxins could lead to the development of new treatments that will specifically target the pathophysiologic processes of anemia consecutive to CKD, such as the newly marketed erythropoiesis-stimulating agents.

Activation of lysophosphatidic acid receptors regulate differentiation of hematopoietic stem cells in myeloid lineage

Lysophosphatidic acid (LPA) is a membrane‐derived lysophospholipid that exists in plasma and platelet. It exerts its functions through activating various LPA receptors (LPARs), which belong to the family of G‐protein coupled receptors (GPCRs). Activation of LPARs has been implicated for important roles during stem cell differentiation. However, how LPA affects human hematopoietic stem cell (HSC) differentiation and underlying lineages remains elusive. In our previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis in human HSC and zebrafish. In the present study, pharmacological approach was adopted to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. Treatment of LPA2 agonist GRI977143 suppressed erythropoiesis, whereas activation of LPA3 by 2S‐OMPT promoted RBC differentiation both in vitro and in vivo. Furthermore, our results demonstrated a negative role of LPA3 during platelet differentiation both in vitro and in vivo. Moreover, the expression pattern of LPA receptors correlated to the hematopoietic transcriptional factors GATA1 and GATA2 in different stages of myeloid progenitors. In addition, manipulation of these GATA factors affected the expression level of LPA2 and LPA3. By luciferase assays, we demonstrated that the promoter region of LPAR2 and LPAR3 were regulated by these GATA factors. Mutation of identified GATA‐Binding‐Sites (GBS) in the region abrogate the luciferase activities, suggested that LPA2and LPA3 are regulated by GATA factors. In addition, chromatin immunoprecipitation assays confirmed the directly interaction between GATA factors and promoter region of LPARs. Taken together, our results suggested that the expression level of LPA2 and LPA3, which may be determined by GATA factors, is a regulatory switch for the lineage commitment in myeloid progenitors. The pharmacological activation of LPA receptor subtypes represents a novel strategyfor regulating erythropoiesis.LPA receptors are expressed at different stages during myeloid differentiation. GATA switch, which is an important molecular switch in hematopoiesis, cause the difference of expressional pattern of LPA receptor in different lineage.Figure 1

Focusing-free impedimetric differentiation of red blood cells and leukemia cells: A system optimization

A focusing-free microfluidic impedimetric cell detection system is developed. The effect of the channel dimensions, solution conductivity, excitation voltage, and particle size on impedimetric signal outputs were optimized to increase the sensitivity of the system. Conventional microfabrication techniques were adapted to obtain low height, resealable microchannels. The geometry optimization was performed by a combination of analytical, numerical and experimental approaches. The results demonstrate that reliable impedimetric particle differentiation can be achieved without any labeling or particle focusing. The system parameters were studied and rule-of-thumb design criteria were provided. Finally, using the developed system, red blood cells and leukemia cells were experimentally detected and differentiated. Thanks to its simplicity, the focusing-free cell differentiation system may find applications in several cellular diagnostic uses.

Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a “hematopoietic cell forming complex (HCFC).” From this HCFC, CD43+ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34+/CD45+ progenitors with high erythroid colony-forming potential and CD36+ erythroid precursors. A total of 1.5 × 107 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA+ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

Mutations in the zebrafish hmgcs1 gene reveal a novel function for isoprenoids during red blood cell development

AbstractErythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is 1 potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wild-type siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal 2 novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.