Primitive Erythropoiesis and Osteogenesis Are Differentially Impaired In Rpl5 and Rps19 Mutant Murine Embryonic Stem Cell Models Of Diamond Blackfan Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3704-3704
Author(s):  
Brian M. Dulmovits ◽  
Sharon A. Singh ◽  
Tracie Goldberg ◽  
Sebastien Didier ◽  
Jeffrey M. Lipton ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Alizarin Red ◽  
Diamond Blackfan Anemia ◽  
Mutant Cells

Abstract Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 798-805
Author(s):  
Hideo Harigae ◽  
Naruyoshi Suwabe ◽  
Peter H. Weinstock ◽  
Mayumi Nagai ◽  
Hiroyoshi Fujita ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aminolevulinate Synthase ◽  
E Gene ◽  
In The Wild ◽  
Mutant Cells

The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

scholarly journals Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 798-805 ◽  
Author(s):  
Hideo Harigae ◽  
Naruyoshi Suwabe ◽  
Peter H. Weinstock ◽  
Mayumi Nagai ◽  
Hiroyoshi Fujita ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aminolevulinate Synthase ◽  
E Gene ◽  
In The Wild ◽  
Mutant Cells

Abstract The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

scholarly journals Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ping Zhou ◽  
Jia-Min Shi ◽  
Jing-E Song ◽  
Yu Han ◽  
Hong-Jiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Pluripotent Stem Cells ◽  
Cell Counting ◽  
Cell Type ◽  
Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

Specific Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem (ES) Cells with Abortive Ribosome Assembly.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1088-1088
Author(s):  
Tracie A. Goldberg ◽  
Adrianna Henson ◽  
Sharon Singh ◽  
Abdallah Nihrane ◽  
Jeffrey Michael Lipton ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Embryoid Bodies ◽  
Ribosome Assembly ◽  
Trap Cell

Abstract Abstract 1088 Poster Board I-110 Background Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes, characterized by erythroid hypoplasia, congenital anomalies and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, RPL35a), which somehow triggers apoptosis of erythroid precursors. There is a marked variation in phenotype among members of the same family and also between subsets of patients with different mutations. Methods We studied primary and secondary in vitro differentiation of two murine ES gene trap cell lines with mutations in Rps19: S17-10H1, in which Rps19 is disrupted by insertion of the ROSAFARY gene trap vector between exons 2 and 3; and YHC074, in which the pGT0Lxf gene trap vector is inserted between exons 3 and 4 and whose growth is feeder cell-independent. For primary differentiation and generation of embryoid bodies (EBs), the ES cells were cultured in a serum-supplemented methylcellulose-based medium containing stem cell factor (SCF). After 7 days, the cultures were fed with a medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 13 for hematopoietic percentage. Secondary (hematopoietic) differentiation was performed on day 9 EBs. EBs were harvested and disrupted with collagenase, and the disrupted cells were suspended in a serum-supplemented methylcellulose-based medium with SCF, IL-3, IL-6 and epo. Hematopoietic colonies were counted on day 10. Results Decreased expression of Rps19 protein was confirmed by Western blot analysis in both S17-10H1 and YHC074 gene trap cell lines. We focused on YHC074 because its growth is feeder-independent, and it expresses approximately 50% of normal Rps19 levels. By polysome analysis, we found a selective reduction in the 40S subunit peak in mutant YHC074 cells as compared to parental controls. By Northern blot assays, we also found a relative increase in the 21S pre-rRNA to 18S rRNA ratio in mutant YHC074 cells. The viability of undifferentiated ES cells was not significantly different from parental control cells in the first 72 hours of culture; however, there was a significantly decreased number of EBs, particularly hematopoietic EBs, following primary differentiation (Fig. 1). Furthermore, when day 9 EBs were induced to secondary (hematopoietic) differentation, there was a significant decrease in the ratio of erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation in mutant YHC074 cells. In order to confirm these results in an isogenic background, we stably transfected S17-10H1 cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones were found to overexpress Rps19 and were further studied in secondary differentiation experiments. There was a significant decrease in erythroid and myeloid colony formation and in BFU-E size from mutant S17-10H1 cells when compared to the Rps19-overexpressing clone, suggesting a direct relationship between the levels of Rps19 protein and hematopoietic growth and differentiation. Conclusion Using two ES cell lines with slightly different Rps19 mutations and genetic backgrounds, we have recapitulated the major DBA erythroid growth and differentiation defect, as well as the defect in ribosome assembly and rRNA processing caused by Rps19 haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

Functional Analysis of FOXO3A Involved in Erythroid Differentiation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4731-4731
Author(s):  
Hai Wang ◽  
Yadong Yang ◽  
Hongzhu QU ◽  
Xiuyan Ruan ◽  
Zhaojun Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Expression Level ◽  
Homologous Gene ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Central Node

Abstract Abstract 4731 FOX (Forkhead box) proteins are a family of transcription factors that emerged as playing an important role in the embryonic development, cell cycle, carbohydrate and fatty acid metabolism and immune response. It was found that FOXO3A (also known as FOXO3) involved in erythroid differentiation, yet the mechanism for regulating hematopoietic stem cells (HSCs) differentiation is unknown. We analyzed the dynamics of genome-wide transcriptome (mRNA-Seq) of human undifferentiated embryonic stem cells (HESC), erythroid cells derived from ES cells (ESER), human fetal erythroid liver cells (FLER) and peripheral CD34+derived erythroid cells (PBER) using high throughput sequencing technology. The transcriptome analysis showed that FOXO3 was barely expression in HESC while was observably up-regulated in ESER. However, FOXO3 was down-regulated in FLER and PBER compare with ESER, the erythroid cells at early developmental stage. We presumed that FOXO3 plays an important role in primitive erythropoiesis and built up the interactions network in which FOXO3 acts as a central node by Gene Ontology (GO), correlation analysis and Ingenuity Pathways Analysis (IPA). In addition, we analyzed the profiles of histone methylation in the four types of cells by ChIP-Seq to study the chromatin conformation in the vicinity of FOXO3. More histone 3 lysine 4 (H3K4) trimethylation was found near the promoter region of FOXO3 in ESER compared with the other cells, which is coincided with the mRNA-seq results. We performed a series of experiment to identify the roles of FOXO3 in regulating erythroid differentiation. The results showed that the expression level of ε and γ globin were up-regulated in FOXO3-over-expressed 293T and Hela cells and the expression level of FOXO1 and CAT in predicted network were increased by quantitative real-time PCR detection. In addition, when FOXO3 knocked down in K562 cells, the expression level of ε and γ globin were down-regulated. The expression level of CAT, BCL2L1 and other factors in predicted network, were also decreased. These results indicate FOXO3 plays an important role in globin expression and identify the credibility of our predicted networks in which FOXO3 acts as a central node. FOXO3 binding sites (GTAAACA or ATAAACA) were predicted on the upstream of CAT and BCL2L1. We are trying to prove CAT or BCL2L1 is a direct FOXO3 target in vitro and in vivo. In conclusion, we have demonstrated FOXO3 plays a key role in erythroid differentiation and globin expression. We will further determine the enriched profiles of FOXO3 by ChIP-seq in HESC, ESER, FLER and PBER to find more targets of FOXO3. Since the zebrafish is a powerful model system for investigating vertebrate hematopoiesis. We will identify the role of Foxo3b, the homologous gene of human FOXO3, in erythroid differentiation and study the dynamic transcriptomes of Foxo3b morphants in zebrafish. We are trying to make a whole picture to elaborate the molecular mechanism of FOXO3 involved in regulation of erythroid differentiation. Disclosures: No relevant conflicts of interest to declare.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3846-3846
Author(s):  
Ji-Yoon Noh ◽  
Shilpa Gandre-Babbe ◽  
Yuhuan Wang ◽  
Vincent Hayes ◽  
Yu Yao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell

Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1857-1863 ◽  
Author(s):  
Kenji Kitajima ◽  
Makoto Tanaka ◽  
Jie Zheng ◽  
Hilo Yen ◽  
Ayuko Sato ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Hematopoietic Differentiation ◽  
Macrophage Differentiation ◽  
Conditional Gene Expression

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

scholarly journals Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds

2004 ◽  
Vol 2004 (4) ◽  
pp. 203-210 ◽  
Author(s):  
G. R. Chaudhry ◽  
D. Yao ◽  
A. Smith ◽  
A. Hussain
Keyword(s):  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Pcr Analysis ◽  
Specific Marker ◽  
Es Cell ◽  
Alizarin Red ◽  
Osteogenic Cells ◽  
Differentiated Cells

An approach for 3D bone tissue generation from embryonic stem (ES) cells was investigated. The ES cells were induced to differentiate into osteogenic precursors, capable of proliferating and subsequently differentiating into bone-forming cells. The differentiated cells and the seeded scaffolds were characterized using von Kossa and Alizarin Red staining, electron microscopy, and RT-PCR analysis. The results demonstrated that ES-derived bone-forming cells attached to and colonized the biocompatible and biodegradable scaffolds. Furthermore, these cells produced bone nodules when grown for 3–4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not an ES cell-specific marker,oct-4. These findings suggest that ES cell can be used for in vitro tissue engineering and cultivation of graftable skeletal structures.

scholarly journals Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

2020 ◽  
Author(s):  
Ping Zhou ◽  
Jia-Min Shi ◽  
Jing-E Song ◽  
Yu Han ◽  
Hong-Jiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Pluripotent Stem Cells ◽  
Cell Counting ◽  
Cell Type ◽  
Alizarin Red

Abstract Background: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed.Methods: Monolayer cultured human embryonic stem cells and human induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week.Results: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrated the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded.Conclusions: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a step-wise induction system in the future.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-2-SCI-2
Author(s):  
Stefan Karlsson ◽  
Johan Flygare ◽  
Pekka Jaako ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

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