Developmental Stage-Specific Changes in Protein Synthesis Differentially Sensitize Hematopoietic Stem Cells and Erythroid Progenitors to Impaired Ribosome Biogenesis

AbstractRibosomopathies encompass a collection of human genetic disorders that often arise from mutations in ribosomal proteins or ribosome biogenesis factors. Despite ubiquitous requirement of ribosomes for protein synthesis, ribosomopathies present with tissue- and cell-type-specific disorders, and blood is particularly affected. Several ribosomopathies present with congenital anemias and bone marrow failure, and accordingly, erythroid lineage cells and hematopoietic stem cells (HSCs) are preferentially impaired by ribosomal dysfunction. However, the factors that influence this cell-type-specific sensitivity are incompletely understood. Here, we show that protein synthesis rates change during HSC and erythroid progenitor ontogeny. Fetal HSCs exhibit significantly higher protein synthesis than adult HSCs. Despite protein synthesis differences, reconstituting activity of both fetal and adult HSCs is severely disrupted by a ribosomal mutation (Rpl24Bst/+). In contrast, fetal erythroid lineage progenitors exhibit significantly lower protein synthesis than their adult counterparts. Protein synthesis declines during erythroid differentiation, but the decline starts earlier in fetal differentiation than in adults. Strikingly, the Rpl24Bst/+ mutation impairs fetal, but not adult erythropoiesis, by impairing proliferation at fetal erythroid progenitor stages with the lowest protein synthesis relative to their adult counterparts. Thus, developmental and cell-type-specific changes in protein synthesis can sensitize hematopoietic cells to impaired ribosome biogenesis.Key PointsFetal HSCs synthesize much more protein per hour than young adult HSCs in vivoFetal erythroid progenitors synthesize much less protein per hour than young adult erythroid progenitors in vivoDifferences in protein synthesis dynamics distinguish fetal and adult erythroid differentiationA ribosomal mutation that reduces protein synthesis impairs fetal and adult HSCsReduced protein synthesis impairs fetal but not adult erythroid progenitors

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1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Experimental Hematology ◽

10.1016/j.exphem.2020.09.015 ◽

2020 ◽

Vol 88 ◽

pp. S22

Author(s):

Wei Tong ◽

Kaosheng Lv ◽

Charles Aruljothi ◽

Chujie Gong ◽

Xu Han ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ribosome Biogenesis ◽

Hematopoietic Stem

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Developmental Stage Dependent Response to Proliferation in Hematopoietic Stem Cells

Experimental Hematology ◽

10.1016/j.exphem.2018.06.263 ◽

2018 ◽

Vol 64 ◽

pp. S77

Author(s):

Satish Khurana ◽

Atreyi Biswas ◽

Irene Roy ◽

Sarah Schouteden ◽

Joerg Huelsken ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Developmental Stage ◽

Hematopoietic Stem

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Linkage between the mechanisms of thrombocytopenia and thrombopoiesis

Blood ◽

10.1182/blood-2015-07-607903 ◽

2016 ◽

Vol 127 (10) ◽

pp. 1234-1241 ◽

Cited By ~ 29

Author(s):

Koji Eto ◽

Shinji Kunishima

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Developmental Stage ◽

Genetic Defects ◽

Homeostatic Regulation ◽

Hematopoietic Stem ◽

The Past ◽

Genes Encoding ◽

Stage Process ◽

Platelet Release

Abstract Thrombocytopenia is defined as a status in which platelet numbers are reduced. Imbalance between the homeostatic regulation of platelet generation and destruction is 1 potential cause of thrombocytopenia. In adults, platelet generation is a 2-stage process entailing the differentiation of hematopoietic stem cells into mature megakaryocytes (MKs; known as megakaryopoiesis) and release of platelets from MKs (known as thrombopoiesis or platelet biogenesis). Until recently, information about the genetic defects responsible for congenital thrombocytopenia was only available for a few forms of the disease. However, investigations over the past 15 years have identified mutations in genes encoding >20 different proteins that are responsible for these disorders, which has advanced our understanding of megakaryopoiesis and thrombopoiesis. The underlying pathogenic mechanisms can be categorized as (1) defects in MK lineage commitment and differentiation, (2) defects in MK maturation, and (3) defect in platelet release. Using these developmental stage categories, we here update recently described mechanisms underlying megakaryopoiesis and thrombopoiesis and discuss the association between platelet generation systems and thrombocytopenia.

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Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

10.1101/256024 ◽

2018 ◽

Author(s):

Tatyana Grinenko ◽

Anne Eugster ◽

Lars Thielecke ◽

Beata Ramazs ◽

Anja Krueger ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Division ◽

Hematopoietic Stem Cells ◽

Embryonic Stem ◽

Erythroid Progenitors ◽

Multipotent Stem Cells ◽

Hematopoietic Stem ◽

Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

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Bile Acids Protect Expanding Hematopoietic Stem Cells from Unfolded Protein Stress in Fetal Liver

Blood ◽

10.1182/blood.v126.23.897.897 ◽

2015 ◽

Vol 126 (23) ◽

pp. 897-897

Author(s):

Valgardur Sigurdsson ◽

Hajime Takei ◽

Svetlana Soboleva ◽

Visnja Radulovic ◽

Roman Galeev ◽

...

Keyword(s):

Stem Cells ◽

Protein Synthesis ◽

Hematopoietic Stem Cells ◽

Er Stress ◽

Bile Acids ◽

Stress Responses ◽

Fetal Liver ◽

Synthesis Rate ◽

Protein Synthesis Rate ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are effectively expanded in fetal liver (FL), while they are maintained in a dormant state in adult bone marrow (BM). However, developmental mechanisms allowing this have not been fully explained. BM-HSCs have the lowest protein synthesis rate within the blood hierarchy, even under forced self-renewal divisions. In addition, HSCs are vulnerable to and quickly activate endoplasmic reticulum (ER) stress responses fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). Of note, we have seen that FL-HSCs have low levels of ER stress related genes despite their high proliferation status without an increase in heat shock protein levels, strongly indicating that other factor(s) block ER stress elevation. This raises the question how HSCs deal with the higher protein-folding requirement during expansion in the FL. Here we demonstrate that bile acids (BAs) are required to eliminate ER stress in the FL and are essential for proper expansion of FL-HSCs. Measurement of protein synthesis rate using OP-puro incorporation revealed that protein synthesis was enhanced in FL-HSCs, whereas BM-HSCs have half the rate of other populations in BM. Mass spectrometry analyses showed that BAs in the FL were all taurine conjugated while 30% of BA in the adult liver was taurine-conjugated, and the main proportion was taurocholic acid (TCA) that is known for its low toxicity. In the FL we also detected secondary BAs (e.g. TDCA), requiring intestinal bacteria in the production process, suggesting that FL BAs are a mixture of fetal and maternal BAs. Reduction of BA levels using GW4064, a chemical inhibitor of BA synthesis, significantly decreased the number of HSCs (6.6 fold decrease compared to vehicle treatment). This decrease was due to increased apoptosis caused by elevated ER stress levels. Similarly, dual deletion of Cyp27a1, a key BA synthetic enzyme, in both mother and fetus severely decreased total cellularity (2.0 fold decrease compared to littermate heterozygotes) and number of HSCs (6.8 fold decrease) in FL due to increased ER stress and subsequent apoptosis. Interestingly, FL of homozygotes grown in heterozygous mothers did not show any significant differences compared to littermate heterozygotes, suggesting that the contribution of maternal BA in FL is critical for HSCs. In both models, ER stress-oriented apoptosis and reduction in cellularity were most pronounced within the HSC population, indicating that stem cells are particularly sensitive to BA levels during development in FL. Importantly, injection of TCA or Salubrinal, an ER stress inhibitor, rescued the effects of BA reduction in both models. These data strongly suggest that BAs are required to block ER stress elevation in expanding FL-HSCs. ER stress and protein aggregation are closely linked together in number of pathological diseases like AlzheimerÕs- and HuntingtonÕs disease. Quantification of aggregated proteins (aggresomes) revealed that Cyp27a1 KO FL-HSCs from homozygote mothers contained significantly higher amount of aggresomes (2.0 fold), while KO FL-HSCs from heterozygote mothers showed no increase. Higher levels of aggregated proteins were most pronounced within the HSC population and BA suppressed formation of aggresomes during in vitro culture. This leads to reduction of ER stress and the maintenance of functional HSCs. Finally, transplantation assay showed that TCA can support functional HSCs ex vivo for up to 14 days. These findings propose a novel role for BA as a critical part of fetal hematopoiesis supporting expansion of HSC. Maternal and fetal BA coordinately contribute to this natural chaperone regulation. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Releasing the brake on protein synthesis in hematopoietic stem cells

Cell Stem Cell ◽

10.1016/j.stem.2021.06.003 ◽

2021 ◽

Vol 28 (7) ◽

pp. 1183-1185

Author(s):

Craig M. Forester ◽

Davide Ruggero

Keyword(s):

Stem Cells ◽

Protein Synthesis ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem

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Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Blood ◽

10.1182/blood.v126.23.1174.1174 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1174-1174

Author(s):

Darlene Monlish ◽

Angela Herman ◽

Molly Romine ◽

Sima Bhatt ◽

Laura G. Schuettpelz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Tlr2 Agonist ◽

Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM3CSK4, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM3CSK4 led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2-/- + Tlr2+/+ bone marrow transplanted into Tlr2+/+ or Tlr2-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM3CSK4, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM3CSK4-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

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Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells

10.1101/856559 ◽

2019 ◽

Author(s):

Peng Gao ◽

Changya Chen ◽

Elizabeth D. Howell ◽

Yan Li ◽

Joanna Tober ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Hematopoietic Stem Cells ◽

Developmental Stage ◽

Regulatory Networks ◽

Developmental Stages ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Transcriptional Regulatory ◽

Computational Analyses

AbstractHematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-Seq and histone mark ChIP-Seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in the mouse. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries (dorsal aorta, umbilical and vitelline), an enriched population of pre-hematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. We observed dynamic and combinatorial epigenetic changes that mark regulatory DNA sequences including gene promoters and enhancers. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo at the developmental stages where they are required to control their target genes, without prior priming in earlier stages. We constructed developmental-stage-specific transcriptional regulatory networks during HSC ontogeny by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm. Our computational analyses predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors whose regulon activities correlate with the emergence of pre-HSCs. We validated roles for the broadly expressed transcription factors SP3 and MAZ in arterial hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

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Yolk sac, but not hematopoietic stem cell–derived progenitors, sustain erythropoiesis throughout murine embryonic life

Journal of Experimental Medicine ◽

10.1084/jem.20201729 ◽

2021 ◽

Vol 218 (4) ◽

Author(s):

Francisca Soares-da-Silva ◽

Laina Freyer ◽

Ramy Elsaid ◽

Odile Burlen-Defranoux ◽

Lorea Iturri ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Fetal Liver ◽

Yolk Sac ◽

Lineage Tracing ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Embryonic Life

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell–derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac–derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.

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