Complete Spatial Mapping of Steady-State and Stress Erythropoiesis

The anatomy of differentiation in the bone marrow (BM) is poorly understood due to lack of markers to image stepwise HSPC differentiation. We analyzed 250+ cell surface molecules in all hematopoietic progenitors and identified 56 differentially expressed markers in at least one HSPC that can be "mixed and matched" to prospectively image any HSPC of interest in the bone marrow. We used this data to develop a pipeline to map stepwise erythropoiesis in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BFU-E activity was restricted to Pre-MegE and Pre- CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image stepwise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in mouse sternum and interrogated the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulations. We found erythroid progenitors show no specific association with HSC, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 8 CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they downregulate CD117 and upregulate CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To examine the clonal architecture of erythropoiesis we used Ubc-creERT2:confetti mice where a tamoxifen pulse leads to irreversible expression of GFP, CFP, YFP or RFP. Four weeks later we found that the CFU-E strings are oligoclonal with each clone contributing 2-6 CFU-E to the string. The budding erythroblasts clusters are similarly organized. These indicate that different CFU-E are serially recruited to the same sinusoidal spot where they self-renew 1-2 times and then undergo terminal differentiation. We then tracked how this architecture changed in response to stress (hemorrhage). Two days after bleeding we found that Pre-Meg-E and Pre-CFU-E numbers and locations were unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) suggesting increased self-renewal. Unexpectedly, fate mapping showed that the size of CFU-E clones did not increase when compared to steady-state. These results indicate that all CFU-E expand in respond to stress and that this is mediated via increased recruitment and differentiation of upstream progenitors. In summary we have found 56 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image and fate map all steps of red blood cell maturation in situ; demonstrated that terminal erythropoiesis emerges from strings of sinusoidal CFU-E, and revealed the clonal architecture of normal and stress erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis

Blood ◽

10.1182/blood-2009-09-242487 ◽

2010 ◽

Vol 115 (18) ◽

pp. 3686-3694 ◽

Cited By ~ 31

Author(s):

Soizic Guihard ◽

Denis Clay ◽

Laurence Cocault ◽

Nathalie Saulnier ◽

Paule Opolon ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Serum Levels ◽

Negative Regulator ◽

Erythroid Progenitors ◽

Stress Erythropoiesis ◽

Extracellular Signal ◽

Mitogen Activated Protein

Abstract The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1−/− mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)–dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1−/− spleens. The ERK1−/− phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.

Download Full-text

BMP4/Madh5 Dependent Signaling Is Required for the Expansion of a Population of Stress Erythroid Progenitors in the Fetal Liver.

Blood ◽

10.1182/blood.v106.11.4195.4195 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4195-4195

Author(s):

Robert F. Paulson ◽

Prashanth Porayette

Keyword(s):

Bone Marrow ◽

Steady State ◽

Fetal Liver ◽

Erythroid Progenitors ◽

Acute Anemia ◽

Erythroid Response ◽

Stress Erythropoiesis ◽

And Control ◽

Flexed Tail

Abstract Fetal liver hematopoiesis is primarily erythropoiesis, which robustly produces erythrocytes to meet the growing need of the developing embryo. In many ways fetal liver erythropoiesis resembles stress erythropoiesis in the adult, where in response to acute anemia, a unique population of stress erythroid progenitors is rapidly expanded in the spleen. The development of these stress progenitors requires BMP4/Madh5 dependent signals. Spleen stress progenitors exhibit properties that are distinct from bone marrow steady state progenitors in that they are able to rapidly form large BFU-E colonies, which require only Epo stimulation for their generation. Mice mutant at the flexed-tail locus exhibit a defective stress erythroid response because of a mutation in Madh5. In addition to this defect, flexed-tail mice also exhibit a severe fetal-neonatal anemia. We have analyzed fetal liver erythropoiesis in flexed-tail and control embryos. We show that BMP4 is expressed in the fetal liver and its expression correlates with the time of maximum erythropoiesis. In flexed-tail mutant embryos the expression is delayed and this correlates with both a delay and a defect in the expansion of erythroid progenitors. Our analysis also shows that the fetal liver contains two types of erythroid progenitors. One type exhibits the properties of stress BFU-E found in the adult spleen, which are compromised in flexed-tail embryos and a second type that is similar to bone marrow steady state BFU-E. These data demonstrate that BMP4 dependent signaling drives the expansion of erythroid progenitors in the fetal liver in a manner similar to stress erythropoiesis in the adult spleen.

Download Full-text

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

EPO-Dependent Recovery of Late-Stage Erythroid Progenitors in the Marrow Precedes Splenic Expansion: Insights From a Sublethal Radiation Model

Blood ◽

10.1182/blood.v118.21.180.180 ◽

2011 ◽

Vol 118 (21) ◽

pp. 180-180

Author(s):

Scott A Peslak ◽

Jesse Wenger ◽

Amali P Epa ◽

Jeffrey C Bemis ◽

Paul D Kingsley ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Late Stage ◽

Erythroid Cell ◽

Functional Evaluation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Irradiation Injury ◽

Radiation Induced ◽

Sublethal Irradiation

Abstract Abstract 180 Erythropoiesis is a robust process of cellular expansion and maturation that occurs in the bone marrow and spleen of mice. Following clastogenic injury such as total body irradiation (TBI), erythroblasts are severely depleted in these organs, resulting in loss of reticulocyte output and the development of a mild anemia (Peslak et al., Exp. Hematol. 2011). However, the mechanistic and microenvironmental factors underlying erythroid recovery following sublethal TBI are poorly understood. To this end, we utilized colony assays to quantify erythroid progenitors, which consist of immature d7 erythroid burst-forming units (BFU-E) and more mature d3 BFU-E and erythroid colony forming units (CFU-E). Imaging flow cytometry was used to quantify erythroblast precursors. We found that d7 BFU-E undergo a slow, incomplete recovery during the first 10 days post-4 Gy TBI of C57Bl/6 mice. In contrast, d3 BFU-E exhibit a robust recovery beginning at 4 days post-TBI that is immediately followed by a rapid increase in CFU-E numbers to over 200 percent of steady-state levels. This initial erythroid progenitor recovery is followed by a wave of erythroid precursor maturation and red cell formation that occurs in close association with macrophages in the bone marrow. These erythroblast islands undergo a rapid synchronous expansion that peaks at 6 days post-TBI, suggesting that the bone marrow microenvironment plays a role in the recovery of the erythron from sublethal TBI. We hypothesized that erythropoietin (EPO), the primary regulator of erythroid survival and proliferation, mediates the rapid, specific expansion of late-stage erythroid progenitors following radiation injury. We found that plasma EPO levels increase 13-fold 4 days after 4 Gy TBI, temporally correlated with expansion of d3 BFU-E. Furthermore, maintenance of steady-state hematocrit levels following TBI prevented EPO induction and blocked expansion of late-stage erythroid progenitors, while exogenous EPO administered at 1 hour post-radiation specifically advanced recovery of late-stage progenitors. These data indicate that EPO is required for expansion of d3 BFU-E and CFU-E following radiation-induced marrow depletion. During times of acute hypoxia, such as the severe anemia induced by bleeding or phenylhydrazine exposure, EPO production is rapidly upregulated and splenic stress erythropoiesis is induced. Surprisingly, splenic erythropoiesis is absent during the rapid initial recovery of erythropoiesis in the bone marrow at 4–6 days post-TBI. However, a massive expansion of CFU-E begins at 7–8 days post-4 Gy TBI in spleen. EPO administration at 4 days following 4 Gy TBI significantly enhances late-stage progenitor recovery exclusively in the marrow, indicating that erythroid progenitors are not present in spleen at the time of rapid bone marrow expansion and that late-stage erythroid progenitor recovery initiates in the marrow and subsequently proceeds to the spleen. Furthermore, we found that erythroid progenitors transiently emerge in the bloodstream at 6–8 days post-TBI, following marrow recovery and prior to initiation of splenic erythropoiesis. These data are consistent with endogenous migration of the erythron from the bone marrow to the spleen during recovery from radiation-induced erythroid injury. Taken together, our data indicate that recovery from sublethal irradiation injury is regulated primarily by the EPO-induced expansion of late-stage erythroid progenitors in the bone marrow. This form of clastogenic injury is critically different from bleeding or hemolysis, which preserve bone marrow and splenic erythroblasts and induce expansion of splenic erythroid stress progenitors. Sublethal irradiation injury thus provides a unique model for the in vivo study of endogenous erythroid recovery. This model may be clinically useful for the functional evaluation of therapeutic factors that regulate or modulate erythroid cell maturation. Disclosures: Bemis: Litron Laboratories: Employment, Patents & Royalties.

Download Full-text

Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽

10.1182/blood-2021-152898 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 288-288

Author(s):

Suhita Ray ◽

Linda Chee ◽

Nicholas T. Woods ◽

Kyle J Hewitt

Keyword(s):

Steady State ◽

Hemolytic Anemia ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Interacting Proteins ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Capping Protein ◽

Sam Domain ◽

Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output

Haematologica ◽

10.3324/haematol.2019.234542 ◽

2020 ◽

Vol 105 (11) ◽

pp. 2561-2571 ◽

Cited By ~ 2

Author(s):

Sofie Singbrant ◽

Alexander Mattebo ◽

Mikael Sigvardsson ◽

Tobias Strid ◽

Johan Flygare

Keyword(s):

Steady State ◽

Progenitor Cells ◽

Fluorescent Protein ◽

Chromatin Accessibility ◽

Transcriptional Analysis ◽

Specific Marker ◽

Erythroid Progenitor ◽

Responsive Evaluation ◽

Stress Erythropoiesis

Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress-BFU-Es with a 100-fold improved enrichment compared to state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1- stress-BFU-Es provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-Es express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-Es, and are BMP-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-Es compared to steady-state BFU-Es. Our findings offer molecular insight to the unique capacity of stress-BFU-Es to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents.

Download Full-text

Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis

Blood ◽

10.1182/blood-2007-09-115121 ◽

2008 ◽

Vol 111 (8) ◽

pp. 4375-4385 ◽

Cited By ~ 57

Author(s):

Laura Gutiérrez ◽

Saho Tsukamoto ◽

Mikiko Suzuki ◽

Harumi Yamamoto-Mukai ◽

Masayuki Yamamoto ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Erythroid Progenitors ◽

Hematopoietic Development ◽

Stress Erythropoiesis ◽

Adult Mice ◽

Aplastic Crisis ◽

Excessive Proliferation ◽

Erythropoietic Response

Abstract The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre–mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre–mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

Download Full-text

Erythroblastic islands: niches for erythropoiesis

Blood ◽

10.1182/blood-2008-03-077883 ◽

2008 ◽

Vol 112 (3) ◽

pp. 470-478 ◽

Cited By ~ 287

Author(s):

Joel Anne Chasis ◽

Narla Mohandas

Keyword(s):

Bone Marrow ◽

Terminal Differentiation ◽

Erythroid Progenitors ◽

Stress Erythropoiesis ◽

Transmission Electron ◽

Erythroid Precursors ◽

Erythroid Development ◽

Anemia Of Inflammation

Abstract Erythroblastic islands, the specialized niches in which erythroid precursors proliferate, differentiate, and enucleate, were first described 50 years ago by analysis of transmission electron micrographs of bone marrow. These hematopoietic subcompartments are composed of erythroblasts surrounding a central macrophage. A hiatus of several decades followed, during which the importance of erythroblastic islands remained unrecognized as erythroid progenitors were shown to possess an autonomous differentiation program with a capacity to complete terminal differentiation in vitro in the presence of erythropoietin but without macrophages. However, as the extent of proliferation, differentiation, and enucleation efficiency documented in vivo could not be recapitulated in vitro, a resurgence of interest in erythroid niches has emerged. We now have an increased molecular understanding of processes operating within erythroid niches, including cell-cell and cell-extracellular matrix adhesion, positive and negative regulatory feedback, and central macrophage function. These features of erythroblast islands represent important contributors to normal erythroid development, as well as altered erythropoiesis found in such diverse diseases as anemia of inflammation and chronic disease, myelodysplasia, thalassemia, and malarial anemia. Coupling of historical, current, and future insights will be essential to understand the tightly regulated production of red cells both in steady state and stress erythropoiesis.

Download Full-text

Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche

Blood Advances ◽

10.1182/bloodadvances.2019000375 ◽

2019 ◽

Vol 3 (14) ◽

pp. 2205-2217 ◽

Cited By ~ 9

Author(s):

Siyang Hao ◽

Jie Xiang ◽

Dai-Chen Wu ◽

James W. Fraser ◽

Baiye Ruan ◽

...

Keyword(s):

Steady State ◽

Essential Role ◽

Metabolic Enzymes ◽

Metabolic Status ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Growth Differentiation Factor 15 ◽

Stress Erythropoiesis ◽

Murine Spleen

Abstract Anemic stress induces the proliferation of stress erythroid progenitors in the murine spleen that subsequently differentiate to generate erythrocytes to maintain homeostasis. This process relies on the interaction between stress erythroid progenitors and the signals generated in the splenic erythroid niche. In this study, we demonstrate that although growth-differentiation factor 15 (Gdf15) is not required for steady-state erythropoiesis, it plays an essential role in stress erythropoiesis. Gdf15 acts at 2 levels. In the splenic niche, Gdf15−/− mice exhibit defects in the monocyte-derived expansion of the splenic niche, resulting in impaired proliferation of stress erythroid progenitors and production of stress burst forming unit-erythroid cells. Furthermore, Gdf15 signaling maintains the hypoxia-dependent expression of the niche signal, Bmp4, whereas in stress erythroid progenitors, Gdf15 signaling regulates the expression of metabolic enzymes, which contribute to the rapid proliferation of stress erythroid progenitors. Thus, Gdf15 functions as a comprehensive regulator that coordinates the stress erythroid microenvironment with the metabolic status of progenitors to promote stress erythropoiesis.

Download Full-text

Complete Spatial Mapping of Steady-State Erythropoiesis

Blood ◽

10.1182/blood-2019-129122 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 274-274

Author(s):

Qingqing Wu ◽

Jizhou Zhang ◽

Courtney Johnson ◽

Anastasiya Slaughter ◽

Margot Lindsay May ◽

...

Keyword(s):

Cell Surface ◽

Blood Cell ◽

Red Blood Cell ◽

Differentially Expressed ◽

Cell Maturation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Mature Erythrocyte

Lack of markers to image the different progenitors has limited analyses of interactions between HSPC and their offspring. To overcome this we analyzed the expression of 250+ cell surface molecules for which antibodies are commonly available in all hematopoietic progenitors. We found 76 differentially expressed markers in at least one HSPC showing medium to bright fluorescence suggesting that many commonly used cell surface markers can be used to prospectively image HSPC in the bone marrow. We focused in erythropoiesis as it has not been possible to image step-wise red blood cell maturation in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BM BFU-E activity was restricted to Pre-MegE and CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image step-wise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells in the BM (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in the murine sternal BM and interrogate the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell in a mouse sternum. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulation. We found that Pre-MegE and Pre-CFU-E are closer to each other than predicted from random (average Pre-CFU-E to Pre-MegE distance= 92.3 µm vs. 156.7 µm random, p=0.028) but never adjacent indicating that Pre-CFU-E migrate away from Pre-MegE (0% of Pre-CFU-E adjacent to PreMegE). We also found that CFU-E were not adjacent to pre CFU-E, instead 7-8 CFU-E align to form "CFU-E strings" along the central BM (74% of CFU-E found in strings vs. 17.5% in random p<0.0001). Early erythroblasts form elongated clusters (66% early erythroblasts found within 10µm of another vs 10% in random p<0.0001) that emerge from these CFU-E strings like buds on a tree branch. Each of these early erythroblasts buds is enveloped by a large cluster of late erythroblasts, a reticulocyte cluster, and a mature erythrocyte cluster (68% of early erythroblasts buds form this 4-cluster structure vs.0% of random, P<0.0001). A recent study suggested that BM endothelial cells regulate erythroid progenitors via SCF. We found that the CFU-E strings sit on top of central BM sinusoids (average CFU-E to sinusoid distance= 0.8µm observed vs. 8.562μm random; P<0.0001) but are selectively depleted from arterioles (average CFU-E to arterioles distance=176 µm observed vs. 90.98 µm random, P<0.0001). In contrast downstream erythroid cells are farther away from sinusoids (average Early-Erythroblast to sinusoid distance=5.995 µm vs.8.224 µm random, P<0.0001; Late-erythroblast: 6.552 µm vs.9.053 µm random, P<0.0001; Reticulocytes: 6.013 µm vs.9.844 µm random, P<0.0001; Erythrocytes: 6.520 µm vs.8.986 µm random, P<0.0001). These suggest a model in which CFU-E progenitors are selectively recruited to sinusoids where they self-renew to generate strings of progenitors from which immature erythroblasts arise and mature while migrating away from the sinusoid. In summary we have found 76 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image all steps of red blood cell maturation; demonstrated that erythropoiesis takes place in highly organized 4-cluster structures emerging from strings of sinusoidal CFU-E, and demonstrated that sinusoids are the exclusive site of erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text