Nestin+ Pericytes In The Fetal Liver Are Necessary To Maintain HSCs

Abstract Although most hematopoietic stem cells (HSCs) are quiescent under homeostasis in the adult bone marrow, they are actively proliferating during development. Definitive HSCs, marked by the ability to repopulate a lethally irradiated adult mouse, are first detectable in the aorta-gonad-mesonephros region around E10.5, and then colonize the fetal liver (FL) to expand in this organ until E15 when hematopoietic activity shifts to the fetal bone marrow. The role of the microenvironment, or niche, in the regulation of HSCs in the FL, a site of physiological expansion, is unclear. Fetal liver sinusoidal cells as well as hepatic progenitors have been proposed as sources of supportive signals that drive the exponential expansion of HSCs, however, exact cell type(s) supporting HSC expansion have not been defined. Since stromal cells marked by Nestin-GFP form niches for HSCs in the BM, we have hypothesized that similar cells exist in the FL. Indeed, a rare population of Nestin+ cells (CD31-CD45-Ter119-) comprising 0.02 ± 0.01% of nucleated FL cells was isolated. FL Nestin+ cells express surface markers similar to their BM counterparts, including PDGFRα, CD51, and endoglin. Since BM Nestin+ cells are enriched in mesenchymal stem and progenitor cell (MSPC) activity, we next tested this activity in FL Nestin+ cells. We found that the entire CFU-F forming capacity of the FL was contained within FL Nestin+ cells, further suggesting similarities of these cells between these two hematopoietic organs. In addition, FL Nestin+ cells were enriched for the HSC maintenance genes SCF and CXCL12, suggesting that they may also serve as the HSC niche in the FL. FL Nestin+ cells were localized on the abluminal side of large-bore arteries and expressed the pericyte markers, α-smooth muscle actin and NG2. To investigate the function of the FL Nestin+NG2+ cells in vivo, we generated a genetic depletion mouse model of NG2+ cells (NG2-cre / inducible diphtheria toxin-A (iDTA) transgenic mice). We found that E14.5 NG2-Cre;iDTA mouse embryos developed normally compared to fl-DTA control littermates. The frequency and absolute numbers of HSCs per FL, however, were reduced by about 40% in NG2-Cre;iDTA embryos (control / depleted; 0.01146 ± 0.0008% / 0.00660 ± 0.0008% of nucleated cells, p=0.0015; 606 ± 76 / 377 ± 48 HSCs/FL, p=0.03, , N=6-7 per group) suggesting that Nestin+ cells are required to maintain HSCs/progenitors in vivo. To interrogate further the relationship between FL Nestin+ cells and HSCs, we adopted the re-aggregate organ culture assay (Sheridan, Genesis 2009). Sorted lineage- FL hematopoietic progenitor cells and FL parenchymal cells either with or without sorted Nestin+ cells were re-aggregated and cultured for 7 days in serum-free, cytokine-free, media. After 7 days of culture, we found that CD150+ CD48− CD41− Lineage− HSCs were maintained in re-aggregates containing Nestin+ cells, but not when Nestin+ cells were absent. To confirm that FL Nestin+ cells were essential to maintain functional HSCs, re-aggregated cells cultured for 7 days were transplanted together with competitor bone marrow into lethally irradiated mice. We found that the contribution to the peripheral blood at 8 weeks post-transplant was only observed in the re-aggregated group containing Nestin+ cells. These preliminary data indicate that factors derived from FL Nestin+ cells are required to maintain HSCs in re-aggregate cultures. These findings suggest that HSCs inhabit similar microenvironments in temporally and spatially distinct hematopoietic organs. Further studies on differences between Nestin+ cells in these tissues may shed light on the mechanisms that determine the finely tuned quiescence, self-renewal and differentiation of HSCs. Disclosures: No relevant conflicts of interest to declare.

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The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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Suppression of CXCL12 Production by Bone Marrow Osteoblasts Is a Common and Critical Pathway for Cytokine-Induced Mobilization.

Blood ◽

10.1182/blood.v110.11.220.220 ◽

2007 ◽

Vol 110 (11) ◽

pp. 220-220

Author(s):

Matthew J. Christopher ◽

Matthew J. Hilton ◽

Fanxin Long ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Fetal Liver ◽

Common Mechanism ◽

Critical Pathway ◽

Cxcl12 Expression ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Protease Activation

Abstract There is strong evidence that CXCL12 (stromal-derived factor-1)/CXCR4 signaling is a key regulator of hematopoietic stem and progenitor cell (HSPC) trafficking in the bone marrow. CXCL12 protein and mRNA expression in the bone marrow are markedly reduced with G-CSF treatment. We and others recently showed that G-CSF treatment results in a marked loss of mature endosteal and trabecular osteoblasts. Since osteoblasts are a major source of CXCL12, this observation provides a potential mechanism by which G-CSF downregulates CXCL12 expression in the bone marrow. To test this hypothesis, we performed RNA in situ studies for CXCL12. These studies show that CXCL12 is highly expressed in endosteal osteoblasts as well as in scattered cells in the bone marrow in untreated mice. In mice treated with G-CSF, there is a near complete loss of CXCL12 signal along the endosteum. Together, these data suggest a model in which G-CSF induced suppression of osteoblasts leads to a decrease in CXCL12 expression in the bone marrow, disrupting CXCR4 signaling and ultimately leading to HSPC mobilization. This model raises several important questions. Many different hematopoietic cytokines can induce HSPC mobilization. Is loss of CXCL12 expression by osteoblasts a common mechanism by which cytokines induce HSPC mobilization? To address this question, we studied HSPC mobilization by Flt3 ligand (Flt3L) and stem cell factor (SCF), two potent mobilizing hematopoietic cytokines. Treatment with Flt3L or SCF resulted in a significant decrease in bone marrow CXCL12 protein and mRNA expression. Moreover, the decrease in CXCL12 expression was accompanied by a loss in trabecular osteoblasts [number osteoblasts per mm bone ± SEM: 5.1 ± 0.1 (control), 3.1 ± 0.4 (Flt3L), 1.8 ± 0.8 (SCF); n=2, p<0.05]. There is considerable evidence implicating other pathways in HSPC mobilization, including upregulation of protease activity in the bone marrow and downregulation of critical adhesion molecules on HSPC and their supporting stromal cells. What is the relative importance of each of these pathways to mobilization in vivo? To address this question, we established chimeras containing CXCR4−/− hematopoietic cells by transplanting CXCR4−/− fetal liver cells into irradiated syngeneic recipients. Consistent with previous reports, CXCR4−/− chimeras display constitutive mobilization of HSPC (number CFU-C/ml blood ± SEM: 12,080 ± 2,904 compared with 120 ± 25 for control chimeras; n=7–9). Surprisingly, G-CSF treatment of these mice did not result in an increase in circulating HSPC (9,511 ± 3,044; n=7–9). This lack of response to G-CSF was not simply due to a loss of mobilizable HSPC in the bone marrow, as treatment with AMD15057, a specific VLA4 antagonist, induced robust mobilization in CXCR4−/− chimeras (30,600 ± 3,790; n=6–9, p<0.01 compared with G-CSF-treated chimeras). Together, these findings suggest that: suppression of CXCL12 expression by osteoblasts is a common mechanism of cytokine-induced HSPC mobilization; this mechanism represents the principal pathway by which G-CSF mobilizes HSPC, with other pathways (e.g. protease activation) operating upstream or downstream from this pathway; and VLA-4 antagonism, in contrast, provides a distinct mechanism by which HSPC may be mobilized from the bone marrow.

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Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽

10.1182/blood.v120.21.1224.1224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1224-1224

Author(s):

Junke Zheng ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Actin Binding ◽

Wild Type ◽

Cell Fates ◽

Hematopoietic Stem ◽

Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽

10.1182/blood.v120.21.4089.4089 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4089-4089

Author(s):

Yanyan Zhang ◽

Hadjer Abdelouahab ◽

Aline Betems ◽

Monika Wittner ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Survival Time ◽

Median Survival ◽

Median Survival Time ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 4089 The receptor CXCR4 and its ligand SDF-1 play major physiological roles especially on hematopoietic stem cells homing and retention. Many studies have implicated CXCR4 in the invasion by tumor cells of organs that produce SDF-1. In acute myeloid leukemia, the physiological role of CXCR4 is not fully understood. We used retrovirus to express MLL-ENL oncogene in CXCR4+/+ and CXCR4−/− hematopoietic primitive cells (Lin- isolated from fetal liver) and showed that CXCR4 is dispensable for generation of immortalized colonies in vitro. To determine CXCR4 function in vivo, CXCR4+/+ and CXCR4−/− transformed cells were transplanted into lethally irradiated mice. Whatever their phenotype, the recipient developed a myelo-monocytique leukemia characterized by their expression of Gr-1 and Mac-1. As expected, all recipients of MLL-ENL transduced CXCR4+/+ cells were moribund within 35 to 80 days post transplant (median survival time: 50 days). Strikingly, recipients of MLL-ENL transduced CXCR4−/− cells showed significantly increased lifespan, with a median survival time of 90 days. The cellularity of the peripheral blood of recipients of MLL-ENL transduced cells displayed considerable increases over time although this increase was much lower in CXCR4−/− than in CXCR4+/+ chimera. Bone marrow of MLL-ENL transduced CXCR4−/− chimera had moderately decreased numbers of mononuclear cells. There were important numbers of leukemic CD45.2+/Gr1+/Mac1+/c-kit+ cells in spleen from MLL-ENL CXCR4+/+ chimera which suggested that CXCR4 is important for leukemic progenitors cells retention in the bone marrow and especially in the spleen. The homing capacity of transduced CXCR4+/+ cells is comparable to the CXCR4−/− cells. Finally, more DNA damages were found in the BM cells of MLL-ENL CXCR4−/− chimera. All these results were confirmed by treating of MLL-ENL CXCR4+/+ chimera with CXCR4 inhibitor (TN140). These results demonstrated that in absence of CXCR4, the cells transduced by oncogene MLL-ENL are capable of generating leukemia in the recipients. However, mice transplanted with MLL-ENL transduced CXCR4−/− FL cells developed acute myeloid leukemia with reduced aggressiveness and organ infiltration, which is associated with induced differentiation and DNA instability. These results indicated that the MLL-ENL progenitors are dependent on CXCR4 for their maintenance in the BM and spleen suggesting that CXCR4 inhibitors might have potential therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

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hnRNP K: A Tumor Suppressor or Oncogene?

Blood ◽

10.1182/blood.v124.21.259.259 ◽

2014 ◽

Vol 124 (21) ◽

pp. 259-259

Author(s):

Miguel Gallardo ◽

Hun Ju Lee ◽

Carlos E. Bueso-Ramos ◽

Xiaorui Zhang ◽

Laura R. Pageon ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Conflicts Of Interest ◽

Chromosome 9 ◽

Wild Type ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Hnrnp K ◽

Differentiation And Proliferation

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene. The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML. To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K. Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression). Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-122449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2488-2488 ◽

Cited By ~ 1

Author(s):

José Gabriel Barcia Durán

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Hematopoietic Stem ◽

Interleukin Receptors ◽

Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

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Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽

10.1182/blood-2021-153296 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 20-20

Author(s):

James Bartram ◽

Baobao (Annie) Song ◽

Juying Xu ◽

Nathan Salomonis ◽

H. Leighton Grimes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Functional Decline ◽

Functional Heterogeneity ◽

Hematopoietic Stem ◽

Donor Cells ◽

Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

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Spatial Regulation of Thrombopoiesis in the Bone Marrow

Blood ◽

10.1182/blood-2018-99-109545 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-23-SCI-23

Author(s):

David Stegner ◽

Judith van Eeuwijk ◽

Maximilian G Gorelashvili ◽

Oguzhan Angay ◽

Mike Friedrich ◽

...

Keyword(s):

Bone Marrow ◽

Fluorescence Microscopy ◽

Conflicts Of Interest ◽

Blood Platelets ◽

Marrow Cell ◽

Light Sheet ◽

Computational Simulations ◽

Hematopoietic Stem ◽

Light Sheet Fluorescence Microscopy

Abstract Blood platelets play key roles in hemostasis and thrombosis and are the second most abundant cell type in the circulation. Due to their short life span of only a few days, anuclear platelets are continuously replenished and thus provide a classic system to study hematopoiesis. In mammals, platelets are produced by megakaryocytes (MKs) that are predominantly residing in the bone marrow (BM). MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Unfortunately, previous studies on megakaryopoiesis were often limited by 2D imaging and cutting artefacts when analyzing bone sections, potentially resulting in underestimation of MK-to-vessel contacts and MK volumes. We studied megakaryopoiesis by visualizing MKs in their 3D environment. To this end, murine bones were simultaneously stained for MKs and endothelial cells, fixed, chemically cleared and imaged by Light Sheet Fluorescence Microscopy (LSFM). Thus, we achieved 3D-reconstructions of the complete and intact bone with subcellular resolution. Through imaging of MKs in the intact BM, we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. We developed and compared different image processing pipelines and simulation scenarios for precise identification of MKs in 3D light-sheet fluorescence microscopy of uncut murine bones. By combining in vivo two-photon microscopy and in situ LSFM with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. To complement limited imaging approaches computational simulations represent an important, well-controllable tool. Typically, simulation studies use artificial meshes as templates to minimize the computational effort or due to the lack of experimental data. Unfortunately, such simplified artificial templates for MKs and the vasculature can bias simulations and lead to misinterpretations as we show here. Using the segmented cell and vessel objects of true 3D images can overcome those limitations providing a simulation framework that has the prerequisites to maximally reflect the physiological situation. Thus, imaging and simulations go hand in hand when the respective 3D cell and vessel objects perfectly serve as biological templates for advanced simulations. We demonstrate reliable whole-bone analysis in silico, and found that MKs influence neutrophil and HSC migration as biomechanical restrainers modulating cell mobility and extravasation. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche (1). Furthermore, we identify MKs as biomechanical restraints for bone marrow cell mobilization. As MKs themselves do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts. (1) Stegner D, van Eeuwijk JMM, Angay O, Gorelashvili MG, Semeniak D, Pinnecker J, Schmithausen P, Meyer I, Friedrich M, Dütting S, Brede C, Beilhack A, Schulze H, Nieswandt B, Heinze KG. Thrombopoiesis is spatially regulated by the bone marrow vasculature, Nat Commun. 2017 8(1):127. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽

10.1182/blood-2020-141437 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Na Yoon Paik ◽

Grace E. Brown ◽

Lijian Shao ◽

Kilian Sottoriva ◽

James Hyun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fetal Liver ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

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