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2.
Nature ; 569(7755): 222-228, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30971824

RESUMO

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.


Assuntos
Medula Óssea/irrigação sanguínea , Microambiente Celular , Hematopoese , Células-Tronco Hematopoéticas , Análise de Célula Única , Nicho de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/citologia , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células-Tronco/genética , Estresse Fisiológico/genética , Transcriptoma/genética
3.
Stem Cell Rev Rep ; 20(4): 1135-1149, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38438768

RESUMO

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.


Assuntos
Células Endoteliais , Integrases , Tamoxifeno , Animais , Camundongos , Células Endoteliais/metabolismo , Integrases/metabolismo , Integrases/genética , Tamoxifeno/farmacologia , Medula Óssea/metabolismo , Camundongos Transgênicos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Hematopoese
4.
Nat Commun ; 14(1): 2018, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-37037837

RESUMO

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Netrina-1/genética , Células-Tronco Hematopoéticas/fisiologia , Células da Medula Óssea , Envelhecimento/genética , Nicho de Células-Tronco
5.
J Exp Med ; 217(6)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32289154

RESUMO

Aging leads to a decline in hematopoietic stem and progenitor cell (HSPC) function. We recently discovered that aging of bone marrow endothelial cells (BMECs) leads to an altered crosstalk between the BMEC niche and HSPCs, which instructs young HSPCs to behave as aged HSPCs. Here, we demonstrate aging leads to a decrease in mTOR signaling within BMECs that potentially underlies the age-related impairment of their niche activity. Our findings reveal that pharmacological inhibition of mTOR using Rapamycin has deleterious effects on hematopoiesis. To formally determine whether endothelial-specific inhibition of mTOR can influence hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that their HSPCs displayed attributes of an aged hematopoietic system. Transcriptional profiling of HSPCs from mTOR(ECKO) mice revealed that their transcriptome resembled aged HSPCs. Notably, during serial transplantations, exposure of wild-type HSPCs to an mTOR(ECKO) microenvironment was sufficient to recapitulate aging-associated phenotypes, confirming the instructive role of EC-derived signals in governing HSPC aging.


Assuntos
Envelhecimento/fisiologia , Células Endoteliais/metabolismo , Hematopoese , Serina-Treonina Quinases TOR/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Microambiente Celular , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Sirolimo/farmacologia , Transcrição Gênica
6.
Nat Commun ; 11(1): 666, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015345

RESUMO

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.


Assuntos
Células Endoteliais/metabolismo , Hematopoese/fisiologia , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Antígenos CD , Medula Óssea , Caderinas , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Inflamação , Masculino , Camundongos , Transdução de Sinais , Transplante Autólogo
7.
J Clin Invest ; 127(11): 4163-4178, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29035282

RESUMO

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.


Assuntos
Células Endoteliais/fisiologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Envelhecimento , Animais , Medula Óssea/irrigação sanguínea , Transplante de Medula Óssea , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/transplante , Camundongos Endogâmicos C57BL , Microvasos/patologia , Lesões Experimentais por Radiação/prevenção & controle , Tolerância a Radiação
8.
Nat Commun ; 7: 13829, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28000664

RESUMO

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.


Assuntos
Células Endoteliais/metabolismo , Hematopoese , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Pancitopenia/terapia , Nicho de Células-Tronco
9.
Stem Cell Reports ; 5(5): 881-894, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26441307

RESUMO

Hematopoietic stem cells (HSCs) inhabit distinct microenvironments within the adult bone marrow (BM), which govern the delicate balance between HSC quiescence, self-renewal, and differentiation. Previous reports have proposed that HSCs localize to the vascular niche, comprised of endothelium and tightly associated perivascular cells. Herein, we examine the capacity of BM endothelial cells (BMECs) to support ex vivo and in vivo hematopoiesis. We demonstrate that AKT1-activated BMECs (BMEC-Akt1) have a unique transcription factor/cytokine profile that supports functional HSCs in lieu of complex serum and cytokine supplementation. Additionally, transplantation of BMEC-Akt1 cells enhanced regenerative hematopoiesis following myeloablative irradiation. These data demonstrate that BMEC-Akt1 cultures can be used as a platform for the discovery of pro-HSC factors and justify the utility of BMECs as a cellular therapy. This technical advance may lead to the development of therapies designed to decrease pancytopenias associated with myeloablative regimens used to treat a wide array of disease states.


Assuntos
Células Progenitoras Endoteliais/citologia , Hematopoese , Nicho de Células-Tronco , Animais , Citocinas/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/transplante
10.
Exp Hematol ; 42(11): 976-986.e3, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25179751

RESUMO

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.


Assuntos
Medula Óssea/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Adesão Celular , Linhagem Celular Transformada , Proliferação de Células , Microambiente Celular , Células Clonais , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
11.
Cell Rep ; 4(5): 1022-34, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-24012753

RESUMO

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Endoteliais/metabolismo , Hematopoese/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células Endoteliais/citologia , Homeostase , Proteína Jagged-1 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Serrate-Jagged , Transdução de Sinais
12.
Microsc Res Tech ; 75(3): 325-33, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21823204

RESUMO

Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1 µm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.


Assuntos
Colículos Superiores/citologia , Peixe-Zebra/anatomia & histologia , Animais , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica
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