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1.
Dev Cell ; 59(9): 1110-1131.e22, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38569552

RESUMO

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo
3.
Blood Adv ; 7(14): 3366-3377, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-36809781

RESUMO

Hematopoietic stem cells (HSCs) are a rare type of hematopoietic cell that can entirely reconstitute the blood and immune system after transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases; however, it remains a high-risk therapy because of its potential side effects, including poor graft function and graft-versus-host disease (GVHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution in low-cell dose grafts. Here, we demonstrate that the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures can be improved using physioxic culture conditions. Single-cell transcriptomic analysis helped confirm the inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GVHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, and highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Animais , Camundongos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Transplante Homólogo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/metabolismo
4.
Methods Mol Biol ; 2524: 291-297, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35821480

RESUMO

The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that arise from using embryonic stem cells. Understanding the in vivo behavior of iPSCs, as well as their derivatives, requires the monitoring of their localization, proliferation, and viability after transplantation. Bioluminescence imaging (BLI) gives investigators a non-invasive and sensitive means for spatio-temporal tracking in vivo. For scientists working within the field of iPSCs, this protocol provides a walk-through on how to conduct in vitro and in vivo experiments with an iPSCs constitutively expressing luciferase.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Embrionárias , Humanos , Luciferases/genética
5.
J Immunol ; 199(5): 1567-1571, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28760883

RESUMO

NK cells play a critical role in host defense against viruses. In this study, we investigated the role of NKG2D in the expansion of NK cells after mouse CMV (MCMV) infection. Wild-type and NKG2D-deficient (Klrk1-/- ) Ly49H+ NK cells proliferated robustly when infected with MCMV strains engineered to allow expression of NKG2D ligands, which enhanced the response of wild-type NK cells. Naive NK cells exclusively express NKG2D-L, which pairs only with DAP10, whereas NKG2D-S expressed by activated NK cells pairs with DAP10 and DAP12, similar to Ly49H. However, NKG2D alone was unable to drive robust expansion of Ly49H- NK cells when mice were infected with these MCMV strains, likely because NKG2D-S was only transiently expressed postinfection. These findings demonstrate that NKG2D augments Ly49H-dependent proliferation of NK cells; however, NKG2D signaling alone is inadequate for expansion of NK cells, likely due to only transient expression of the NKG2D-DAP12 complex.


Assuntos
Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Imunidade Inata , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Ligação Proteica , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais
6.
J Biol Chem ; 290(36): 22298-308, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26221034

RESUMO

Recruitment of circulating monocytes and neutrophils to infection sites is essential for host defense against infections. Here, we identified a previously unannotated gene that encodes an immunoglobulin-like receptor, designated CD300H, which is located in the CD300 gene cluster. CD300H has a short cytoplasmic tail and associates with the signaling adaptor proteins, DAP12 and DAP10. CD300H is expressed on CD16(+) monocytes and myeloid dendritic cells. Ligation of CD300H on CD16(+) monocytes and myeloid dendritic cells with anti-CD300H monoclonal antibody induced the production of neutrophil chemoattractants. Interestingly, CD300H expression varied among healthy subjects, who could be classified into two groups according to "positive" and "negative" expression. Genomic sequence analysis revealed a single-nucleotide substitution (rs905709 (G → A)) at a splice donor site on intron 1 on either one or both alleles. The International HapMap Project database has demonstrated that homozygosity for the A allele of single nucleotide polymorphism (SNP) rs905709 ("negative" expression) is highly frequent in Han Chinese in Beijing, Japanese in Tokyo, and Europeans (A/A genotype frequencies 0.349, 0.167, and 0.138, respectively) but extremely rare in Sub-Saharan African populations. Together, these results suggest that CD300H may play an important role in innate immunity, at least in populations that carry the G/G or G/A genotype of CD300H.


Assuntos
Imunidade Inata/imunologia , Família Multigênica , Receptores Imunológicos/imunologia , Sequência de Aminoácidos , Animais , Povo Asiático/genética , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Frequência do Gene , Genótipo , Células HEK293 , Humanos , Imunidade Inata/genética , Immunoblotting , Camundongos , Dados de Sequência Molecular , Neutrófilos/imunologia , Neutrófilos/metabolismo , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Células U937 , População Branca/genética
7.
Nat Commun ; 5: 4710, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25134989

RESUMO

Inflammatory monocytes play an important role in host defense against infections. However, the regulatory mechanisms of transmigration into infected tissue are not yet completely understood. Here we show that mice deficient in MAIR-II (also called CLM-4 or LMIR2) are more susceptible to caecal ligation and puncture (CLP)-induced peritonitis than wild-type (WT) mice. Adoptive transfer of inflammatory monocytes from WT mice, but not from MAIR-II, TLR4 or MyD88-deficient mice, significantly improves survival of MAIR-II-deficient mice after CLP. Migration of inflammatory monocytes into the peritoneal cavity after CLP, which is dependent on VLA-4, is impaired in above mutant and FcRγ chain-deficient mice. Lipopolysaccharide stimulation induces association of MAIR-II with FcRγ chain and Syk, leading to enhancement of VLA-4-mediated adhesion to VCAM-1. These results indicate that activation of MAIR-II/FcRγ chain by TLR4/MyD88-mediated signalling is essential for the transmigration of inflammatory monocytes from the blood to sites of infection mediated by VLA-4.


Assuntos
Movimento Celular/fisiologia , Inflamação/patologia , Integrina alfa4beta1/fisiologia , Monócitos/patologia , Receptores Imunológicos/fisiologia , Receptores de Imunoglobulina Polimérica/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Ceco , Adesão Celular/fisiologia , Modelos Animais de Doenças , Feminino , Inflamação/etiologia , Inflamação/fisiopatologia , Ligadura , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologia , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Peritonite/etiologia , Peritonite/patologia , Peritonite/fisiopatologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores de Imunoglobulina Polimérica/deficiência , Receptores de Imunoglobulina Polimérica/genética , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética
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