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1.
Methods Mol Biol ; 2567: 205-232, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255704

RESUMO

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time points post-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6 × BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Animais , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Camundongos SCID , Camundongos Endogâmicos NOD , Modelos Teóricos
2.
Biomaterials ; 285: 121569, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35567999

RESUMO

Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.


Assuntos
Hemangioblastos , Animais , Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Mesonefro , Camundongos , Peixe-Zebra
3.
Blood Cells Mol Dis ; 91: 102594, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34520986

RESUMO

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.


Assuntos
Quimiocinas CXC/metabolismo , Células Eritroides/citologia , Granulócitos/citologia , Macrófagos/citologia , Células-Tronco/citologia , Animais , Proliferação de Células , Células Cultivadas , Células Eritroides/metabolismo , Granulócitos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Pontos de Checagem da Fase S do Ciclo Celular , Células-Tronco/metabolismo
4.
Stem Cell Rev Rep ; 17(4): 1083-1090, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34255283

RESUMO

We wish to suggest the possibility there is a link between the brain and hematopoiesis in the bone marrow and that in the future it may be possible to use such information for better understanding of the regulation of hematopoiesis, and for efficacious treatment of hematopoietic disorders.


Assuntos
Medula Óssea , Encéfalo , Hematopoese , Medula Óssea/fisiologia , Encéfalo/fisiologia , Humanos
5.
Blood ; 137(6): 775-787, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32881992

RESUMO

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.


Assuntos
Células-Tronco Hematopoéticas/citologia , Receptores de GABA-B/fisiologia , Animais , Linfócitos B/patologia , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Medula Óssea/inervação , Medula Óssea/metabolismo , Transplante de Medula Óssea , Divisão Celular , Linhagem da Célula , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Linfopenia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Quimera por Radiação , Receptores de GABA-B/deficiência , Receptores de GABA-B/genética , Nicho de Células-Tronco
6.
Stem Cells ; 22(6): 1003-14, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15536191

RESUMO

Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.


Assuntos
Antígenos CD34/biossíntese , Sangue Fetal/citologia , Antígenos CD15/biossíntese , Células Mieloides/citologia , Proteômica/métodos , Células-Tronco/citologia , Carcinoma Embrionário/metabolismo , Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Neuroblastoma/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , RNA Nuclear Pequeno/química , Transdução de Sinais
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