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1.
JCI Insight ; 7(9)2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35531956

RESUMO

Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais
2.
Sci Rep ; 12(1): 7181, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35504940

RESUMO

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.


Assuntos
Sistemas CRISPR-Cas , Proteína I de Ligação a Poli(A) , Regiões 3' não Traduzidas , Animais , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Proteína I de Ligação a Poli(A)/metabolismo , Poliadenilação , RNA Mensageiro/genética
3.
Cell Stem Cell ; 29(5): 760-775.e10, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35523139

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.


Assuntos
Apresentação do Antígeno , Células-Tronco Hematopoéticas , Diferenciação Celular , Linfócitos T
4.
Methods Mol Biol ; 2429: 103-124, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507158

RESUMO

Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.


Assuntos
Hemangioblastos , Células-Tronco Pluripotentes , Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas , Humanos
5.
Methods Mol Biol ; 2429: 281-306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507169

RESUMO

Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , RNA Guia/genética , RNA Guia/metabolismo
6.
Semin Hematol ; 59(1): 13-20, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35491054

RESUMO

Bone marrow failure (BMF) syndromes are a heterogeneous group of benign hematological conditions characterized by uni- or multi-lineage marrow and/or peripheral blood cytopenia(s), and can be classified in constitutional or acquired syndromes based on pathophysiology. In inherited diseases, germline mutations occur in the hematopoietic stem and progenitor cell (HSPC) compartment causing a progressive loss of normal hematopoiesis, while in acquired syndromes, HPSC compartment disruption can be caused by an extrinsic direct damage by external cytotoxic agents on the stem cell pool or by an autoimmune attack against HSPCs. Aplastic anemia is an acquired immune-mediated BMF syndrome where marrow disruption is driven by a cytotoxic T cell-mediated autoimmune attack against HSPCs sustained by type I interferons that polarize the immune system toward T helper 1 responses in early phases and then toward T helper 17 and effector memory CD8+ T cell in late stage and severe disease. Cytokines and chemokines also play a crucial role in driving immune responses and HSPC growth inhibition and apoptosis, as described for interferon-γ and tumor necrosis factor α. In this review, we summarize current knowledge on acquired aplastic anemia pathophysiology.


Assuntos
Anemia Aplástica , Anemia Aplástica/genética , Medula Óssea/patologia , Transtornos da Insuficiência da Medula Óssea , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndrome
7.
Semin Hematol ; 59(1): 21-29, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35491055

RESUMO

Severe aplastic anemia, a disease characterized by pancytopenia and a hypocellular marrow, is treatable by either immunosuppressive therapy (IST) or hematopoietic stem cell transplant. Much is understood about the immune-mediated pathophysiology of AA now, but the inciting factor remains elusive. Many groups around the globe contributed to understanding the disease pathophysiology and optimizing the IST regimen. Horse antithymocyte globulin and cyclosporine, the initial IST regimen, achieved a hematologic response rate in about 60% to 65% of treated patients, with less than 10% achieving a complete count recovery. However, adding a thrombopoietin receptor agonist, eltrombopag (EPAG), to IST improved these response rates to nearly 80% and an unprecedented increase in complete response to almost 40%. The latest report indicates that a high-risk clonal evolution to myeloid malignancies is not increased with hematopoietic stem cell stimulation by adding EPAG in the front line setting. Despite the great success of IST and EPAG in improving early outcomes, relapse remains a problem. Further optimization of upfront therapy and treatment protocol is needed to prevent relapses and decrease clonal evolution rates for even better long-term results.


Assuntos
Anemia Aplástica , Anemia Aplástica/tratamento farmacológico , Células-Tronco Hematopoéticas , Humanos , Recidiva , Indução de Remissão
8.
Semin Hematol ; 59(1): 47-53, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35491059

RESUMO

Advances in molecular technologies accelerated investigations of the hematopoietic stem cell (HSC) compartment in aplastic anemia (AA). Initially, stem cell biology approaches indicated a profound depletion of HSC pool, while studies of paroxysmal nocturnal hemoglobinuria (PNH), X-chromosome inactivation and cytogenetics provided the first evidence for the presence of clonality in the context of a contracted HSC compartment. More recently, the introduction of deep NGS allowed a more precise assessment of clonal expansions in AA. NGS studies demonstrated that the acquisition of somatic defects, including mutations and copy number alterations, can occur prior to and without a strict consequence of a later evolution of post-AA myelodysplastic syndrome (MDS). Such mutant clones may simply correspond to clonal hematopoiesis of indeterminate potential (CHIP) prematurely uncovered by the depletion of polyclonal "normal" HSCs. However, clonal expansions may also correspond to adaptive responses extrinsically or intrinsically enhancing clonal fitness. These processes could lead to adaptive (clonal non-malignant) or maladaptive (post-AA MDS) attempts at reconstitution of hematopoiesis. The spectrum of molecular events provides many clues as to the evolutionary forces driving the pathogenesis of AA and also valuable lessons as to the function of normal and malignant hematopoiesis. This article summarizes current thinking, controversies and dilemmas in interpreting molecular data obtained from the studies of AA.


Assuntos
Anemia Aplástica , Hemoglobinúria Paroxística , Síndromes Mielodisplásicas , Anemia Aplástica/genética , Hematopoese , Células-Tronco Hematopoéticas , Hemoglobinúria Paroxística/genética , Hemoglobinúria Paroxística/patologia , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia
9.
Cancer Discov ; 12(5): OF6, 2022 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-35491633

RESUMO

VCAM1 acts as an immune checkpoint for haplotype-mismatched HSC bone marrow entry and engraftment.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Tolerância Imunológica
10.
Stomatologiia (Mosk) ; 101(2): 80-86, 2022.
Artigo em Russo | MEDLINE | ID: mdl-35362708

RESUMO

The aim of the work is to attract the attention of specialists: dentists, oncologists, hematologists to thorough sanitation of the oral cavity of patients preparing for chemotherapy treatment, to transplantation of hematopoietic stem cells. Two clinical cases described in the article were observed at the R.M. Gorbacheva First Saint-Petersburg State Medical University from 2010 to 2019. They confirm the possibility of the occurrence of infectious complications with damage to the maxillofacial region caused by rare pathogens of invasive mycosis, which debuted as an odontogenic inflammatory process. The success of the treatment of Invasive Mycosis depends on early diagnosis and antimycotic therapy; active surgical tactics in relation to the affected tissues in a controlled course of the underlying disease and the restoration of effective hematopoiesis.


Assuntos
Mucormicose , Células-Tronco Hematopoéticas , Humanos , Boca , Mucormicose/tratamento farmacológico , Mucormicose/cirurgia
11.
Nature ; 604(7906): 534-540, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35418685

RESUMO

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.


Assuntos
Células Endoteliais , Células-Tronco Hematopoéticas , Diferenciação Celular , Endotélio , Feminino , Hematopoese , Humanos , Mesonefro , Gravidez
12.
J Transl Med ; 20(1): 184, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468789

RESUMO

Cellular therapies have become an important part of clinical care. The treatment of patients with cell therapies often involves the collection of autologous cells at the medical center treating the patient, the shipment of these cells to a centralized manufacturing site, and the return of the cryopreserved clinical cell therapy to the medical center treating the patient for storage until infusion. As this activity grows, cell processing laboratories at many academic medical centers are involved with many different autologous products manufactured by several different centralized laboratories. The handling of these products by medical center-based cell therapy laboratories is complicated and resource-intensive since each centralized manufacturing laboratory has unique methods for labeling, storing, shipping, receiving, thawing, and infusing the cells. The field would benefit from the development of more uniform practices. The development of a coordinating center similar to those established to facilitate the collection, shipping, and transplantation of hematopoietic stem cells from unrelated donors would also be beneficial. In summary, the wide range of practices involved with labeling, shipping, freezing, thawing, and infusing centrally manufactured autologous cellular therapies lack efficiency and consistency and puts patients at risk. More uniform practices are needed.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco Hematopoéticas , Criopreservação/métodos , Células-Tronco Hematopoéticas , Humanos , Transplante Autólogo
13.
Nat Commun ; 13(1): 2048, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440586

RESUMO

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)- (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level.


Assuntos
Células-Tronco Hematopoéticas , Análise de Célula Única , Antígenos CD34 , Moléculas de Adesão Celular , Linhagem da Célula , Receptor de Proteína C Endotelial , Humanos
14.
Commun Biol ; 5(1): 396, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484199

RESUMO

Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders and malignancies. SIRT1 has been implicated in healthy aging, but its role in HSC aging is poorly understood. Surprisingly, we found that Sirt1 knockout improved the maintenance of quiescence of aging HSCs and their functionality as well as mouse survival in serial bone marrow transplantation (BMT) recipients. The majority of secondary and tertiary BMT recipients of aging wild type donor cells developed B/myeloid mixed phenotype acute leukemia (MPAL), which was markedly inhibited by Sirt1 knockout. SIRT1 inhibition also reduced the growth and survival of human B/myeloid MPAL cells. Sirt1 knockout suppressed global gene activation in old HSCs, prominently the genes regulating protein synthesis and oxidative metabolism, which may involve multiple downstream transcriptional factors. Our results demonstrate an unexpected role of SIRT1 in promoting HSC aging and age-dependent MPAL and suggest SIRT1 may be a new therapeutic target for modulating functions of aging HSCs and treatment of MPAL.


Assuntos
Leucemia , Sirtuína 1 , Envelhecimento/genética , Animais , Células-Tronco Hematopoéticas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Camundongos , Fenótipo , Sirtuína 1/genética , Sirtuína 1/metabolismo
15.
BMC Infect Dis ; 22(1): 352, 2022 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-35397492

RESUMO

BACKGROUND: Invasive fungal diseases (IFD) remain a major complication of allogeneic hematopoietic stem cell transplantation (alloHSCT) and are associated with high mortality rates in patients receiving alloHSCT. Antifungal prophylaxis is increasingly being used in the management of IFDs in patients receiving alloHSCT. METHODS: A post-hoc analysis of the cross-sectional observational AFHEM study was carried out to describe the use of antifungal drugs in real-life clinical practice in alloHSCT recipients hospitalized in French hematological units. RESULTS: A total of 147 alloHSCT recipients were enrolled; most were adults (n = 135; 92%) and had received alloHSCT < 6 months prior to enrollment (n = 123; 84%). Overall, 119 (81%) patients received a systemic antifungal therapy; of these, 95 (80%) patients received antifungal prophylaxis. Rates of patients receiving systemic antifungal treatment were similar irrespective of transplant time, neutropenic, and graft-versus-host disease status. Among patients on systemic antifungal treatment, 83 (70%) received an azole, 22 (18%) received an echinocandin, and 16 (13%) received a polyene. CONCLUSIONS: This work provides evidence of the antifungal strategies used in alloHSCT recipients hospitalized in French hematological units. Unlike earlier studies, the AFHEM study showed that prophylaxis appears to be the leading antifungal strategy used in alloHSCT recipients in France.


Assuntos
Hematologia , Transplante de Células-Tronco Hematopoéticas , Adulto , Antifúngicos/uso terapêutico , Estudos Transversais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas , Humanos , Transplante Homólogo/efeitos adversos
16.
Development ; 149(8)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35438172

RESUMO

Hofbauer cells (HBCs) are tissue macrophages of the placenta thought to be important for fetoplacental vascular development and innate immune protection. The developmental origins of HBCs remain unresolved and could implicate functional diversity of HBCs in placenta development and disease. In this study, we used flow cytometry and paternally inherited reporters to phenotype placenta macrophages and to identify fetal-derived HBCs and placenta-associated maternal macrophages in the mouse. In vivo pulse-labeling traced the ontogeny of HBCs from yolk sac-derived erythro-myeloid progenitors, with a minor contribution from fetal hematopoietic stem cells later on. Single-cell RNA-sequencing revealed transcriptional similarities between placenta macrophages and erythro-myeloid progenitor-derived fetal liver macrophages and microglia. As with other fetal tissue macrophages, HBCs were dependent on the transcription factor Pu.1, the loss-of-function of which in embryos disrupted fetoplacental labyrinth morphology, supporting a role for HBC in labyrinth angiogenesis and/or remodeling. HBC were also sensitive to Pu.1 (Spi1) haploinsufficiency, which caused an initial deficiency in the numbers of macrophages in the early mouse placenta. These results provide groundwork for future investigation into the relationship between HBC ontogeny and function in placenta pathophysiology.


Assuntos
Macrófagos , Placenta , Animais , Feminino , Células-Tronco Hematopoéticas , Camundongos , Células Progenitoras Mieloides , Gravidez , Saco Vitelino
17.
Development ; 149(8)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35452096

RESUMO

Previously, we have demonstrated that a subpopulation of microglia, known as Hoxb8 microglia, is derived from the Hoxb8 lineage during the second wave (E8.5) of yolk sac hematopoiesis, whereas canonical non-Hoxb8 microglia arise from the first wave (E7.5). Hoxb8 microglia have an ontogeny distinct from non-Hoxb8 microglia. Dysfunctional Hoxb8 microglia cause the acquisition of chronic anxiety and an obsessive-compulsive spectrum-like behavior, trichotillomania, in mice. The nature and fate of the progenitors generated during E8.5 yolk sac hematopoiesis have been controversial. Herein, we use the Hoxb8 cell lineage reporter to define the ontogeny of hematopoietic cells arising during the definitive waves of hematopoiesis initiated in the E8.5 yolk sac and aorta-gonad-mesonephros (AGM) region. Our murine cell lineage analysis shows that the Hoxb8 cell lineage reporter robustly marks erythromyeloid progenitors, hematopoietic stem cells and their progeny, particularly monocytes. Hoxb8 progenitors and microglia require Myb function, a hallmark transcription factor for definitive hematopoiesis, for propagation and maturation. During adulthood, all immune lineages and, interestingly, resident macrophages in only hematopoietic/lymphoid tissues are derived from Hoxb8 precursors. These results illustrate that the Hoxb8 lineage exclusively mirrors murine definitive hematopoiesis.


Assuntos
Hematopoese , Saco Vitelino , Animais , Linhagem da Célula , Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Mesonefro , Camundongos
18.
Hematology ; 27(1): 476-487, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35413231

RESUMO

OBJECTIVE: The interplay between intrinsic and extrinsic elements involved in the physiology of hematopoietic cells is not completely understood. In the present study, we analyzed the transcriptional profiles of human cord blood-derived hematopoietic stem cells (HSCs), as well as myeloid (MPCs) and erythroid (EPCs) progenitors, and assessed their proliferation and expansion kinetics in vitro. METHODS: All cell populations were obtained by cell-sorting, and were cultured in liquid cultures supplemented with different cytokine combinations. Their gene expression profiles were determined by RNA microarrays right after cell-sorting, before culture. RESULTS: HSCs showed the highest proliferation and expansion capacities in culture, and were found to be more closely related, in transcriptional terms, to MPCs than to EPCs. This correlated with the fact that after 30 days, only cultures initiated with HSCs and MPCs were sustained. Expression of cell cycle and cell division-related genes was enriched in EPCs. Such cells showed significantly higher proliferation than MPCs, however, their expansion potential was reduced, so that cultures initiated with EPCs declined after 15 days and became exhausted by day 30. Proliferation and expansion of HSCs and EPCs were higher in the presence of a cytokine combination that favors erythropoiesis, whereas the growth of MPCs was higher under a cytokine combination that favors myelopoiesis. CONCLUSION: This study shows a correlation between the transcriptional profiles of HSCs, MPCs, and EPCs, and their respective in vitro growth under particular culture conditions. These results may be relevant in the development of ex vivo systems for the expansion of hematopoietic cells for clinical application.


Assuntos
Citocinas , Células-Tronco Hematopoéticas , Antígenos CD34/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/genética , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Transcriptoma
19.
Cell Death Dis ; 13(4): 412, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484113

RESUMO

In acquired immune aplastic anemia (AA), pathogenic cytotoxic Th1 cells are activated and expanded, driving an immune response against the hematopoietic stem and progenitor cells (HSPCs) that provokes cell depletion and causes bone marrow failure. However, additional HSPC defects may contribute to hematopoietic failure, reflecting on disease outcomes and response to immunosuppression. Here we derived induced pluripotent stem cells (iPSCs) from peripheral blood (PB) erythroblasts obtained from patients diagnosed with immune AA using non-integrating plasmids to model the disease. Erythroblasts were harvested after hematologic response to immunosuppression was achieved. Patients were screened for germline pathogenic variants in bone marrow failure-related genes and no variant was identified. Reprogramming was equally successful for erythroblasts collected from the three immune AA patients and the three healthy subjects. However, the hematopoietic differentiation potential of AA-iPSCs was significantly reduced both quantitatively and qualitatively as compared to healthy-iPSCs, reliably recapitulating disease: differentiation appeared to be more severely affected in cells from the two patients with partial response as compared to the one patient with complete response. Telomere elongation and the telomerase machinery were preserved during reprogramming and differentiation in all AA-iPSCs. Our results indicate that iPSCs are a reliable platform to model immune AA and recapitulate clinical phenotypes. We propose that the immune attack may cause specific epigenetic changes in the HSPCs that limit adequate proliferation and differentiation.


Assuntos
Anemia Aplástica , Células-Tronco Pluripotentes Induzidas , Anemia Aplástica/genética , Anemia Aplástica/patologia , Transtornos da Insuficiência da Medula Óssea , Diferenciação Celular , Células-Tronco Hematopoéticas/patologia , Humanos
20.
Cell Death Dis ; 13(4): 337, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35414137

RESUMO

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34+ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.


Assuntos
Vesículas Extracelulares , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Vesículas Extracelulares/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
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