RESUMEN

Acute myeloid leukaemia (AML) is a severe haematological neoplasm that originates from the transformation of haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs). The bone marrow (BM) microenvironment, particularly that of mesenchymal stromal cells (hMSCs), plays a crucial role in the maintenance of HSCs. In this context, we explored whether alterations in the secretome of hMSCs derived from AML patients (hMSC-AML) could impact HSC gene expression. Proteomic analysis revealed that the secretome of coculture assays with hMSC-AMLs and HSC from healthy donor is altered, with increased levels of secretory leukocyte protease inhibitor (SLPI), a protein associated with important processes for maintenance of the haematopoietic niche that has already been described to be altered in several tumours. Increased SLPI expression was also observed in the BM plasma of AML patients. Transcriptome analysis of HSCs cocultured with hMSC-AML in comparison with HSCs cocultured with hMSC-HD revealed altered expression of SLPI target genes associated with the cell cycle, proliferation, and apoptosis. Important changes were identified, such as increased expression levels of CCNA2, CCNE2, CCND2, CD133 and CDK1 and decreased levels of CDKN2A and IGFBP3, among others. Overall, these findings suggest that the altered secretome of coculture assays with hMSC-AMLs and HSC from healthy donor, particularly increased SLPI expression, can contribute to gene expression changes in HSCs, potentially influencing important molecular mechanisms related to AML development and progression.

Asunto(s)

Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Inhibidor Secretorio de Peptidasas Leucocitarias , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Técnicas de Cocultivo , Transcriptoma , Femenino , Masculino , Perfilación de la Expresión Génica , Persona de Mediana Edad , Proteómica/métodos , Regulación Leucémica de la Expresión Génica , Anciano , Adulto , Proliferación Celular/genética

RESUMEN

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

Asunto(s)

Anemia de Células Falciformes , Células Madre Mesenquimatosas , Humanos , Animales , Ratones , Médula Ósea , Ratones Endogámicos C57BL , Células Madre Hematopoyéticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular

RESUMEN

Although multiple myeloma (MM) is a neoplasm that leads affected individuals to death, little is known about why some patients survive much longer than others. In this context, we investigated the transcriptomic profile of bone marrow hematopoietic stem cells obtained from MM patients and compared the clinical outcomes of death and survival six months after bone marrow transplantation. The leukapheresis products of 39 patients with MM eligible for autologous transplantation were collected and analyzed. After extraction, the RNA was analyzed using the GeneChip Human Exon 1.0 Array method. The transcriptome profile was analyzed in silico, and the differentially expressed signaling pathways of interest were validated. The results showed a difference in the expression of inflammation-related genes, immune response processes, and the oxidative stress pathway. The in silico study also pointed out the involvement of the NFκB transcription factor in the possible modulation of these genes. We chose to validate molecules participating in these processes, including the cytokines TNF-α, IFN-γ, and TGF-ß1; in addition, we measured the levels of oxidative stress mediators (pro-oxidant profile and the total antioxidant capacity). TNF-α levels were significantly reduced in patients who died and were over 50 years old at diagnosis, as well as in patients with plasmacytoma. Increased TNF-α was detected in patients with very high levels of ß2-microglobulin. IFN-γ reduction was observed in patients with a complete response to treatment compared to those with a very good response. Patients with plasmacytoma who died also had an increased pro-oxidant profile. These data show the profile of inflammatory response markers that are altered in patients with MM who die quickly and serve as a basis for the development of future studies of markers to predict better survival in this disease.

Asunto(s)

Mediadores de Inflamación , Mieloma Múltiple , Transcriptoma , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/mortalidad , Mieloma Múltiple/metabolismo , Persona de Mediana Edad , Masculino , Femenino , Transcriptoma/genética , Mediadores de Inflamación/metabolismo , Anciano , Estrés Oxidativo , Adulto , Células de la Médula Ósea/metabolismo , Análisis de Supervivencia , FN-kappa B/metabolismo , Inflamación/metabolismo , Inflamación/genética , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Despite their biological importance, the role of stem cells in human aging remains to be elucidated. In this work, we applied a machine learning methodology to GTEx transcriptome data and assigned stemness scores to 17,382 healthy samples from 30 human tissues aged between 20 and 79 years. We found that ~60% of the studied tissues exhibit a significant negative correlation between the subject's age and stemness score. The only significant exception was the uterus, where we observed an increased stemness with age. Moreover, we observed that stemness is positively correlated with cell proliferation and negatively correlated with cellular senescence. Finally, we also observed a trend that hematopoietic stem cells derived from older individuals might have higher stemness scores. In conclusion, we assigned stemness scores to human samples and show evidence of a pan-tissue loss of stemness during human aging, which adds weight to the idea that stem cell deterioration may contribute to human aging.

Asunto(s)

Envejecimiento , Senescencia Celular , Humanos , Envejecimiento/fisiología , Anciano , Persona de Mediana Edad , Adulto , Femenino , Senescencia Celular/fisiología , Células Madre/metabolismo , Masculino , Proliferación Celular , Adulto Joven , Transcriptoma , Aprendizaje Automático , Células Madre Hematopoyéticas/metabolismo

RESUMEN

The bone marrow is responsible for producing an incredible number of cells daily in order to maintain blood homeostasis through a process called hematopoiesis. Hematopoiesis is a greatly demanding process and one entirely dependent on complex interactions between the hematopoietic stem cell (HSC) and its surrounding microenvironment. Zinc (Zn2+) is considered an important trace element, playing diverse roles in different tissues and cell types, and zinc finger proteins (ZNF) are proteins that use Zn2+ as a structural cofactor. In this way, the ZNF structure is supported by a Zn2+ that coordinates many possible combinations of cysteine and histidine, with the most common ZNF being of the Cys2His2 (C2H2) type, which forms a family of transcriptional activators that play an important role in different cellular processes such as development, differentiation, and suppression, all of these being essential processes for an adequate hematopoiesis. This review aims to shed light on the relationship between ZNF and the regulation of the hematopoietic tissue. We include works with different designs, including both in vitro and in vivo studies, detailing how ZNF might regulate hematopoiesis.

Asunto(s)

Factores de Transcripción , Dedos de Zinc , Factores de Transcripción/metabolismo , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis , Médula Ósea

RESUMEN

Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Esfingomielina Fosfodiesterasa , Animales , Humanos , Ratones , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Esfingolípidos/metabolismo

RESUMEN

The cross talk between extrinsic niche-derived and intrinsic hematopoietic stem cell (HSC) factors controlling HSC maintenance remains elusive. Here, we demonstrated that amphiregulin (AREG) from bone marrow (BM) leptin receptor (LepR+) niche cells is an important factor that mediates the cross talk between the BM niche and HSCs in stem cell maintenance. Mice deficient of the DNA repair gene Brca2, specifically in LepR+ cells (LepR-Cre;Brca2fl/fl), exhibited increased frequencies of total and myeloid-biased HSCs. Furthermore, HSCs from LepR-Cre;Brca2fl/fl mice showed compromised repopulation, increased expansion of donor-derived, myeloid-biased HSCs, and increased myeloid output. Brca2-deficient BM LepR+ cells exhibited persistent DNA damage-inducible overproduction of AREG. Ex vivo treatment of wild-type HSCs or systemic treatment of C57BL/6 mice with recombinant AREG impaired repopulation, leading to HSC exhaustion. Conversely, inhibition of AREG by an anti-AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2fl/fl mice rescued HSC defects caused by AREG. Mechanistically, AREG activated the phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, promoted HSC cycling, and compromised HSC quiescence. Finally, we demonstrated that BM LepR+ niche cells from other DNA repair-deficient and aged mice also showed persistent DNA damage-associated overexpression of AREG, which exerts similar negative effects on HSC maintenance. Therefore, we identified an important factor that regulates HSCs function under conditions of DNA repair deficiency and aging.

Asunto(s)

Trastornos por Deficiencias en la Reparación del ADN , Receptores de Leptina , Ratones , Animales , Anfirregulina/genética , Anfirregulina/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Ratones Endogámicos C57BL , Células Madre Hematopoyéticas/metabolismo , Envejecimiento/genética , Trastornos por Deficiencias en la Reparación del ADN/metabolismo , Nicho de Células Madre/genética , Mamíferos/metabolismo

RESUMEN

Hematopoiesis occurs in different anatomical niches throughout the life of the individual. The first hematopoietic extra-embryonic stage is replaced by a intra-embryonic stage that occurs in a region that is adjacent to the dorsal aorta. Then, the prenatal hematopoietic function is continued by the liver and spleen, and later by the bone marrow. The objective of the present work was to describe the morphological characteristics of hepatic hematopoiesis in the alpaca and to analyze the proportion of the hematopoietic compartment of the organ and the cell types, at different times of ontogeny. Sixty-two alpaca samples were collected from the municipal slaughterhouse of Huancavelica, Perú. They were processed by routine histological techniques. Hematoxylin-eosin staining, special dyes, immunohistochemical techniques and supplementary analyses by lectinhistochemistry, were performed. The prenatal liver is an important structure in the expansion and differentiation of hematopoietic stem cells. Their hematopoietic activity was characterized by four stages: initiation, expansion, peak, and involution. The liver started its hematopoietic function at 21 days EGA and it was maintained until shortly before birth. Differences were found in the proportion and morphology of the hematopoietic tissue in the different groups corresponding to each gestational stage.

Asunto(s)

Camélidos del Nuevo Mundo , Embarazo , Animales , Femenino , Hematopoyesis , Hígado , Células Madre Hematopoyéticas/metabolismo , Médula Ósea

RESUMEN

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Asunto(s)

Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Ratones , Animales , Células Madre Hematopoyéticas/metabolismo , Trasplante de Médula Ósea , Huesos

RESUMEN

INTRODUCTION: Although plerixafor in association with granulocyte colony-stimulating factor (G-CSF) can improve mobilization and collection of hematopoietic stem cells (HSC) by leukapheresis, cost may limit its clinical application. The present study systematically reviews economic evaluations of plerixafor plus G-CSF usage compared to G-CSF alone and compares different strategies of plerixafor utilization in multiple myeloma and lymphoma patients eligible for autologous HSC transplantation. AREAS COVERED: Relevant economic evaluations, partial or complete, were searched on PubMed, Embase, LILACS, and Cochrane Central Register of Controlled Trials for a period ending 30 June 2021. This systematic review was reported following the PRISMA Statement. Six economic evaluations were included, considering the use of upfront or just-in-time plerixafor compared to G-CSF alone or other plerixafor strategies. Most comparisons showed both increased cost and health benefits with the addition of plerixafor. Most analyses favored just-in-time plerixafor compared to upfront plerixafor, with a probable preference for broader cutoffs for just-in-time plerixafor initiation. EXPERT OPINION: Plerixafor is a potentially cost-effective technology in the mobilization of HSC in patients with multiple myeloma and lymphomas eligible for autologous HSC transplantation. There is a decreased number of leukapheresis sessions and remobilizations and a higher yield of CD34+ cells.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Compuestos Heterocíclicos , Linfoma , Mieloma Múltiple , Humanos , Mieloma Múltiple/terapia , Movilización de Célula Madre Hematopoyética , Leucaféresis , Análisis Costo-Beneficio , Trasplante Autólogo , Compuestos Heterocíclicos/metabolismo , Linfoma/terapia , Linfoma/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Factor Estimulante de Colonias de Granulocitos , Bencilaminas/metabolismo

RESUMEN

GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

Asunto(s)

Factor de Transcripción GATA1 , Células Madre Hematopoyéticas , Enfermedades Neuroinflamatorias , Animales , Ratones , Diferenciación Celular , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Factor de Transcripción GATA1/metabolismo

RESUMEN

The use of non-cryopreserved hematopoietic stem cells (HSC) can be an alternative to the traditional cryopreserved infusions of HSCs in autologous stem cell transplantation (aHSCT). After high-dose melphalan conditioning (HDM), we sought to compare time to engraftment, overall survival, and safety in multiple myeloma (MM) patients undergoing a first aHSCT after high-dose melphalan conditioning (HDM). We conducted a cohort study from March 2018 to December 2019. Of all autologous transplants performed during this period, 105 were for MM as the first consolidation. Fifty-one patients received a cryopreserved graft; the remaining 54 patients received a fresh infusion. General clinical characteristics were similar between these two groups. Cell viability was higher in non-cryopreserved grafts (95% vs. 86% p < 0.01). Four deaths occurred during hospitalization in the cryopreserved group, one in the non-cryopreserved group. The cumulative incidence of neutrophil and platelet engraftment on D + 25 was higher in the non-cryopreserved compared to the cryopreserved group (98% vs 90% p < 0.01 and 96.2% vs 72.54% p < 0.01 respectively). Additionally, the hospital length of stay was reduced by 4 days for patients for the non-cryopreserved cohort. In summary, the use of non-cryopreserved HSCs after HDM is safe and effective compared to patients who received a cryopreserved graft.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Mieloma Múltiple , Autoinjertos , Estudios de Cohortes , Células Madre Hematopoyéticas/metabolismo , Humanos , Melfalán , Acondicionamiento Pretrasplante , Trasplante Autólogo

RESUMEN

Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell-cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta-gonad-mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community.

Asunto(s)

Células Madre Hematopoyéticas , Pez Cebra , Animales , Diferenciación Celular , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Mesonefro/metabolismo , Ratones , Pez Cebra/genética

RESUMEN

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.

Asunto(s)

Citocinas , Células Madre Hematopoyéticas , Antígenos CD34/metabolismo , Proliferación Celular , Células Cultivadas , Citocinas/genética , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Transcriptoma

RESUMEN

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to alleviate hematologic symptoms in monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi anemia (FA). However, for FA and other BMF syndromes, insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein LNK (SH2B3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs results in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhances cytokine-mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment of FANCD2-depleted FA-like HSCs was markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

Asunto(s)

Anemia de Fanconi , Trasplante de Células Madre Hematopoyéticas , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD34/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/terapia , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones

RESUMEN

Hematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematologic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNγ) as a potential mediator. Interestingly, improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNγ activity. Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of engraftment failure is high.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Donantes de Tejidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/inmunología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Perfilación de la Expresión Génica/métodos , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Depleción Linfocítica/métodos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos

RESUMEN

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Asunto(s)

Células Madre Hematopoyéticas/metabolismo , Inflamación/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estrés Fisiológico/fisiología , Transactivadores/metabolismo , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Homeostasis/fisiología , Inmunidad Innata/fisiología , Ratones , Ratones Endogámicos C57BL

RESUMEN

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1ß (IL-1ß), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1ß exposure engendered potent selection for Cebpa loss. Chronic IL-1ß augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1ß, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-1beta/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula/fisiología , Expresión Génica/fisiología , Células HEK293 , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inflamación/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo

RESUMEN

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Standard induction therapy leads to complete remission in approximately 50% to 75% of patients. In spite of favorable primary response rates, only 20% to 30% of patients enjoy long-term disease free survival. Identifying proteins involved in prognosis is important for proposing biomarkers that can aid in the clinical management of the disease. The aim of this study was to construct a protein-protein interaction (PPI) network based on serum proteins associated with unfavorable prognosis of AML, and analyze the biological pathways underlying molecular complexes in the network. We identified 16 candidate serum proteins associated with unfavorable prognosis (in terms of poor response to treatment, poor overall survival, short complete remission, and relapse) in AML via a search in the literature: IL2RA, FTL, HSP90AA1, D2HGDH, PLAU, COL18A1, FGF19, SPP1, FGA, PF4, NME1, TNF, ANGPT2, B2M, CD274, LGALS3. The PPI network was constructed with Cytoscape using association networks from String and BioGRID, and Gene Ontology enrichment analysis using the ClueGo pluggin was performed. The central protein in the network was found to be PTPN11 which is involved in modulating the RAS-ERK, PI3K-AKT and JAK-STAT pathways, as well as in hematopoiesis, and in the regulation of apoptotic genes. Therefore, a dysregulation of this protein and/or of the proteins connected to it in the network leads to the defective activation of these signaling pathways and to a reduction in apoptosis. Together, this could cause an increase in the frequency of leukemic cells and a resistance to apoptosis in response to treatment.

Asunto(s)

Biomarcadores de Tumor/genética , Proteínas Sanguíneas/genética , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Supervivencia sin Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/sangre , Quinasas MAP Reguladas por Señal Extracelular/genética , Ontología de Genes , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Quinasas Janus/sangre , Quinasas Janus/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Anotación de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/sangre , Fosfatidilinositol 3-Quinasas/genética , Pronóstico , Mapeo de Interacción de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/sangre , Proteínas Proto-Oncogénicas c-akt/sangre , Proteínas Proto-Oncogénicas c-akt/genética , Inducción de Remisión , Factores de Transcripción STAT/sangre , Factores de Transcripción STAT/genética , Transducción de Señal , Proteínas ras/sangre , Proteínas ras/genética

RESUMEN

Recent evidence suggests that abnormalities involving CD4+T lymphocytes are associated with the pathophysiology of osteonecrosis (ON); however, few studies have addressed the CD4+T cells in ON related to sickle cell disease (SCD/ON). In addition, T cells producing multiple cytokines simultaneously are often present in the inflammatory milieu and may be implicated in the immune response observed in SCD/ON. In the present study, we aimed to characterize the functional status of CD4+T cells in SCD by simultaneously determining the frequency of IFN-γ +, IL-4+, and IL-17+ CD4+T in cell cultures under exogenous stimuli. Peripheral blood mononuclear cells (PB-MNCs) from 9 steady-state SCD patients, 15 SCD/ON patients, and 19 healthy controls had functional status of CD4+T cells analyzed. Bone marrow mononuclear cells (BM-MNCs) from 24 SCD/ON patients (SCD BM) and 18 patients with ON not related to SCD (non-SCD BM) were also analyzed. We found that PB-MNC of SCD patients with or without ON presented significantly reduced TCD4+, TCD8+, and TCD4+ naïve cell frequencies and increased frequency of circulating CD4+T cells able to simultaneously produce IFN-γ +/IL4+ and IL-17+/IL4+ compared to healthy controls. Conversely, the polyclonal stimulation of BM-MNC induced an increased frequency of CD4+IFN-γ + and CD4+IL-17+ in SCD BM compared to non-SCD BM. The increased proportion of CD4+ T cells able to produce a broad spectrum of proinflammatory cytokines after a strong stimulus indicates that the immune system in SCD/ON patients presents an expressive pool of partially differentiated cells ready to take on effector function. It is possible that this increased subpopulation may extend to inflammatory sites of target organs and may contribute to the maintenance of inflammation and the pathophysiology of osteonecrosis in sickle cell disease.

Asunto(s)

Anemia de Células Falciformes/inmunología , Anemia de Células Falciformes/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Osteonecrosis/inmunología , Osteonecrosis/metabolismo , Adolescente , Adulto , Femenino , Células Madre Hematopoyéticas/metabolismo , Hemoglobinas/metabolismo , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Masculino , Persona de Mediana Edad , Adulto Joven