RESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon in which hematopoietic stem cells acquire mutations in a select set of genes commonly mutated in myeloid neoplasia which then expand clonally. Current sequencing assays to detect CHIP mutations are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or to serial sequencing. In this study, an affordable (approximately US $8 per sample), accurate, and scalable sequencing assay for CHIP is introduced and validated. The efficacy of the assay was demonstrated by identifying CHIP mutations in a cohort of 456 individuals with DNA collected at multiple time points in Vanderbilt University's biobank and quantifying clonal expansion rates over time. A total of 101 individuals with CHIP/clonal cytopenia of undetermined significance were identified, and individual-level clonal expansion rate was calculated using the variant allele fraction at both time points. Differences in clonal expansion rate by driver gene were observed, but there was also significant individual-level heterogeneity, emphasizing the multifactorial nature of clonal expansion. Additionally, mutation co-occurrence and clonal competition between multiple driver mutations were explored.
Assuntos
Hematopoiese Clonal , Mutação , Humanos , Hematopoiese Clonal/genética , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Adulto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/economia , Análise Custo-Benefício , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Evolução Clonal/genética , Idoso de 80 Anos ou mais , Hematopoese/genéticaRESUMO
Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency characterized by a defect in nicotinamide adenine dinucleotide phosphate oxidase required for phagocytosis. Hematopoietic stem cell (HSC) transplantation is currently the only curative treatment, but it is ladened with morbidities and mortality. Gene therapy is a promising treatment for CGD. However, if not properly designed, the gene therapy approach may not be successful. We engineered lentiviral vectors (LVs) carrying a universal promoter (EF1a) and two myeloid-specific promoters (miR223 and CD68) to drive the expression of green fluorescence protein (GFP) or CYBB, one of the key defective genes causing CGD. Tissue-specific LV expression was investigated in vitro and in a CGD mouse model. We compared GFP expression in both myeloid differentiated and undifferentiated HSCs. The CGD mice were transplanted with LV-modified mouse HSCs to investigate expression of CYBB and restoration of reactive oxygen species. The LV promoters were further compared under low and high-transgenic conditions to assess safety and therapeutic efficacy. A pneumonia disease model based on pathogenic Staphylococcus aureus challenge was established to assess the survival rate and body weight change. All three promoters demonstrated ectopic CYBB expression in vitro and in vivo. The EF1a promoter showed the highest expression of GFP or CYBB in transduced cells, including HSCs without cytotoxicity, whereas the LV-miR223 showed the highest transgene delivery efficiency with high myeloid specificity. Importantly, under low-transgenic condition, only the LV-EF1a-CYBB showed high antibacterial activity in vivo.
Assuntos
Doença Granulomatosa Crônica , Transplante de Células-Tronco Hematopoéticas , MicroRNAs , Camundongos , Animais , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Terapia Genética , Vetores Genéticos/genética , MicroRNAs/genéticaRESUMO
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.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Linfoma , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/terapia , Mobilização de Células-Tronco Hematopoéticas , Leucaférese , Análise Custo-Benefício , Transplante Autólogo , Compostos Heterocíclicos/metabolismo , Linfoma/terapia , Linfoma/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Fator Estimulador de Colônias de Granulócitos , Benzilaminas/metabolismoRESUMO
The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various "front end" strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.
Assuntos
Criopreservação , Células-Tronco Hematopoéticas/metabolismo , Modelos Biológicos , Estresse Fisiológico , Linhagem Celular , Sobrevivência Celular , Congelamento , HumanosRESUMO
BACKGROUND: Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs. METHODS AND RESULTS: HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs + hypoxia (MSC + Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres + hypoxia (Sph-MSC + Hyp), co-culturing with MSC spheres + cytokines (Sph-MSC + Cyto). After 10 days, total nucleated cell (TNC) and CD34+/CD38- cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38- cell count, CFU, and LTC-IC were higher in the Sph-MSC + Hyp and Sph-MSC + Cyto groups as compared with those of the MSC + Hyp group (P < 0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC + Cyto and Sph-MSC + Hyp groups. CONCLUSION: Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs.
Assuntos
Técnicas de Cocultura/métodos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Antígenos CD34/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Hipóxia Celular/fisiologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura/economia , Análise Custo-Benefício , Citocinas/metabolismo , Sangue Fetal/citologia , Humanos , Receptores CXCR4RESUMO
Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Calreticulina/antagonistas & inibidores , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Terapia de Alvo Molecular , Trombocitemia Essencial/terapia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/fisiologia , Éxons/genética , Mutação da Fase de Leitura , Técnicas de Introdução de Genes , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Esplenomegalia/etiologia , Trombocitemia Essencial/sangue , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , TranscriptomaRESUMO
Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/patologia , Animais , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Heterogeneidade Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Cadeias de Markov , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq , Análise de Célula ÚnicaRESUMO
BACKGROUND: Successful peripheral blood stem cell (PBSC) collection depends on optimal timing of apheresis, as usually determined by flow cytometry CD34-positive (+) cell count in peripheral blood (PB). Since this method is costly and labour-intensive, we evaluated the use of the Hematopoietic Progenitor Cell count programme on a Sysmex® XN haematologic analyser (XN-HPC) as a rapid and inexpensive alternative for predicting CD34+ cell count in PB samples. MATERIALS AND METHODS: Haematopoietic progenitor cell and CD34+ cell counts were compared using 273 PB samples collected from 78 healthy donors and 72 patients who underwent PBSC transplantation. We assessed the effectiveness of the XN-HPC in safely predicting pre-harvest CD34+ counts. The most efficient cut-off values of XNHPC were identified. We also evaluated the imprecision (coefficient of variation, CV) and functional sensitivity. RESULTS: Imprecision of the XN-HPC count was <6.3% on daily measurement of three levels of quality control material. Functional sensitivity was 8.9×106/L. A cut-off value of ≥62×106/L XN-HPC for multiple myeloma (MM) patients and ≥30×106/L for all other subjects had both 100% specificity and 100% positive predictive value for identifying samples with CD34+ cells ≥20×106/L. An XN-HPC threshold of <13×106/L identified preharvest CD34+ cell count <10×106/L with 100% sensitivity and 100% negative predictive value. DISCUSSION: The XN-HPC is a fast, easy and inexpensive test that can safely improve apheresis workflow thus possibly replacing other more expensive CD34 counts currently performed and promoting optimal timing of PBSC collection.
Assuntos
Antígenos CD34/metabolismo , Remoção de Componentes Sanguíneos/métodos , Células-Tronco Hematopoéticas/metabolismo , Transplante de Células-Tronco de Sangue Periférico/métodos , Células-Tronco de Sangue Periférico/metabolismo , Remoção de Componentes Sanguíneos/instrumentação , Contagem de Células , Células-Tronco Hematopoéticas/citologia , Humanos , Mieloma Múltiplo/metabolismo , Células-Tronco de Sangue Periférico/citologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Our ability to track cellular dynamics in humans over time in vivo has been limited. Here, we demonstrate how somatic mutations in mitochondrial DNA (mtDNA) can be used to longitudinally track the dynamic output of hematopoietic stem and progenitor cells in humans. Over the course of 3 years of blood sampling in a single individual, our analyses reveal somatic mtDNA sequence variation and evolution reminiscent of models of hematopoiesis established by genetic labeling approaches. Furthermore, we observe fluctuations in mutation heteroplasmy, coinciding with specific clinical events, such as infections, and further identify lineage-specific somatic mtDNA mutations in longitudinally sampled circulating blood cell subsets in individuals with leukemia. Collectively, these observations indicate the significant potential of using tracking of somatic mtDNA sequence variation as a broadly applicable approach to systematically assess hematopoietic clonal dynamics in human health and disease.
Assuntos
Evolução Clonal/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/genética , Mosaicismo , Mutação , DNA Mitocondrial , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
During cytokine- or chemotherapy-induced hematopoietic stem cell (HSC) mobilization, a highly proteolytic microenvironment can be observed in the bone marrow that has a strong influence on adhesive and chemotactic interactions of HSC with their niches. The increase of proteases during mobilization goes along with a decrease of endogenous protease inhibitors. Prominent members of the proteases involved in HSC mobilization belong to the families of matrix metalloproteinases and cathepsins, which are able to degrade chemokines/cytokines, extracellular matrix components, and membrane-bound adhesion receptors. To determine the functional activity of different proteolytic enzymes, zymographic analyses with different substrates and pH conditions can be employed. An involvement of cysteine cathepsins can be determined by the "active site labeling" technique using a modified inhibitor irreversibly binding to the active center of the enzymes. Intact or degraded chemokines and cytokines, which fall into the range between 1000 and 20,000 Da, can readily be detected by MALDI-TOF analysis. These three methods can help to detect proteolytic activities directly involved in the mobilization process.
Assuntos
Citocinas/química , Células-Tronco Hematopoéticas/citologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Domínio Catalítico , Catepsinas/química , Catepsinas/metabolismo , Linhagem Celular , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nicho de Células-TroncoRESUMO
Priming haematopoietic stem/progenitor cells (HSPCs) in vitro with specific chromatin modifying agents and cytokines under serum-free-conditions significantly enhances engraftable HSC numbers. We extend these studies by culturing human CD133+ HSPCs on nanofibre scaffolds to mimic the niche for 5-days with the HDAC inhibitor Scriptaid and cytokines. Scriptaid increases absolute Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSPC numbers, while concomitantly decreasing the Lin-CD38-CD34+CD45RA-CD90- subset. Hypothesising that Scriptaid plus cytokines expands the CD90+ subset without differentiation and upregulates CD90 on CD90- cells, we sorted, then cultured Lin-CD34+CD38-CD45RA-CD90- cells with Scriptaid and cytokines. Within 2-days and for at least 5-days, most CD90- cells became CD90+. There was no significant difference in the transcriptomic profile, by RNAsequencing, between cytokine-expanded and purified Lin-CD34+CD38-CD45RA-CD49f+CD90+ cells in the presence or absence of Scriptaid, suggesting that Scriptaid maintains stem cell gene expression programs despite expansion in HSC numbers. Supporting this, 50 genes were significantly differentially expressed between CD90+ and CD90- Lin-CD34+CD38-CD45RA-CD49f+ subsets in Scriptaid-cytokine- and cytokine only-expansion conditions. Thus, Scriptaid treatment of CD133+ cells may be a useful approach to expanding the absolute number of CD90+ HSC, without losing their stem cell characteristics, both through direct effects on HSC and potentially also conversion of their immediate CD90- progeny into CD90+ HSC.
Assuntos
Doenças Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidroxilaminas/farmacologia , Quinolinas/farmacologia , Transcriptoma/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Meios de Cultura Livres de Soro , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , RNA-Seq , Análise de Célula Única , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismoRESUMO
Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation.
Assuntos
Diferenciação Celular , Eritropoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Animais , Biomarcadores , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Imunofenotipagem , Camundongos , Modelos BiológicosRESUMO
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
Assuntos
Hemoglobina Fetal/genética , Filtração/métodos , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Ribonucleoproteínas/genética , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Eletroporação/métodos , Feminino , Hemoglobina Fetal/metabolismo , Filtração/economia , Filtração/instrumentação , Terapia Genética/economia , Terapia Genética/instrumentação , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Membranas Artificiais , Camundongos , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Transplante AutólogoRESUMO
In umbilical cord blood transplantation (UCBT), the number of cluster of differentiation (CD)34+ cells and colonyforming units (CFUs) in the cord blood (CB) graft positively correlate with patient survival. Therefore, these parameters are currently used for quality assessment of the cryopreserved CB cells in the attached segment that is considered representative of the CB in the main bag prior to UCBT. Since aldehyde dehydrogenase (ALDH) activity is high in hematopoietic stem cells, the number of ALDHbright (ALDHbr) cells was examined in comparison with the number of CD34+ cells and CFUs for the quality assessment of CB units. In the cryopreserved main bag, the number of ALDHbr cells in the CB unit exhibited positive correlation with the number of CD34+ cells, and with CFUgranulocytes/macrophages and total CFU counts. Furthermore, the concentration of ALDHbr cells in the cryopreserved attached segment was not significantly different compared to that of the main bag, suggesting that the attached segment is representative of the main bag. In conclusion, the present study suggested that ALDHbr cell counts in the cryopreserved attached segments may serve as a quality assessment indicator for CB units prior to UCBT.
Assuntos
Aldeído Desidrogenase/genética , Diferenciação Celular/genética , Criopreservação , Sangue Fetal/enzimologia , Antígenos CD34/genética , Linhagem da Célula/genética , Ensaio de Unidades Formadoras de Colônias , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/genética , Granulócitos/citologia , Granulócitos/enzimologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/enzimologia , Células-Tronco/metabolismoRESUMO
Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.
Assuntos
Quimiotaxia/efeitos dos fármacos , Filgrastim/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Neutrófilos/metabolismo , Doadores de Tecidos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fagocitose/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacosAssuntos
Mobilização de Células-Tronco Hematopoéticas , Linfoma/terapia , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico , Benzilaminas , Contagem de Células , Análise Custo-Benefício , Ciclamos , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/administração & dosagem , Humanos , Mieloma Múltiplo/diagnóstico , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Transplante de Células-Tronco de Sangue Periférico/métodos , Resultado do TratamentoRESUMO
BACKGROUND: Cryopreserved hematopoietic progenitor cell (HPC) grafts are widely infused to patients with malignant and nonmalignant conditions. Despite reduction of immediate side effects linked to dimethyl sulfoxide (DMSO), cell debris-containing grafts and comparable hematopoietic engraftment between washed and unwashed cryopreserved products, bedside infusion of thawed HPC grafts is still preferred. Introduction of automated devices is important for standardization and consistency of graft manipulation. Additionally, these techniques are likely to be useful for the delivery of innovative cell-based medicinal products that are currently under development. METHODS: In this study, we evaluated three consecutive versions of the Lovo device (Fresenius Kabi) for automated washing of thawed HPC products. A total of 42 HPC products intended for destruction were used. Measured outcomes included viable CD34+ cell recovery, viability, total processing time and post-washing stability. RESULTS: Preliminary data using the prototype Lovo 0.0 to process a single HPC unit showed better recovery and viability of CD34+ cells using a two-cycle than a three-cycle wash, with >95% DMSO elimination. The Lovo 1.0 performed equally well. When simultaneously processing two HPC units, the upgraded Lovo 2.0 device demonstrated comparable CD34+ recovery, DMSO elimination efficiencies and time-saving capacity. Furthermore, washed cell products were stable for 4 hours at room temperature. DISCUSSION: Lovo device satisfies clinically relevant issues: ability to efficiently wash two HPC units simultaneously and compatibility with transport to nearby transplantation centers.
Assuntos
Criopreservação/instrumentação , Dimetil Sulfóxido/isolamento & purificação , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/metabolismo , Criopreservação/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , TemperaturaRESUMO
The toxic effects of combined cisplatin/docetaxel therapy cycles on erythroid and granulocytic hematopoietic lineages as well as their intercycle recovery were examined in patients with stage III-IV non-small-cell lung carcinoma. Responsiveness of the blood system to this therapy remained at a high level. Combined therapy pronouncedly activated the key elements of the erythroid and granulocytic hematopoietic lineages leading to accumulation of immature and mature myelokaryocytes in the bone marrow, enlargement of the medullary pool of mature neutrophils, and increase in the count of medullary erythroid and granulocytic precursor cells under conditions of their accelerated maturation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Granulócitos/metabolismo , Neoplasias Pulmonares/metabolismo , Antraciclinas/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cisplatino/farmacologia , Dissacarídeos/farmacologia , Docetaxel , Doxorrubicina/farmacologia , Eritropoese/efeitos dos fármacos , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrolídeos/farmacologia , Compostos de Nitrosoureia/farmacologia , Compostos Organoplatínicos/farmacologia , Taxoides/farmacologiaRESUMO
OBJECTIVE: Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method. MATERIALS AND METHODS: In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units. RESULTS: Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant. CONCLUSION: In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.
Assuntos
Aldeído Desidrogenase/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/análise , Células Cultivadas , Ensaios Enzimáticos/economia , Ensaios Enzimáticos/métodos , Sangue Fetal/metabolismo , Citometria de Fluxo/economia , Granulócitos/citologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Fatores de TempoRESUMO
Clinical gene therapy has been practiced for more than a quarter century and the first products are finally gaining regulatory/marketing approval. As of 2016, there have been 11 haemophilia gene therapy clinical trials of which six are currently open. Each of the ongoing phase 1/2 trials is testing a variation of a liver-directed adeno-associated viral (AAV) vector encoding either factor VIII (FVIII) or factor IX (FIX) . As summarized herein, the clinical results to date have been mixed with some perceived success and a clear recognition of the immune response to AAV as an obstacle to therapeutic success. We also attempt to highlight promising late-stage preclinical activities for AAV-FVIII where, due to inherent challenges with manufacture, delivery and transgene product biosynthesis, more technological development has been necessary to achieve results comparable to what has been observed previously for AAV-FIX. Finally, we describe the development of a stem cell-based lentiviral vector gene therapy product that has the potential to provide lifelong production of FVIII and provide a functional 'cure' for haemophilia A. Integral to this program has been the incorporation of a blood cell-specific gene expression element driving the production of a bioengineered FVIII designed for optimal efficiency. As clearly outlined herein, haemophilia remains at the forefront of the rapidly advancing clinical gene therapy field where there exists a shared expectation that transformational advances are on the horizon.