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One of the hallmarks of cancer is the expansion and accumulation of highly immunosuppressive myeloid cells known as myeloid-derived suppressor cells (MDSCs). To study MDSCs biology, differentiation from hematopoietic progenitor cells (HPC) is an useful tool to elucidate the biological and biochemical mechanisms associated with acquisition of immune suppressive activity and expansion in cancer. Although this is one of the protocols performed to study immune suppressive myeloid cells, differentiation of MDSCs from HPC is a method that allows to modify conditions of the supernatants used. In this protocol, we outline the process of differentiating HPCs into MDSCs in vitro using tumor explant supernatants to recapitulate the tumor microenvironment.
Assuntos
Células Supressoras Mieloides , Neoplasias , Animais , Camundongos , Células-Tronco Hematopoéticas , Diferenciação Celular , Microambiente TumoralRESUMO
BACKGROUND: Autologous stem cell transplantation is the standard procedure for multiple myeloma and the grafts are usually cryopreserved. Previous studies reported advantages in the use of fresh peripheral blood stem cells (PBSC) autotransplantation compared to cryopreservation of the grafts. This study compared the transplant-related outcomes of two graft preservation methods: fresh storage (4°C/72 h) and cryopreservation (-80°C). STUDY DESIGN AND METHODS: We performed an analysis of 45 patients with multiple myeloma under autotransplantation (17 fresh and 28 cryopreserved) from 2017 to 2021. Fresh PBSC were maintained in the refrigerator for three days in a concentration up to 300 × 103 TNC/µL. Cryopreserved PBSC were concentrated by plasma reduction after centrifugation (950 g/10 min/4°C) and an equal volume of cryoprotection solution was added for a final concentration of 300 × 103 TNC/µL, 5% DMSO, 6% hydroxyethyl starch, and 3% human albumin. RESULTS: Neutrophil engraftment was significantly faster with fresh PBSCs (10 vs. 11.5 days, p = 0.045). Adverse effects were more common in cryopreserved PBSC transplantation (75% vs. 35.3% patients; p = 0.013). Post transplantation hospital stay was 20 and 22 days for fresh and cryopreserved PBSCs respectively (p = 0.091). There was no difference in platelet engraftment time (10.5 days for both; p = 0.133), number of antibiotics used after transplantation (3 for fresh and 2.5 for cryopreserved; p = 0.828), days of antibiotic use after transplantation (12.2 days for fresh and 13.3 days for cryopreserved, p = 0.579), and overall survival (p = 0.736). CONCLUSION: The infusion of fresh PBSC refrigerated for up to three days is effective and safe for autologous transplantation in patients with multiple myeloma, which is a useful alternative to cryopreserved PBSC.
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Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Células-Tronco de Sangue Periférico , Antibacterianos , Criopreservação/métodos , Dimetil Sulfóxido , Humanos , Mieloma Múltiplo/terapia , Albumina Sérica Humana , Amido , Transplante Autólogo/métodosRESUMO
OBJECTIVES: This study investigated whether intramyocardial bone marrow-derived hematopoietic progenitor cells (BMCs) increase coronary flow reserve (CFR) in ischemic myocardial regions where direct revascularization was unsuitable. BACKGROUND: Patients with diffuse coronary artery disease frequently undergo incomplete myocardial revascularization, which increases their risk for future adverse cardiovascular outcomes. The residual regional ischemia related to both untreated epicardial lesions and small vessel disease usually contributes to the disease burden. METHODS: The MiHeart/IHD study randomized patients with diffuse coronary artery disease undergoing incomplete coronary artery bypass grafting to receive BMCs or placebo in ischemic myocardial regions. After the procedure, 78 patients underwent cardiovascular magnetic resonance (CMR) at 1, 6, and 12 months and were included in this cardiac magnetic resonance substudy with perfusion quantification. Segments were classified as target (injected), adjacent (surrounding the injection site), and remote from injection site. RESULTS: Of 1,248 segments, 269 were target (22%), 397 (32%) adjacent, and 582 (46%) remote. The target had significantly lower CFR at baseline (1.40 ± 0.79 vs 1.64 ± 0.89 in adjacent and 1.79 ± 0.79 in remote; both P < 0.05). BMCs significantly increased CFR in target and adjacent segments at 6 and 12 months compared with placebo. In target regions, there was a progressive treatment effect (27.1% at 6 months, P = 0.037, 42.2% at 12 months, P = 0.001). In the adjacent segments, CFR increased by 21.8% (P = 0.023) at 6 months, which persisted until 12 months (22.6%; P = 0.022). Remote segments in both the BMC and placebo groups experienced similar improvements in CFR (not significant at 12 months compared with baseline). CONCLUSIONS: BMCs, injected in severely ischemic regions unsuitable for direct revascularization, led to the largest CFR improvements, which progressed up to 12 months, compared with smaller but persistent CFR changes in adjacent and no improvement in remote segments.
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Doença da Artéria Coronariana , Reserva Fracionada de Fluxo Miocárdico , Células da Medula Óssea , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/terapia , Humanos , Espectroscopia de Ressonância Magnética , Miocárdio , Perfusão , Valor Preditivo dos TestesRESUMO
Taro (Colocasia esculenta) corm is traditionally consumed as a medicinal plant to stimulate immune responses and restore a health status. Tarin, a taro lectin, is considered responsible for the immunomodulatory effects of taro. In the present study, in order to investigate the effects of tarin on bone marrow hematopoietic population, murine cells were stimulated with tarin combined with a highly enriched conditioned medium containing either IL-3 or GM-CSF. Cells challenged with tarin proliferated in a dose-dependent manner, evidenced by the increase in cell density and number of clusters and colonies. Tarin exhibited a cytokine-mimetic effect similar to IL-3 and GM-CSF, increasing granulocytic cell lineage percentages, demonstrated by an increase in the relative percentage of Gr-1+ cells. Tarin does not increase lymphocytic lineages, but phenotyping revealed that the relative percentage of CD3+ cells was increased with a concomitant decrease in CD19+ and IL-7Rα+ cells. Most bone marrow cells were stained with tarin-FITC, indicating non-selective tarin binding, a phenomenon that must still be elucidated. In conclusion, taro corms contain an immunomodulatory lectin able to boost the immune system by promoting myeloid and lymphoid hematopoietic progenitor cell proliferation and differentiation.
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BACKGROUND: The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS: This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS: The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION: The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Laboratórios , Células-Tronco de Sangue Periférico/fisiologia , Prática Profissional , Adulto , Idoso , Sobrevivência Celular , Estudos de Coortes , Criopreservação/normas , Feminino , Congelamento/efeitos adversos , Mobilização de Células-Tronco Hematopoéticas/normas , Transplante de Células-Tronco Hematopoéticas/normas , Células-Tronco Hematopoéticas/citologia , Humanos , Laboratórios/normas , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico , Células-Tronco de Sangue Periférico/citologia , Prática Profissional/normas , Estudos Retrospectivos , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Transplante Autólogo , Resultado do TratamentoRESUMO
BACKGROUND: Cell adhesion molecules (CAMs) expressed on hematopoietic progenitor cells (HPCs), endothelial cells, and stromal cells play a pivotal role in the mobilization of CD34+ cells. Herein, we conducted a non-randomized peripheral blood stem cell (PBSC) mobilization study aimed to compare the potential differences in the expressions of several CAMs and chemokines on CD34+ cells obtained from bone marrow aspirate before and after HPC mobilization from patients with hematologic malignancies and healthy donors. METHODS: Three-color cytofluorometric analysis was used to compare the expressions of CAMs and chemokines in the bone marrow before and after mobilization. RESULTS: For all studied groups, CAM expression among those with good and poor yields of CD34+ cells was significantly correlated with VCAM-1 (P=0.007), CD44 (P=0.027), and VLA-4 (P=0.014) expressions. VCAM-1 (P=0.001), FLT-3 (P=0.001), CD44 (P=0.011), VLA-4 (P=0.001), and LFA-1 (P=0.001) expressions were higher before HPC mobilization than after HPC mobilization. By contrast, the expression of CXCR4 significantly varied before and after mobilization only among those with successful PBSC mobilization (P=0.002). CONCLUSION: We attempted to identify particular aspects of CAMs involved in CD34+ cell mobilization, which is a highly complex mechanism that involves adhesion molecules and matrix metalloproteases. The mechanism by which CD34+ cell mobilization is activated through proteolytic enzymes is not fully understood. We believe that CXCR4, VLA-4, CD44, and VCAM-1 are the most important molecules implicated in HPC mobilization, particularly because they show a correlation with the yield of CD34+ cells collected via large volume leukapheresis.
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Evidence presented over the last few years indicates that the hematopoietic stem cell (HSC) compartment comprises not just one but a number of different cell populations. Based on HSCs' proliferation and engraftment potential, it has been suggested that there are two classes of HSC, with long- and short-term engraftment potential. HSC heterogeneity seems to involve differentiation capacities as well, since it has been shown that some HSC clones are able to give rise to both myeloid and lymphoid progeny, whereas others are lymphoid deficient. It has been recognized that HSC function depends on intrinsic cell regulators, which are modulated by external signals. Among the former, we can include transcription factors and non-coding RNAs as well as epigenetic modifiers. Among the latter, cytokines and extracellular matrix molecules have been implicated. Understanding the elements and mechanisms that regulate HSC populations is of significant relevance both in biological and in clinical terms, and research in this area still has to face several complex and exciting challenges.
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O vírus da imunodeficiência humana causa infecção persistente apesar da terapia com antirretrovirais. Isso leva a crer que existem outros reservatórios virais além dos já conhecidos. Estudos recentes mostram que o vírus pode invadir as células progenitoras hematopoiéticas, causar infecção ativa assim como latente e morte celular. Essas célulasinfectadas latentemente podem persistir por vários anos e contribuir para a viremia residual e a persistência viral. A morte celular causada pela invasão e replicação do vírus pode estar associada a anormalidades hematopoiéticas. Palavras-chave. Células progenitoras hematopoiéticas; vírus da imunodeficiência adquirida; reservatórios virais
Human immunodeficiency virus (HIV) causes persistent infection despite antiretroviral therapy. This suggests the existence of other viral reservoirs beyond those already known. Recent studies show that HIV can invade hematopoietic progenitor cells, generating active infection as well as latent infection and cell death. The latently infected HPCs may persist for several years and contribute to residual viremia and viral persistence. Cell death caused by the invasion and replication of the virus may be associated with hematopoietic abnormalities.
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Los productos de células progenitoras hematopoyéticas (CPH) representan una fuente potencial de infección en pacientes inmunosuprimidos que reciben infusión de CPH como parte de su tratamiento. La probabilidad de contaminación de cada producto difiere según la técnica de colecta y el procesamiento aplicado. En este trabajo hemos realizado un análisis retrospectivo de los resultados de cultivos microbiológicos de 1707 productos de CPH obtenidos de sus tres fuentes (médula ósea, sangre periférica y sangre de cordón umbilical) con el objetivo de determinar la proporción de unidades contaminadas. Además, fueron comparadas las distintas técnicas de colecta y las diferentes manipulaciones a las que fueron sometidos los productos. Por otro lado, se analizaron las posibles fuentes de contaminación según el microorganismo identificado y se evaluó la supervivencia de los mismos luego del descongelamiento. La prevalencia de productos de CPH con cultivos microbiológicos positivos reportados en este estudio (5,2%) se corresponde con lo descripto en la literatura. No encontramos diferencias significativas al comparar los productos según la fuente de la cual provenían las CPH. Tampoco hubo diferencias según los procedimientos aplicados a cada unidad. Los microorganismos aislados en los productos de CPH fueron los esperados de acuerdo a la fuente de la cual provenían las células. Pudo comprobarse que algunos de ellos son capaces de sobrevivir a los procesos de criopreservación y descongelamiento. La estricta adhesión a las normas de buenas prácticas de manufactura y buenas prácticas tisulares es un requisito para minimizar los riesgos de introducir microorganismos contaminantes. Disponer de un producto de CPH seguro es fundamental para el éxito de un trasplante.
Hematopoietic stem cell products (HSCP) represent a potential source of infection for immunosuprressed patients that receive HSCP infusion as part of their treatment. The probability of contamination of a HSCP product depends on the collection technique as well as the processing performed. In this work we have carried out a retrospective analysis of the results of 1707 microbiological cultures of HSCP products obtained from three different sources: bone marrow, peripheral blood and umbilical cord blood. We determined the proportion of HSCP units that were contaminated by microorganisms. Furthermore we compared the results obtained with different collection techniques and with the distinct manipulations that were used for processing. Moreover we analysed the possible sources of contamination related to the microorganisms identified and we evaluated the survival of them after thawing. The proportion of HSCP products with positive microbiological cultures obtained in this study (5,2%) correlates with that reported by other authors. We have not found significant differences between the results achieved with HSCP products from different sources. There were neither differences depending on the procedures applied. The isolated microorganisms from the HSCP products were the expected in accordance with the source of the cells. It could be demonstrated that some of them were capable of surviving the cryopreservation and thawing processes. Adherence to good manufacture practices and good tissue practices regulations is critical for minimizing the risks of introducing contaminant microorganisms. A safe HSCP product is essential for the success of a transplant.