RESUMEN
CONTEXT: Recently, umbilical cord blood (UCB) has been recognized as a suitable potential source of hematopoietic stem/progenitor cells (HSPCs) for transplantation. Lengthy thrombocytopenia after UCB transplantation is a major problem because of insufficient megakaryocyte (Mk) progenitors, which results in delayed platelet recovery. Frequent allogenic platelet transfusion leads to resistance to platelet units and higher risk of transmission of pathogenic agent. OBJECTIVE: Ex vivo expansion of HSPCs and their differentiation to Mk progenitors on aminated PES nanofiber could lead to faster platelet recovery after UCB transplantation. MATERIALS AND METHODS: CD34 cells were positively enriched using the MidiMACS system. CD34(+) cells were seeded onto conventional culture and aminated PES scaffold. The proliferation of CD34(+) cells, and also their differentiation into Mk progenitors, were evaluated. We used the flow cytometric method for analyzing CD41 and CD61 markers and real-time PCR for the expression level of transcription factors, as NF-E2 and GATA-1. RESULTS: This study indicated increased CD34(+) cell population in aminated PES compared to the conventional system. After differentiation, the amount of CD41/CD61-expressing cells and the quantity of NF-E2 expression level increased in the aminated PES versus the 2-D system. The quantity of GATA-1 expression level was reduced on CD41/CD61(+) cells compared to CD34(+) cells, with no difference between the aminated PES and the conventional system. DISCUSSION: Aminated PES nanofiber could have more effect on the proliferation of CD34(+) cells and Mk differentiation than the conventional culture. CONCLUSION: Injection of the expanded cells and differentiated Mk progenitors, along with the transplantation of UCB stem cells might accelerate recovery of platelets and decrease the period of thrombocytopenia after transplantation.
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Antígenos CD34 , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Sangre Fetal , Células Madre Hematopoyéticas , Megacariocitos , Nanofibras/química , Andamios del Tejido/química , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Megacariocitos/citología , Megacariocitos/metabolismoRESUMEN
BACKGROUND: Dendritic cells (DCs) play a critical role in the immune response and are a candidate for immunotherapy in cancer. Since gibbon ape leukemia virus (GALV) transduction of CD34+ cells is reasonably efficacious, we assessed the efficacy of GALV transduction of CD34+ derived DCs as a possible approach to creating genetically modified DCs for immunotherapy. METHODS: Peripheral blood CD34+ cells were transduced with retroviruses obtained from the PG13/LN C8 cell line, with the neomycin gene as a marker gene. After prestimulation of hematopoietic cells for 24 hours with 10 ng/mL interleukin (IL)-3, 10 ng/mL IL-6, 100 ng/mL stem cell factor, 100 ng/mL granulocyte-macrophage colony stimulating factor and 8 Amicrog/mL protamine sulfate, the cells were cultured in a transforming media prior to differentiating into DCs by GM-CSF, TNF-a and IL-4. Immunophenotyping analyses for confirmation of the generated DCs, colony formation assay and PCR were done for the expression of neomycin gene in the transduced cells. RESULTS: Titration of viral vectors indicated a transduction efficiency of 1×10(5) CFU/mL. Transduction efficiency for the CD34+ cells transformed to DCs was 45% and 38% before and after DC differentiation, respectively. Additionally, a mean (SEM) of 26.9% (11.4%) and 41.4% (11.8%) of the genetically modified DCs were positive for CD86+ HLA-DR and CD1a+CD14, respectively CONCLUSION: This study showed that the majority of transduced CD34+ cells were successfully differentiated into cells identical to DCs according to morphology and immunophenotyping features, which could be a potential application in immunotherapy.
Asunto(s)
Antígenos CD34 , Células Dendríticas/metabolismo , Inmunoterapia/métodos , Virus de la Leucemia del Gibón/genética , Transducción Genética/métodos , Células Sanguíneas , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Dendríticas/citología , Células Madre Hematopoyéticas/citología , HumanosRESUMEN
BACKGROUND: Recognition of HLA alleles is useful in transplantation and in anthropological and disease studies. Acute lymphoblastic leukemia (ALL) is the most common blood cancer. It is now generally agreed that both genetic and environmental factors play an interactive role in the development of ALL disease. It is unknown whether there exists a restriction to certain MHC genotypes in leukemia like ALL. METHODS: Genetic construct of HLA DRB1 was studied in Iranian normal populations and in patients with acute lymphoblastic leukemia using PCR-SSP method. RESULTS: It was shown that the most common allele in DRB1 locus in normal population was DRB1*11 (20%), whereas DRB1*09 was the least frequent allele (0.9%). Additionally, this study presented the results of HLA-DRB1 typing in 106 ALL patients and compared them with normal individuals. Comparison of the results between the normal population and the patient group revealed that there was allelic association between the DRB1*13 and the disease. Results showed that the difference between the frequencies of DRB1*13 in patients and normal individuals was significant (p=0.04), but there was a moderate difference among the frequencies of DRB1*04, *07, and *09 in childhood (0-15 years) ALL. The frequencies of DRB1*13, *04, and *07 in patients were 2.5, 16, 4.5% and, in normal individuals, were 11.4, 10, and 8.3%, respectively. CONCLUSIONS: It should be concluded that DRB1*13, which showed a decrease in patients, should be protective against acute lymphoblastic leukemia (ALL), whereas DRB1*04, which was moderately increased in patients, could be considered a susceptible allele for childhood ALL.