Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

Combination of epidural electrical stimulation with ex vivo triple gene therapy for spinal cord injury:a proof of principle study

Despite emerging contemporary biotechnological methods such as gene-and stem cell-based therapy,there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury.Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules,including vascular endothelial growth factor(VEGF),glial cell-line derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs.To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury,in this study,rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165,GDNF,NCAM1 at 4 hours after spinal cord injury.Three days after injury,epidural stimulations were given simultaneously above the lesion site at C5(to stimulate the cervical network related to forelimb functions)and below the lesion site at L2(to activate the central pattern generators)every other day for 4 weeks.Rats subjected to the combined treatment showed a limited functional improvement of the knee joint,high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury.However,beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters,and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy.This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy(VEGF,GDNF and NCAM)for treatment of spinal cord injury in rat models.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.2.20.02.18)on February 20,2018.

Functionality of a bicistronic construction containing HEXA and HEXB genes encoding β-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.