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AIM:Toinvestigatetheeffectofnicotinicacidamide(NAA)ontheinfusiondamageofhuman umbilical cord mesenchymal stem cells ( hUC-MSCs) under the condition of instant blood-mediated inflammatory reaction ( IBMIR) .METHODS:Normal peripheral blood without anticoagulant at volume of 2.7 mL was mixed with 0.3 mL phys-iological saline (as blank group), CFSE labeled hUC-MSCs (1 ×106 cells in 0.3 mL as MSC group) and CFSE labeled hUC-MSCs (1 ×106 cells in 0.3 mL) preprocessed with NAA at concentration of 10 mmol/L for 24 h ( as MSC+NAA group) , respectively.The mixture was immediately injected into the improved Chandler Loop model, placed in 37℃water bath, and then started the peristaltic pump at the speed of 20 mL/min for 1 h.The number of CFSE labeled hUC-MSCs, platelets, white blood cells were counted and the concentration of complement C3a was measured before and after cycling, respectively.RESULTS: After 1 h circulation, the platelet dissipation rate were ( 29.96 ±10.88 )% in blank group, (77.76 ±19.29)% in MSC group all and (50.13 ±18.10)% in MSC +NAA group; and the leukocyte counts were (37.82 ±13.81)%in blank group, (64.57 ±17.08)% in MSC group and (41.52 ±17.26)% in MSC+NAA group. Compared with blank group, the differences of the dissipation rates in MSC group and MSC+NAA group all had statistical significance.The hUC-MSCs relative survival rate in MSC+NAA group was higher than that in MSC group.C3a concentra-tions in blank group, MSC group and MSC+NAA group were (206.27 ±58.10), (230.47 ±39.61) and (208.37 ± 40.66) μg/L, respectively.CONCLUSION:Co-circulating the mixture of hUC-MSCs with normal peripheral blood with-out anticoagulant in the improved Chandler Loop for 1 h depletes a large number of hUC-MSCs and blood components, and increases C3a, suggesting that this model can induce IBMIR.NAA has a protective effect on the hUC-MSCs in the infusion damage by inhibiting IBMIR, reducing the wastage of the blood components and enhancing the survival rate of the hUC-MSCs.
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AIM:To investigate the influence of continuous subculturing of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the mRNA expression of all 23 family members of NOD-like receptors (NLRs), and to search for the way of improving the subculture quality of hUC-MSCs and increasing the quantity and safety in the experimental and clinical application .METHODS:Neonatal umbilical cord was collected to isolate and purify the hUC-MSCs with the colla-genase II digestion and adherence screening methods .These cells were continuously subcultured .The hUC-MSCs at pas-sage 3 and passage 28 were identified by flow cytometry and induced differentiation .The mRNA expression of NLRs in the passage 3 and passage 28 hUC-MSCs was detected by RT-qPCR.RESULTS: The cell phenotypes of both passage 3 and passage 28 hUC-MSCs were CD29 +/CD44 +/CD105 +/CD31 -/CD34 -/CD40 -/CD45 -/CD106 -/HLA-DR-, and both of the cells were induced into osteoblasts and adipocytes , which were conformed to the criteria of International Society for Cellular Therapy to define MSCs .All the NLR family members were expressed in passage 3 hUC-MSCs.NOD1, NLRC4, NLRC5, NLRP1, NLRP3, NLRP10, NAIP, NLRX1 and APAF1 at mRNA levels were highly expressed , and the rest were lowly expressed.When hUC-MSCs were subcultured to passage 28, NLRP10 mRNA was increased, NLRC5 mRNA and NLRX1 mRNA were hardly changed , and all of the rest members were decreased .The difference of NLRP1 mRNA expres-sion between passage 3 and passage 28 hUC-MSCs was observed with statistical significance (P<0.05).CONCLU-SION:The effects of subculturing on the expression of NLR family in hUC-MSCs are pleiotropic .It requires further investi-gation to confirm whether these effects are related to the proliferation , differentiation and immunomodulation of MSCs .
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To investigate the expression of the cholinesterase-related cell division controller (CHED) gene in the patients with myelodysplastic syndrome (MDS), CHED gene expression was assayed by RT-PCR and its relative expression rate (RER) was determined by the semi-quantitative RT-PCR analysis in peripheral blood mononuclear cells from 21 patients with MDS, 12 normal individuals served as controls. Results showed that RER of CHED in the patients (2.69 +/- 0.76) was significantly higher than that in controls (1.12 +/- 0.51, P < 0.01), the RER out of 85.7% of the patients was higher than the mean value of the controls, in which three patients developed into acute leukemia; the RER out of 61.9% of the patients was higher than the upper limit of the mean value of the controls; three patients whose RER was lower than the mean value of the controls did not developed into leukemia. These findings suggested that the expression of CHED gene in patients with MDS was significantly higher than in controls.
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AIM: To explore transdifferentiation potential of Sca-1 + cells from murine fetal liver. METHODS: 2?10 3 of Sca-1 + cells from male murine fetal liver were transfused into female mouse irradiated lethally with ? ray from 60 Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS: The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA+/CD - 45 F - 4/80 and NueN +/CD -45 F - 4/80, respectively. CONCLUSION: The evidence is provided for Sca-1 + cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.
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liver.Combined informational analysis showed that the presenting frequency of the cells containing Y chromosome was consistent with the irradiative sensitivity of the organ.CONCLUSION: These findings suggest that the capability of differentiation of Sca-1 positive cells from murine fetal liver was potentially connected to the extent of damage in these organs when transferred in vivo.
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AIM: To study the effect of acute renal failure (ARF) on the differentiated frequency of Sca-1+ cells from murine fetal liver in irradiated mice. METHODS: The Sca-1+ cells from murine fetal liver were isolated with magnetic cell sorting (MACS) technique, the sex of which was identified by PCR. The 2?104 Sca-1+ cells were transplanted into a lethally irradiated ([ 60Co], 8 Gy) inbred female mouse. After 8 weeks, these recipient mice were divided to A, B, and C groups at random (A group: irradiated; B group: ARF; C group: ARF and Sca-1+). The mice in B and C groups were induced to ARF with 50% (V/V) glycerin (11.6 mL/kg). 72 hours later, the mice in C group were injected with the fresh prepared Sca-1+ cells again. 8 weeks later, mice were sacrificed, and their kidneys were taken out, fixed and slices were prepared. Fluorescence in situ hybridization (FISH) of renal slices was performed and the pictures of them were taken and analyzed. RESULTS: The cells containing Y chromosome were found in renal slices from the mice in A, B and C groups, which located in epithelial cells of renal tubules, interstitium, glomeruli, and glomerular margin and increased gradually. The double and encircle zone of Y chromosome cells were found in the slices from the mice in B and C groups separately, which was consist of new renal tubules. The differentiation frequency of Sca-1+ cells in kidney in A, B and C groups were (1.65?0.18)%, (8.58?1.34)% and (18.13?1.91)%, respectively, which showed significant difference between former group and later group (P
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Objective:To explore potential differentiation from antigen-1 positive stem cells (Sca-1 +cells) in murine fetal liver tissue into cardiomyocytes in vivo. Methods: Sca-1 + cells from the murine fetal liver of 14.5 d were separated by Magnetic cell sorting (MACS).The sequence of the sex determination region of the Y chromosome was determined by PCR.Sca-1 + cells (2?10 3) of male mice were transfused to female mice receiving 8-12 weeks irradiation at a lethal dose of ?-ray(10 Gy) from a 60Co source.Two months later,the mice were killed and the hearts were removed to make slices. Fluorescence in situ hybridisation (FISH) tests with a Y chromosome probe were used to detect sex of the cells.Immunohistochemistry with antibodies of desmin,Flt-1,white blood cell common antigen CD45 and macrophage F 4/80 were used to detect tissue characteristics of cardiomyocytes. Results: Cells with Y chromosome were found in the cardiomyocytes of female mice transplanted with Sca-1 + cells,and showed phenotypic characteristics of Desmin +/Flt-1-/CD45-/F- 4/80 at the same time.Conclusion: Sca-1 + cells from the murine fetal liver are mostly hematopoietic stem cells,having the potential of differentiating into cardiomyocytes.