Risk factors of hemorrhagic cystitis after allo-HSCT and therapeutic effects of early transfusion of umbilical cord mesenchymal stem cells / 中华器官移植杂志

Objective:To explore the clinical efficacy and risk factors of umbilical cord mesenchymal stem cells (UCMSCs) infusion at an early stage (i.e.gross hematuria) for hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:The relevant clinical data were retrospectively reviewed for 300 patients undergoing allo-HSCT from January 2016 to July 2021.According to the presence or absence of HC, they were assigned into two groups of HC (n=89) and non-HC (control, n=211). According to whether or not receiving an infusion of UCMSCs, 51 patients of HC degree Ⅱ-Ⅳ were divided into two groups of UCMSC infusion and non-infusion.The risk factors of HC after allo-HSCT were analyzed by χ2 test.Logistic regression was employed for multivariate analysis of P<0.05.Mann-Whitney U test was utilized for statistically analyzing the duration of gross hematuria and urinary tract irritation symptoms and evaluating the clinical efficacy of UCMSCs infusion for HC. Results:Among them, 89 (29.67%) developed HC post-allo-HSCT.Clinical grades were Ⅰ (n=38, 42.70%), Ⅱ (n=36, 40.45%), Ⅲ (n=13, 14.61%) and Ⅳ (n=2, 2.25%). The median occurrence time was 29 (21.5-35.0) days post-allo-HSCT.In univariate analysis, age ≤30 years, haploid transplantation, antithymocyte globulin (ATG), acute graft-versus-host disease (aGVHD), CMV-DNA positive pretreatment significantly boosted the risk of HC ( P<0.05). In multivariate analysis, aGVHD was an independent risk factor for HC ( OR=10.281, 95% CI: 1.606-65.813, P=0.014). Among 89 HC patients, 38 grade Ⅰ patients were complete remission(CR). Among 51 patients of grade Ⅱ-Ⅳ HC, the outcomes were CR (n=48) and non-remission(NR)(n=3). And 24/51 of them received UCMSCs plus conventional treatment.The duration of gross hematuria was shorter in UCMSCs infusion group than that in UCMSCs non-infusion group [12(9-17) vs 17(12.0-26.5) day] and the difference was statistically significant ( P=0.045). And the duration of urinary tract irritation symptoms was shorter in UCMSCs infusion group than that in UCMSCs non-infusion group [18(11-30) vs 27(18.0-35.5) days] and the difference was statistically significant ( P=0.048). Conclusions:Indicated for post-ALLO-HSCT HC, infusion of UCMSCs may significantly shorten the course of disease.Age ≤30 years, haploid transplantation and preconditioning with positive ATG, aGVHD and CMV-DNA may boost the risks of HC post-allo-HSCT.And aGVHD is an independent risk factor for HC after allo-HSCT.

Exosomes secreted from human umbilical cord mesenchymal stem cells promote pancreatic ductal adenocarcinoma growth by transferring miRNAs / 中华肿瘤杂志

Objective: To observe the effects of exosomes derived from human umbilical cord mesenchymal stem cells on the proliferation and invasion of pancreatic cancer cells, and to analyze the contents of exosomes and explore the mechanisms affecting pancreatic cancer cells. Methods: Exosomes extracted from human umbilical cord mesenchymal stem cells were added to pancreatic cancer cells BxPC3, Panc-1 and mouse models of pancreatic cancer, respectively. The proliferative activity and invasion abilities of BxPC3 and Panc-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The expressions of miRNAs in exosomes were detected by high-throughput sequencing. GO and KEGG were used to analyze the related functions and the main metabolic pathways of target genes with high expressions of miRNAs. Results: The results of CCK-8 cell proliferation assay showed that the absorbance of BxPC3 and Panc-1 cells in the hucMSCs-exo group was significantly higher than that in the control group [(4.68±0.09) vs. (3.68±0.01), P<0.05; (5.20±0.20) vs. (3.45±0.17), P<0.05]. Transwell test results showed that the number of invasion cells of BxPC3 and Panc-1 in hucMSCs-exo group was significantly higher than that in the control group (129.40±6.02) vs. (89.40±4.39), P<0.05; (134.40±7.02) vs. (97.00±6.08), P<0.05. In vivo experimental results showed that the tumor volume and weight in the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) group were significantly greater than that in the control group [(884.57±59.70) mm(3) vs. (695.09±57.81) mm(3), P<0.05; (0.94±0.21) g vs. (0.60±0.13) g, P<0.05]. High-throughput sequencing results showed that miR-148a-3p, miR-100-5p, miR-143-3p, miR-21-5p and miR-92a-3p were highly expressed. GO and KEGG analysis showed that the target genes of these miRNAs were mainly involved in the regulation of glucosaldehylation, and the main metabolic pathways were ascorbic acid and aldehyde acid metabolism, which were closely related to the development of pancreatic cancer. Conclusion: Exosomes derived from human umbilical cord mesenchymal stem cells can promote the growth of pancreatic cancer cells and the mechanism is related to miRNAs that are highly expressed in exosomes.

Effect of Acute Myeloid Leukemia Cells on the Proliferation and Apoptosis of Bone Marrow-Derived Mesenchymal Stromal Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of acute myeloid leukemia cells in leukemia-microenvironment on proliferation and apoptosis of bone marrow-derived mesenchymal stromal cells (BM-MSC).@*METHODS@#Acute myeloid leukemia (AML) murine models overexpressing MLL-AF9 were established. The number of BM-MSC of wild type (WT) and AML-derived mice were analyzed by flow cytometry. Morphology and growth differences between WT and AML-derived BM-MSC were analyzed by inverted fluorescence microscope. Proliferation and apoptosis of BM-MSC between these two groups were detected by Brdu and Annexin V/PI.@*RESULTS@#Compared with WT-derived BM-MSC, the number and proliferation rate of AML-derived BM-MSC significantly increased (P<0.01, P<0.001), while apoptosis rate decreased (P<0.05). When cultured in vitro, BM-MSC grew faster under conditional medium.@*CONCLUSION@#AML cells can promote proliferation and inhibit apoptosis of BM-MSC.

Comparative Evaluation of Mouse Bone Marrow Mesenchymal Stromal Cells Characteristics Cultured in Two Different Supplemented Media

@#Introduction: Preclinical studies on mesenchymal stromal cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. In recent years, conflicting data have been reported regarding various aspects of their characteristics, development and differentiation potential, which may be due to arrange of factors. Among the factors worth investigating is the culture medium formulation. Methods: Here we have made a comparative characterization of mouse bone marrow mesenchymal stromal cells (mBM-MSC) that were cultured using two common supplements, MesenCult™ Stimulatory Supplement (MSS) and fetal bovine serum (FBS), under the same experimental conditions at different passages. Clonogenic potential, cumulative population doubling level (CPDL), population doubling time (PDT), immunophenotyping, differentiation, immunosuppression potentials and chromosome analysis of early and late passages mBM-MSC were assessed. Results: Our findings showed that the CPDL, immunophenotype and immunosuppression potential of mBM-MSC were similar. However, variations were seen in their clonogenicity, population doubling time and differentiation efficacy whereby all of these were enhanced in DMSS. These observations suggest that their genetic make-up may be affected by both supplements upon prolonged culture. Interestingly, this conjecture was supported when chromosomal analysis revealed genetic instability of mBM-MSCs cultured in both supplements. Conclusion: In conclusion, culture medium formulation was shown to cause variations and spontaneous transformation in mBM-MSCs raising concerns on the usage of late passages mBMMSCs in fundamental and preclinical downstream experiments.

Effect of quercetin on osteogenic ability of dental pulp mesenchymal cells and repair of calvarial defects in rabbits / 中国药理学通报

Aim To investigate the effect of quercetin on the osteogenic ability of human dental pulp mesenchymal stromal cells (hDPSCs) in vitro and in vivo. Methods hDPSCs were obtained from the pulp tissues of premolar, and the characteristic surface antigens were identified by flow cytometry. Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxicity of quercetin. Alkaline phosphatase (A L P) and alizarin red staining were used to detect the osteogenic ability of cells in vitro. The expression of osteogenic genes was detected by qPCR. Four round calvarial bone defects with a diameter of 8 mm were created in 10 male New Zealand rabbits, and they were differentiated and randomized into four groups. Group A, hDPSCs cultured on Bio-Oss

Adipose tissue-derived mesenchymal stromal cells for treating chronic kidney disease: A pilot study assessing safety and clinical feasibility

BACKGROUND: Chronic kidney disease (CKD) is a growing public health concern, and available treatments are insufficient in limiting disease progression. New strategies, including regenerative cell-based therapies, have emerged as therapeutic alternatives. Results from several groups, including our own, have reported evidence of a supportive role for mesenchymal stromal cells (MSCs) in functional recovery and prevention of tissue damage in murine models of CKD. Prompted by these data, an open pilot study was conducted to assess the safety and efficacy of a single injection of autologous adipose tissue-derived MSCs (AT-MSCs) for treatment of CKD. METHODS: AT-MSCs were infused intravenously into six CKD patients at a dose of 1 million cells/kg. Patients were stabilized and followed for one year prior to MSC infusion and one year following infusion. RESULTS: No patients presented with adverse effects. Statistically significant improvement in urinary protein excretion was observed in AT-MSCs transplanted patients, from a median of 0.75 g/day (range, 0.15–9.57) at baseline to 0.54 g/day (range, 0.01v2.66) at month 12 (P = 0.046). The glomerular filtration rate was not significantly decreased post-infusion of AT-MSCs. CONCLUSION: Findings from this pilot study demonstrate that intravenous infusion of autologous expanded AT-MSCs into CKD patients was not associated with adverse effects and could benefit patients already undergoing standard medical treatment.

Targeted nano-vector for gene delivery into human bone marrow mesenchymal stem cells and cellular MR imaging in vitro / 中国医学影像技术

Objective To explore the feasibility and efficacy of an MRI-visible,targeted,nano-vector which is synthesized by attaching a targeting ligand,the GD2 single chain antibody (scAb GD2),to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging.Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION.The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering.Cytotoxicity of scAbGD2-PEG-g-PEI-SPI-ON was evaluated by CCK-8 assay using hBMSCs.Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry,PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION+ free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established.The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining.MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro.Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs.At the same N/P ratio,the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P＜0.001).At the optimal N/P ratio of 20,scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60 ± 4.50) ％ in hBMSCs.Furthermore,hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2/T2 *-weighted images in vitro.Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRL visible targeted nano vector for gene delivery into hBMSCs.

Targeted nano-vector for gene delivery into human bone marrow mesenchymal stem cells and cellular MR imaging in vitro / 中国医学影像技术

Objective To explore the feasibility and efficacy of an MRI-visible,targeted,nano-vector which is synthesized by attaching a targeting ligand,the GD2 single chain antibody (scAb GD2),to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging.Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION.The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering.Cytotoxicity of scAbGD2-PEG-g-PEI-SPI-ON was evaluated by CCK-8 assay using hBMSCs.Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry,PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION+ free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established.The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining.MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro.Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs.At the same N/P ratio,the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P＜0.001).At the optimal N/P ratio of 20,scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60 ± 4.50) ％ in hBMSCs.Furthermore,hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2/T2 *-weighted images in vitro.Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRL visible targeted nano vector for gene delivery into hBMSCs.

Characterization of Adipose Tissue Mesenchymal Stromal Cell Subsets with Distinct Plastic Adherence

Mesenchymal stromal cells (MSCs) established by in-vitro adherence culture have been widely utilized for various cell therapeutic trials, but potential heterogeneity that can be caused by preparation methods are poorly characterized. In this study, we show that at least two distinct subsets of MSCs with different adherence to plastic surface exist in human adipose tissue-derived stromal vascular fraction (SVF); while 69% of total colony forming units in SVF adhere to the surface before 3 hrs of plating, 13–17% of colonogenic cells adhered to the surface at later period of 15 hr to 1 week after plating. Of note, the late adherent MSCs exhibited higher self-renewal of colony forming cells and higher proliferating potential with comparable level of osteogenic or adipogenic differentiation potential to the early adherence subsets. Moreover, late adherent cells exhibited distinct pattern of paracrine secretome including higher level secretion of cytokines than the early adherent subsets. Taken together, these results suggest the possibility that distinct adherence properties of MSCs can be another parameter of clonal heterogeneity in the subpopulations of adipose tissue MSCs and that it can be an important factor for optimization of MSC preparation for cell therapeutic trials.

The exosome secreted by B16 cells promoted the proliferation and migration of mesenchymal stromal cell / 中华微生物学和免疫学杂志

Objective To investigate the interactions between melanoma-derived exosomes and the microenvironment.Methods The exosomes were isolated from the culture medium of mouse melanoma cells and then co-cuhured with mesenchymal stromal cells (MSC) after identification.Immunofluorescence assay was performed to observe the exosomes engulfed by MSC.CCK-8 and transwell assays were used to evaluate the proliferation and migration of MSC.Effects of the exosomes on the expression of α-smooth muscle actin (α-SMA) in MSC were analyzed by Western blot.Results Co-culture of MSC with melanoma cell-derived exosomes enhanced the proliferation and migration of MSC as well as the expression of α-SMA.All of the changes mediated by the exosomes could be blocked by using the inhibitor of TGF-β receptor.Conclusion Melanoma cell-derived exosomes enhanced the proliferation and migration of MSC as well as the expression of α-SMA through TGF-β signaling pathway,which provided an advantageous microenvironment for melanoma progression.

Canine adipose tissue-derived mesenchymal stem cells ameliorate severe acute pancreatitis by regulating T cells in rats

Severe acute pancreatitis (SAP) is associated with systemic complications and high mortality rate in dogs. Mesenchymal stem cells (MSCs) have been investigated for their therapeutic potential in several inflammation models. In the present study, the effects of canine adipose tissue-derived (cAT)-MSCs in a rat model of SAP induced by retrograde injection of 3% sodium taurocholate solution into the pancreatic duct were investigated. cAT-MSCs labeled with dioctadecyl-3,3,3′-tetramethylindo-carbocyanine perchlorate (1 × 10⁷ cells/kg) were systemically administered to rats and pancreatic tissue was collected three days later for histopathological, quantitative real-time polymerase chain reaction, and immunocytochemical analyses. Greater numbers of infused cAT-MSCs were detected in the pancreas of SAP relative to sham-operated rats. cAT-MSC infusion reduced pancreatic edema, inflammatory cell infiltration, and acinar cell necrosis, and decreased pancreatic expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, -6, -12, -17, and -23 and interferon-γ, while stimulating expression of the anti-inflammatory cytokines IL-4 and IL-10 in SAP rats. Moreover, cAT-MSCs decreased the number of clusters of differentiation 3-positive T cells and increased that of forkhead box P3-positive T cells in the injured pancreas. These results indicate that cAT-MSCs can be effective as a cell-based therapeutic strategy for treatment of SAP in dogs.

Effects of different dosages of BMSC on lung fibrosis in mice / 上海交通大学学报（医学版）

Objective To investigate the effects of different dosages of bone marrow mesenchymal stromal cells (BMSC) on lung fibrosis. Methods BMSCs with red fluorescence protein (RFP) from male FVB mice were cultured in vitro. Twenty-four female wild type FVB mice were randomly divided into four groups: normal group, model group, BMSC 1 group and BMSC 2 group (n = 6). Mouse pulmonary fibrosis models were induced by bleomycin via single intratracheal perfusion. Twenty-four h after model establishment, mice in BMSC 1 group and BMSC 2 group were injected with 1 × 10~6 BMSCs and 2 × 10~6 BMSCs, respectively through vena caudalis for each mouse. All the animals were sacrificed 21 d after model estalishment, and mouse lung tissue samples were obtained. The pathological changes were observed by light microscopy, the hydroxyproline ( Hyp) contents were measured by alkaline hydrolysis assay, the distribution of RFP( + ) BMSCs and quantitation of RFP were analysed by laser scanning confocal microscopy and immunohistochemistry, the expression of surfactant protein A (SP-A) was detected by immunohistochemistry, and the expression of transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) mRNA was detected by Real-time PCR. Results Compared with model group, the pulmonary fibrosis in BMSC 1 group was significantly alleviated, and that of BMSC 2 group became much more severe.A large number of RFP( +) BMSCs were found in fibrosis area of BMSC 2 group,which exhibited morphology similar to fibroblasts. As far as the expression of SP-A was concerned, normal group was higher than BMSC 1 group, BMSC 1 group was higher than BMSC 2 group and model group (P < 0. 05), while there was no significant difference between BMSC 2 group and model group (P >0. 05). Normal group, BMSC I group, model group and BMSC 2 group fell in the increase order by Hyp contents (P <0.01, P <0.05), and BMSC 2 group, BMSC 1 group, model group and normal group fell in the decrease order by expression of TCF-|$ and PDGF mRNA (P < 0.05). Conclusion Proper dose of BMSC has a favourable effect on bleomycin-induced lung fibrosis, while excessive dose of BMSC can aggravate the fibrosis.