[Research Progress on Immunomodulatory Activity and Clinical Application of Mesenchymal Stem Cells-Derived Extracellular Vesicles--Review].

Mesenchymal stem cells (MSC) have been widely used in tissue regeneration and treatment graft versus host disease (GVHD) and immune diseases due to their self-renewal, multi-differentiation and immunoregulatory potential. However, more and more scholars begin to put weight on the MSC -derived extracellular vesicles (MSC-EV) for its regulation of inflammation and immunity. MSC-EV can activate the relevant signal pathways and regulate the function and biological behaviors of cells via acting on target cells and mediating communication between cells. MSC-EV has important potential clinical applications for its powerful immunomodulatory and hematopoietic regulatory functions. It is considered as a potential therapeutic tool to treat autoimmune diseases and GVHD. This paper reviewed the immunomodulatory activity of MSC-EV as well as the research progress of MSC-EV in hematopoietic stem cell transplantation, and discussed its potential clinical applications in the future.

rhTPO combined with chemotherapy and G-CSF for autologous peripheral blood stem cells in patients with refractory/relapsed non-Hodgkin's lymphoma.

OBJECTIVE: The mobilization and collection of sufficient autologous peripheral blood stem cells (APBSCs) are important for the fast and sustained reconstruction of hematopoietic function after autologous transplantation. This study aims to evaluate the mobilization effect and safety of thrombopoietin (TPO) combined with chemotherapy + G-CSF for APBSCs in patients with refractory/relapsed non-Hodgkin's lymphoma. METHODS: A total of 78 patients were included in the present study. After receiving mobilization chemotherapy, all patients were randomly divided into two groups: TPO group (n=40), patients were given subcutaneous injection of rhTPO + G-CSF, and control group (n=38), patients were given subcutaneous injection of G-CSF. The primary endpoint was the total number of obtained CD34+ cells. The secondary endpoints were the mononuclear cell count, the proportion of target and minimum mobilization, the engraftment time of neutrophils and platelets after APBSCT, the number of platelet and red blood cell infusions, the incidence of infectious fever and fever duration, and TPO-related side effects in patients. RESULTS: TPO participation significantly increased the total CD34+ cell count. A higher proportion of patients in the TPO group achieved the minimum and target CD34+ cells, when compared to the control group. TPO-related adverse events were not observed in either of these groups. In addition, there were no significant differences in engraftment time, the number of platelet and red blood cell transfusions, the incidence of infectious fever, and fever duration between these two groups. CONCLUSION: TPO combined with chemotherapy + G-CSF can safely and effectively enhance the mobilization effect for APBSCs in patients with refractory/relapsed non-Hodgkin's lymphoma.

Reduced-dose EPOCH-R chemotherapy for elderly patients with advanced stage diffuse large B cell lymphoma.

The standard treatment in elderly patients with diffuse large B cell lymphoma (DLBCL) has not yet been finely established. We investigated the efficacy and safety of rituximab with a reduced-dose of EPOCH chemotherapy in elderly patients who had advanced DLBCL with high IPI scores. The dose of 70% EPOCH was given to patients aged 75 to 79 years, and dose of 50% to patients aged over 80 years. Thirty-one patients with a median age of 79 years (range 75-86 years) were enrolled. Patients received a median of 6 cycle's chemotherapy. The complete response rate was 71.0%. The 3-year overall survival (OS) and progression-free survival rates were 62.8 and 60.3%, respectively. The most frequent grade 3/4 adverse effects were neutropenia (3 patients, 7 events), febrile neutropenia (3 patients, 5 events), and pulmonary infection (3 patients, 3 events). Our study showed that ECOG score 3-4, bulky disease, ß2-MG > 5.0 mg/L, and loss of any IADL are prognostic factors for OS in univariate analysis. In summary, reduced-dose EPOCH-R chemotherapy for very elderly patients is very effective with acceptable toxicities. Our preliminary study may provide an alternative approach to manage very elderly fit patients with advanced and poor risk DLBCL by first-line treatment with reduced-dose EPOCH-R.

High-dose methotrexate, etoposide, dexamethasone and pegaspargase (MEDA) combination chemotherapy is effective for advanced and relapsed/refractory extranodal natural killer/T cell lymphoma: a retrospective study.

Extranodal natural killer/T cell lymphoma, nasal type (ENK/TCL), is an aggressive and rare hematological malignancy. Patients with advanced and relapsed/refractory disease have very poor outcomes. In this study, we retrospectively assessed the efficacy and safety of MEDA regimen (methotrexate, etoposide, dexamethasone and pegaspargase) in the treatment of advanced and relapsed/refractory ENK/TCL patients. Thirteen patients received a total of 55 cycles of MEDA, with a median of four cycles. At the completion of treatment, the overall response rate was 76.9 %, with a complete response rate of 61.5 %. The 1-year overall survival rate was 69.2 %, and 1-year progression-free survival was 61.5 %. Treatment-related toxicity was monitored in all patients. Grade 3/4 neutropenia occurred in 46.2 % of patients. Serious infections happened in two cases (15.4 %). Grade 3/4 thrombocytopenia occurred in 30.8 % of patients, and 23.1 % received platelet transfusion. Grade 3/4 anemia was observed in 23.1 % of patients. Hepatotoxicity and low fibrinogen were common, but mild. These results show that MEDA regimen is very effective with tolerable adverse effects in the treatment of advanced and relapsed/refractory ENK/TCL. Further prospective trials are expected to validate the efficacy of MEDA in an expanded number of patients.

[Down-regulation of transcription factor PU.1 via abnormal epigenetic modification in chronic myeloid leukemia].

OBJECTIVE: To investigate the underlying mechanism and clinical significance of PU.1 down-expression in chronic myeloid leukemia (CML) patients. METHODS: Different methylation status of PU.1 promoter region containing 20 CpG islands in normal individuals, CML chronic phase and blast crisis patients, complete cytogenetic remission patients after imatinib treatment, and blast crisis bone marrow K562 CML cells was detected by bisulfite sequencing. Semi-quantitative PCR was used to detect the PU.1 mRNA expression in normal controls, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells. Indirect immune fluorescence and Western blot were used to analyze the exprtession of PU.1 protein in normal individuals, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells. RESULTS: Aberrant methylation in the promoter region of transcription factor PU.1 was found in both CML chronic phase and blast crisis phase bone marrow cells, as well as in CML blast K562 cells. Down-expression of PU.1 mRNA and protein levels was found in above cells. No methylation in the promoter region of PU.1 was observed in normal individuals, and the PU.1 mRNA and protein expressions were not reduced at all. Furthermore, high methylation status of bone marrow cells was even observed in the CML patients who acquired complete cytogenetic remission. CONCLUSIONS: The results of our study indicate that the epigenetic modification of PU.1 in CML patients and K562 cell line might be responsible for the down-expression of PU.1. The data suggest that aberrant methylation of PU.1 plays a role in CML pathogenesis, therefore, it might serve as a useful biomarker and potential target in therapy for chronic myeloid leukemia.

Down-regulation of hematopoiesis master regulator PU.1 via aberrant methylation in chronic myeloid leukemia.

The PU.1 transcription factor is a crucial regulator of hematopoiesis, and its expression is altered in various leukemic processes. It has been shown that expression of PU.1 is severely impaired in patients with chronic myeloid leukemia (CML), but the mechanism underlying this effect remains unknown. Through bisulfite sequencing, semi-quantitative PCR, and indirect immunofluorescence and Western blot techniques, we found aberrant methylation in the promoter region of transcription factor PU.1 in CML patients both in the chronic and blast crisis phases, as well as in the CML blast K562 cell line. Of these, several CpG sites were more highly methylated in blast crisis than chronic phase, while no methylation of these sites was observed in healthy individuals. Interestingly, CML patients achieved complete cytogenetic remission under imatinib mesylate treatment, but the aberrant methylation status of PU.1 was not reversed. Down-regulation of PU.1 expression at the mRNA and protein levels was also observed in association with aberrant methylation. Thus, for the first time, we have revealed a potential epigenetic modification of PU.1 in CML, which may be responsible for the down-regulation of PU.1. These data suggest that aberrant methylation of PU.1 may play a role in CML pathogenesis, and may therefore serve as a useful biomarker and potential target for demethylating drugs.

[Effect of TGF-beta1 on biological characteristics of umbilical cord blood hematopoietic progenitor cells during ex-vivo expansion].

To investigate the effects of TGF-beta1 on biological characteristics of hematopoietic progenitor cells (HPC) in umbilical cord blood (UCB) during ex-vivo expansion and feasibility of using it for expansion of UCB HPC, different concentrations of TGF-beta1 were added in the serum-free medium containing a combination of hematopoietic growth factors for expansion of UCB CD133(+) cells and enumeration of nucleated cells (NC), progenitor colonies, immunophenotyping, cell cycle and expression of adhesion molecules of the NC were monitored at every interval. The results showed that total number and expansion of NC from all groups of TGF-beta1 were remarkably less than those in control at each interval. However the content and total numbers as well as expansion of CD34(+), CD133(+), CD34(+)CD38(-) and CD34(+)CD133(+) cells from all groups of TGF-beta1 were more than those in control at each interval during expansion; the plating efficiency and expansion of CFU-GM, CFU-mix and HPP-CFC from NC of TGF-beta1 group were more than those in control at each interval. The contents of cells in G(0)/G(1) phase of NC of TGF-beta1 group at every interval were high. Meanwhile, TGF-beta1 could elevate the expression of some adhesion molecules on NC during expansion such as CD54, CD49d and CD11a, and the contents of CD34(+) cells coexpressing these adhesion molecules in NC of TGF-beta1 group were significantly more than those in control at each interval. In conclusion appropriate dose of TGF-beta1 could accelerate expansion of CD133(+) cells, delay and decrease over-differentiation of HPC, increase the content of HPC in expanded products, upregulate the expression of adhesion molecules on expanded HPC, thus it could promote engraftment of expanded progenitor cells and advantage the ex-vivo expansion of UCB HPC.

[Studies on the dynamics of biological characteristics of CD133+ cells from human umbilical cord blood during short-term culture].

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.