Objective: This study was to investigate the variables in bone marrow harvesting procedure and individual donor factors which can potentially affect the yield of mesenchymal stromal cells (MSC).Methods: We determined the yield of MSC from bone marrow under different clinical conditions by comparing the MSC colony numbers from: (1) donors of different ages; (2) healthy donors and patients with leukemia; (3) bone marrow aspirated at different time points during marrow harvesting; (4) bone marrow harvested by different needles.Results: During the process of harvesting, the number of MSC significantly decreased with increase number of aspiration, from 675/ml at the initial decreased to 60/ml after 100 ml bone marrow aspirated, and 50/ml after 200 ml bone marrow aspirated. The number of MSC retrieved from leukemia patients (99/ml bone marrow) was significantly lower than that of healthy donors (708/ml bone marrow). However, there was no significant difference in growth rate. There was no significant age-related difference of MSC yielded from donors <55 years. And there was no significant difference in MSC number between the samples from single end-holed needle and those from multiple-side-hole needle.Conclusion: The optimal bone marrow samples for MSC collection should be obtained earlier in the process of harvesting procedure. Bone marrow from donors <55 years was equally good as MSC sources. The autologous MSC from leukemia patients can be utilized for in-vitro MSC expansion.

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Background Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transplantation of immunosuppressive human mesenchymal stromal cells (hMSCs) can protect against aGvHD post-HSCT; however, their efficacy is limited by poor engraftment and survival. Moreover, infused MSCs can be damaged by activated complement, yet strategies to minimise complement injury of hMSCs and improve their survival are limited. Methods Human MSCs were derived from bone marrow (BM), adipose tissue (AT) and umbilical cord (UC). In vitro immunomodulatory potential was determined by co-culture experiments between hMSCs and immune cells implicated in aGvHD disease progression. BM-, AT- and UC-hMSCs were tested for their abilities to protect aGvHD in a mouse model of this disease. Survival and clinical symptoms were monitored, and target tissues of aGvHD were examined by histopathology and qPCR. Transplanted cell survival was evaluated by cell tracing and by qPCR. The transcriptome of BM-, AT- and UC-hMSCs was profiled by RNA-sequencing. Focused experiments were performed to compare the expression of complement inhibitors and the abilities of hMSCs to resist complement lysis. Results Human MSCs derived from three tissues divergently protected against aGvHD in vivo. AT-hMSCs preferentially suppressed complement in vitro and in vivo, resisted complement lysis and survived better after transplantation when compared to BM- and UC-hMSCs. AT-hMSCs also prolonged survival and improved the symptoms and pathological features of aGvHD. We found that complement-decay accelerating factor (CD55), an inhibitor of complement, is elevated in AT-hMSCs and contributed to reduced complement activation. We further report that atorvastatin and erlotinib could upregulate CD55 and suppress complement in all three types of hMSCs. Conclusion CD55, by suppressing complement, contributes to the improved protection of AT-hMSCs against aGvHD. The use of AT-hMSCs or the upregulation of CD55 by small molecules thus represents promising new strategies to promote hMSC survival to improve the efficacy of transplantation therapy. As complement injury is a barrier to all types of hMSC therapy, our findings are of broad significance to enhance the use of hMSCs for the treatment of a wide range of disorders.

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Osteoporosis is a very common problem among adolescent and adult patients with thalassaemia major (TM). The pathogenesis of osteoporosis in TM is related to several factors including iron overload. Bone is derived from osteoblasts. And osteoblasts are differentiated from mesenchymal stem cells (MSC). Therefore, iron-overload induced MSC damage may contribute to osteopenia and osteoporosis. The effect of iron-overload on MSC has not been investigated previously. We hypothesize that iron-overload may induce apoptosis in MSC by caspase-dependent pathway, and haematopoietic growth factor thrombopoietin (TPO) and calcium channel blocker amlodipine may have a protective effect on iron-induced apoptosis in these cells. We have shown that iron (FeCl3) reduced hMSCs viability in a dose-dependent manner (0–0.6 mM) (n=5). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM) for 72 hrs (n=4). The expression of active caspase-3 was significantly increased in iron-treated cells (0.15mM, 0.3mM) (n=5). Iron treatment also increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential. TPO exerted protective effect on iron-induced apoptosis in hMSCs. Human MSCs were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/ml) for 72 hrs (n=4). The cell viability was significantly increased with the treatment of TPO. Dot-plot analysis of annexin V/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells. Incubation with TPO also decreased the iron-induced caspase-3 expression. Flow cytometric dot-plot analysis of hMSCs also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO. The population of phospho-Erk1/2 was also significantly increased in TPO-treatment, and the increased phospho-Erk was significantly reversed by the upstream signaling inhibitor PD098059. Calcium channel blocker amlodipine (10−9M) also had a protective effect on iron-induced apoptosis in these cells. Our findings suggest that iron-overload induces apoptosis in hMSCs via the caspase-dependent pathway and that TPO and amlodipine might exert a protective effect on iron-induced apoptosis via the activation of Erk1/2 signaling. The use of either haematopoietic growth factor or calcium channel blocker for the protection of hMSCs from iron induced toxicity is a novel concept. Our study has the potential in minimizing the bone damage induced by iron-overload in patients with thalassaemia major.