The therapeutic potential of bone marrow-derived stromal cells

Since the replacement of the hematopoietic system became feasible through bone marrow (BM) transplantation, the idea of how to replace other organs of the body has been in the forefront of medical research. Scientists have been searching for the ideal stem cell that could be manipulated to differentiate into any tissue. Although the embryonal stem cells seemed to have the ability to do this, the difficulties surrounding their use prevented them from becoming therapeutically useful. Thus, the field turned to adult stem cells, particularly stem cells of BM origin. We have learnt a lot during the last decade about the potential of the BM-derived stromal (also called mesenchymal stem) cells (BMSCs). The first studies suggested them as cell replacement tools, but later it turned out that their usefulness is more likely due to paracrine effects due to a large variety of secreted factors that induce growth and differentiation of the tissue-specific stem cells as well as prevent injured cells from apoptotic death. Finally, a whole new field emerged when many groups confirmed that these cells are also capable of regulating immune function in a so far unknown, dynamic manner. When BMSCs are injected they seem to be able to sense the environment and respond according to the actual need of the organism in order to survive. This plasticity can never be done by the use of any drugs and such a "live" cell therapy could open a whole new chapter in clinical care in the future.     

Recombinant BMP 4/7 fusion protein induces differentiation of bone marrow stem cells

Bone morphogenetic proteins (BMPs) induce differentiation of mesenchymal cells to cartilage and bone. We cloned BMP4 and BMP7 cDNAs from human placenta and fetal cartilage cells, respectively, and used an Escherichia coli expression system to produce recombinant BMP4 and BMP4/7 proteins. Differentiation of primary cultures of bone marrow stem cells (BMSC) treated with BMP4 or BMP4/7 was evaluated by Von Kossa staining and by determining alkaline phosphatase activity and osteocalcin level. BMP4/7-induced BMSC differentiation more potently than BMP4. We showed that BMP4/7 fusion protein expressed in E. coli is biologically active and is a novel strategy to treat bone injury in a clinical setting.     

Hypoxia inhibits the spontaneous calcification of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the popular seed cells for regenerative medicine, and there has been a rapid increase in the number of BM-MSC-based clinical trials. However, the safety of these cells should also be closely studied. In this study, spontaneous calcification of BM-MSCs from rats was evaluated in normoxia (20% O(2)) without osteogenic medium after continuous culture for 21 days; obvious mineralized nodules were observed, which were positive for Alizarin Red, collagen-I (Col-I), osteocalcin (OC) and alkaline phosphatase (ALP), and mainly consisted of C, O and Ca elements. Interestingly, hypoxia (2% O(2)) significantly inhibited this spontaneous calcification. In addition, the ALP and calcium content of rBM-MSCs were sharply reduced. Based on RT-PCR results, the expression of osteogenic genes (Cbfa1/Runx2, Col-I, ALP, and OC) was reduced compared to that in normoxia. These results demonstrate a natural and unique characterization of rat BM-MSCs in normoxia after continuous culture and highlight the inhibiting effects of hypoxia. Finally, this study contributes to the information regarding the application of BM-MSCs in the regeneration of various tissues.     

Effect of mesenchymal stem cells on inhibiting airway remodeling and airway inflammation in chronic asthma

Previous studies proved that bone marrow‐derived mesenchymal stem cells (BMSCs) could improve a variety of immune‐mediated disease by its immunomodulatory properties. In this study, we investigated the effect on airway remodeling and airway inflammation by administrating BMSCs in chronic asthmatic mice. Forty‐eight female BALB/c mice were randomly distributed into PBS group, BMSCs treatment group, BMSCs control group, and asthmatic group. The levels of cytokine and immunoglobulin in serum and bronchoalveolar lavage fluid were detected by enzyme‐linked immunosorbent assay. The number of CD4⁺CD25⁺regulatory T cells and morphometric analysis was determined by flow cytometry, hematoxylin‐eosin, immunofluorescence staining, periodic‐acid Schiff, and masson staining, respectively. We found that airway remodeling and airway inflammation were evident in asthmatic mice. Moreover, low level of IL‐12 and high levels of IL‐13, IL‐4, OVA‐specific IgG1, IgE, and IgG2a and the fewer number of CD4⁺CD25⁺regulatory T cells were present in asthmatic group. However, transplantation of BMSCs significantly decreased airway inflammation and airway remodeling and level of IL‐4, OVA‐specific IgE, and OVA‐specific IgG1, but elevated level of IL‐12 and the number of CD4 + CD25 + regulatory T cells in asthma (P < 0.05). However, BMSCs did not contribute to lung regeneration and had no significant effect on levels of IL‐10, IFN‐Y, and IL‐13. In our study, BMSCs engraftment prohibited airway inflammation and airway remodeling in chronic asthmatic group. The beneficial effect of BMSCs might involved the modulation imbalance cytokine toward a new balance Th1–Th2 profiles and up‐regulation of protective CD4 + CD25 + regulatory T cells in asthma, but not contribution to lung regeneration. J. Cell. Biochem. 114: 1595–1605, 2013. © 2013 Wiley Periodicals, Inc.     

Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.     

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.