Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.

A number of different microRNAs (miRNAs) have been implicated in various autoimmune diseases, including multiple sclerosis (MS). T helper (Th)17 and regulatory T cells (Tregs) have likewise been implicated as key players in MS, and a functional imbalance of these cell types is increasingly recognized as a key etiological factor in the disease. Although specific panels of transcription factors and cytokines are known to regulate the Th17/Treg balance, the role of noncoding RNAs remains poorly understood. The inflammatory cytokine, interleukin (IL)6, appears to play a critical role in both the development of the Th17 response and the inhibition of Treg functions. In this research, an IL6-associated miRNA, miR26a, was identified, and its normally downregulated expression was shown to be highly correlated with disease severity in patients suffering from MS as well as in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE; a well-established animal model of human MS). Using the EAE model system, in vivo silencing of miR26a was found to result in increased expression of Th17-related cytokines and increased severity of EAE, while overexpression of miR26a was found to result in reduced expression of Th17-related cytokines and a milder form of EAE. By contrast, Treg cell-specific transcription factor, Foxp3, was found to be positively correlated with miR26a expression. Finally, miR26a was found to downregulate Th17 and to upregulate Treg cell function through its targeting of IL6. Taken together, our data indicate an important role for miR26a in maintaining the Th17 and Treg cell balance in MS that involves repression of IL6 expression.

In vivo studies have shown that amnion-produced growth factors participate in many diseases that involve angiogenesis, re-epithelialization and immunomodulation. Although human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs) can be obtained from amniotic membranes, there is little information regarding their biological differences. The aim of the present study was to isolate and characterize cells from human amnions, to investigate the biological potential and behavior of these cells on the function of endothelial cells in vivo and in vitro and to examine variations in the expression profile of growth factors in different human amnion-derived cell types. Amnion fragments were enzymatically digested into two cell fractions, which were analyzed by mesenchymal and epithelial cell markers. Human aortic endothelial cells (hAoECs) were cultured with conditioned medium (CdM) collected from hAECs or hAMSCs. We used scratch and Transwell assays to evaluate migration ability; Cell Counting Kit-8 (CCK-8) and cell cycle analysis to evaluate proliferation ability; and a Matrigel tube formation assay to evaluate angiogenesis ability. To detect expression of angiogenesis-related genes, qPCR and enzyme-linked immunosorbent assay (ELISA) analyses were conducted. As stem cells, hAECs and hAMSCs all expressed the stem cell markers SSEA-4, OCT-4 and SOX-2. CdM obtained from hAECs promoted cell migration; CdM obtained from hAMSCs promoted cell proliferation; CdM obtained from hAECs and hAMSCs both promoted angiogenesis in hAoECs. Amnion-derived cells secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, EGF, HB-EGF and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Our results highlight that human amniotic epithelial and mesenchymal stem cells, which showed differences in their soluble factor secretion and angiogenic functions, could be ideal cell sources for regenerative medicine.

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