Differentiation of Mesenchymal Stem Cells Derived From Human Adipose Tissue Into Cholinergic-like Cells; in vitro

Cholinergic associated diseases currently are major cause of neurological and neurodegenerative disabilities. As the drugs are not efficient in improving the suffered tissues, stem cell treatment is considered as an effective strategy for substituting the lost cells. In the current study, we set out to investigate the differentiation properties of human adipose-derived mensenchymal stem cells (AD-MSCs) into cholinergic-like cells by two morphogens; including retinoid acid (RA) and Sonic hedgehog (Shh) using a three- step in vitro procedure. The results were evaluated using Real-time PCR, Flowcytometry and Immunocytochemistry for two weeks. Our data showed that the cells could express cholinergic specific markers; including Islet-1, AChE, SMI-32 and Nestin at the level of mRNA and protein. We could also quantitatively evaluate the expression of Islet-1, AChE and Nestin at 14 days post- induction using flowcytometry. It is concluded that human AD-MSCs are potent type cell to differentiate into cholinergic like cells in the presence of RA and Shh through a three- step protocol; thus it could be a suitable cell candidate for regeneration of cholinergic associated diseases; however, more functional and electrophysiological analysis are needed.

Communications Between Bone Marrow Macrophages and Bone Cells in Bone Remodeling

The mammalian skeleton is a metabolically active organ that continuously undergoes bone remodeling, a process of tightly coupled bone resorption and formation throughout life. Recent studies have expanded our knowledge about the interactions between cells within bone marrow in bone remodeling. Macrophages resident in bone (BMMs) can regulate bone metabolism via secreting numbers of cytokines and exosomes. This review summarizes the current understanding of factors, exosomes, and hormones that involved in the communications between BMMs and other bone cells including mensenchymal stem cells, osteoblasts, osteocytes, and so on. We also discuss the role of BMMs and potential therapeutic approaches targeting BMMs in bone remodeling related diseases such as osteoporosis, osteoarthritis, rheumatoid arthritis, and osteosarcoma.

Author Correction: Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bone Marrow-Derived Mensenchymal Stem Cells Modified by Mouse Interferon-γ Gene Reduce Fibrosis and Improve Function In a Mouse Model of Liver Fibrosis.

Abstract 3768 Objective: To investigate the effect of bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ gene on experimental liver fibrosis in mice. Methods: (1) Mesenchymal stem cells (MSCs) derived from BM in BALB/c mice were cultured and identified by the flow cytometry. (2) The Ad-mIFN-γ, a recombinant adenoviral vector encoding mouse IFN-γ gene obtained by PCR from the vector pORF5-mIFN-γ, was created with the AdEasy Adenoviral Vector System. (3) MSCs were transfected with Ad-mIFN-γ. The transfection efficiency was detected by the flow cytometry at 24 hours after infection to determine the best multiplicity of infection (MOI). (4) The liver fibrosis of BALB/c mice were induced with concanavalin A (Con A) challenge weekly for six times. Then MSCs modified by the recombinant adenovirus encoding mouse IFN-γ gene were infused to BALB/c mice induced with Con A at the 24 hours after the first dose. The behavioral changes of mice were monitored closely. One week after the last dose of Con A, all mice were sacrificed and the fibrosis indexes were assessed by several histopathology methods systemicly. Moreover, hepatic hydroxyproline and serum parameters for liver fibrosis and liver function were evaluated and serum mIFN-γ concentrations were measured, respectively. Results: (1) The flow cytometry confirmed that the cells we obtained were MSCs. (2) The target gene mIFN-γ amplified by PCR from the vector pORF5-mIFN-γ, was identified by sequencing, which proved that the mIFN-γ gene was consisted of 468 nucleotides and was completely the same as the sequence published on GenBank. The target gene mIFN-γ was cut out by double endonucleases and then connected to the shuttle vector pAdTrack-CMV. Then the newly constructed vector was linearized by Pme I following transformed to the E.coli. BJ5183, which has the backbone vector of pAdEasy-1. The correct recombinant pAd-mIFN-γ was selected by endonucleases and by Kanamycin resistance and was transfected into AD-293 cells to obtain the adenoviral vector Ad-mIFN-γ. The Ad-mIFN-γ can be propagated in AD-293 cell line, the titre of which was 3.2×109 pfu/ml. (3) MSCs modified by recombinant adenovirus encoding mIFN-γ (IFN-γ/GFP-MSCs) at different multiplicity of infection (MOI) were detected by the flow cytometry. The optimal MOI for Ad-mIFN-γ was 400 with the transfection efficiency of 80.4%, and the optimal MOI for Ad-control was 200 with the transfection efficiency of 72.3%. (4) The changes of liver tissue and serum parameters for liver fibrosis showed liver fibrosis were reducd more obviously in the group treated with IFN-γ/GFP-MSCs (p<0.05), and liver functions were also improved more obviously in this group (p<0.05). The histopathology study using HE staining, masson trichrome staining and silver staining demonstrated that the structure of liver became much better in mice treated with IFN-γ/GFP-MSCs, with liver fibrosis improved significantly. Conclusion: Bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ can reduce liver fibrosis and improve liver function on immuo-mediated liver fibrosis in mice induced by Con A. Disclosures: No relevant conflicts of interest to declare.

The Influence of Titanium Particles Size on Bone Marrow Mensenchymal Stem Cells Viability

The decrease in bone mass caused by wear debris-induced osteolysis could have been compensated through osteoblasts secreting enough new bone matrix. However, the normal osteoblastic population depends on the regular differentiation of their progenitor cells, the bone marrow mesenchymal stem cells (BMSCs). It is not possible to predict whether wear particles will affect the BMSCs’ viability, and subsequently their differentiation. Furthermore, little is known about the extent to which the sizes of the wear particles loading can impact the viability the most. This study has, therefore, concentrated on the potential mechanism for rat BMSCs’ (rBMSCs) viability influenced by different-sized titanium particle (Ti) loading in vitro.rBMSCs were harvested and loaded with circular Ti particles having three different mean diameters, 0.9, 2.7 and 6.9 .m respectively. The results showed that different-sized titanium particles all inhibited rBMSCs’ proliferation and induced rBMSCs’ apoptosis response , but this influence varied with the size of the Ti particles, their concentration and the duration of loading. The smallest Ti particles (0.9.m) exhibited the earliest and largest suppression on the proliferation and the most powerful induction on the apoptotic response of rBMSCs. qRT-PCR analysis demonstrated that those apoptotic effects were association with the abnormal accentuation of inducible nitric oxide synthase(iNOS) activity. The size of titanium particles generated through wear of a prosthetic device and the osteoblastic progenitor BMSCs may be both important considerations in the development of superior implant technology.