Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND AIMS: Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stem cells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used either immediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate the advantages and drawbacks of their in vitro expansion. METHODS: The phenotype and osteogenic differentiation potential of oAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. Expanded oAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLA antigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expanded oAFMSCs was assessed by qualitative and quantitative methods. RESULTS: Expanded oAFMSCs reduced their proliferative activity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution of pluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of cultured oAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion, oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lower percentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin. CONCLUSIONS: The results presented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticity of oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employing expanded oAFMSCs for therapeutic purposes.

Reduced Osteogenic Differentiation Potential In Vivo in Acute Myeloid Leukaemia Patients Correlates with Decreased BMP4 Expression in Mesenchymal Stromal Cells.

The osteogenic differentiation potential of mesenchymal stromal cells (hMSCs) is an essential process for the haematopoiesis and the maintenance of haematopoietic stem cells (HSCs). Therefore, the aim of this work was to evaluate this potential in hMSCs from AML patients (hMSCs-AML) and whether it is associated with BMP4 expression. The results showed that bone formation potential in vivo was reduced in hMSCs-AML compared to hMSCs from healthy donors (hMSCs-HD). Moreover, the fact that hMSCs-AML were not able to develop supportive haematopoietic cells or to differentiate into osteocytes suggests possible changes in the bone marrow microenvironment. Furthermore, the expression of BMP4 was decreased, indicating a lack of gene expression committed to the osteogenic lineage. Overall, these alterations could be associated with changes in the maintenance of HSCs, the leukaemic transformation process and the development of AML.

Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/ß-catenin signalling pathway via DNA methylation.

OBJECTIVES: Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5-aza2'-deoxycytidine (5-aza-dC) and DKK-1 were used to treat ASCs. Real-time cell analysis and cell counting kit-8 were used to monitor the proliferation of ASCs within and without AGEs. Real-time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/ß-catenin signalling pathway among the different groups. RESULTS: The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs-treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs-treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including ß-Catenin, Lef1 and P-GSK-3ß, were inhibited. After treatment with 5-aza-dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. CONCLUSIONS: Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/ß-catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.

Molecular- and microarray-based analysis of diversity among resting and osteogenically induced porcine mesenchymal stromal cells of several tissue origin.

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches in bone-healing disorders. Although bone marrow-derived MSCs are most frequently used, the knowledge that many other adult tissues represent promising sources for potent MSCs has gained acceptance. In the present study, the osteogenic differentiation potential of porcine skin fibroblasts (FBs), as well as bone marrow- (BMSCs), adipose tissue- (ASCs) and dental pulp-derived stromal cells (DSCs) were evaluated. However, additional application of BMP-2 significantly elevated the delayed osteogenic differentiation capacity of ASC and FB cultures, and in DSC cultures the supplementation of platelet-rich plasma increased osteogenic differentiation potential to a comparable level of the good differentiable BMSCs. Furthermore, microarray gene expression performed in an exemplary manner for ASCs and BMSCs revealed that ASCs and BMSCs use different gene expression patterns for osteogenic differentiation under standard media conditions, as diverse MSCs are imprinted dependent from their tissue niche. However, after increasing the differentiation potential of ASCs to a comparable level as shown in BMSCs, a small subset of identical key molecules was used to differentiate in the osteogenic lineage. Until now, the importance of identified genes seems to be underestimated for osteogenic differentiation. Apparently, the regulation of transmembrane protein 229A, interleukin-33 and the fibroblast growth factor receptor-2 in the early phase of osteogenic differentiation is needed for optimum results. Based on these results, bone regeneration strategies of MSCs have to be adjusted, and in vivo studies on the osteogenic capacities of the different types of MCSs are warranted. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.