Different methylation patterns of RUNX2, OSX, DLX5 and BSP in osteoblastic differentiation of mesenchymal stem cells

Runt-related transcription factor 2 [RUNX2] and osterix [OSX] as two specific osteoblast transcription factors and distal-less homeobox 5 [DLX5] as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein [BSP], osteopontin and collagen type I?1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells [MSCs]. In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR [MSP]. Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction [PCR]. MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role

Ror2 promoter methylation change in osteoblastic differentiation of mesenchymal stem cells

Osteoblasts arise from multipotent mesenchymal stem cells [MSCs] present in the bone marrow stroma and undergo further differentiation to osteocytes or bone cells. Many factors such as proteins present in the Wnt signaling pathway affect osteoblast differentiation. ROR2 is an orphan tyrosine kinase receptor that acts as a co-receptor in the non-canonical Wnt signaling pathway. However, ROR2 has been shown to be regulated by both canonical and non-canonical Wnt signaling pathways, ROR2 expression increases during differentiation of MSCs to osteoblasts and then decreases as cells differentiate to osteocytes. On the other hand, research has shown that ROR2 changes MSC fate towards osteoblasts by inducing osteogenic transcription factor OSTERIX. Here we speculated whether ROR2 gene expression regulation during osteoblastogenesis is epigenetically determined. MSCs from bone marrow were isolated, expanded and characterized in vitro according to standard procedures. ROR2 promoter methylation status was determined using methylation specific PCR in a multipotent state and during differentiation to osteoblasts. We determined that the demethylation process in ROR2 promoter occurs during the differentiation process. The process of demethylation begins at day 8 and continues until 21 days of differentiation. This result is in concordance with previous works on the role of ROR2 on osteoblast differentiation, which have shown an upregulation of ROR2 expression during this process

[3D culture of umblical cord blood-drived CD34 + cells on a DBM scaffold coated by cord blood-derived unrestricted somatic stem cells [USSC]]

Allogeneic transplantation with umbilical cord blood [UCB] in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell [MPC] -unrestricted somatic stem cells [USSC] -was incorporated in an attempt to expand CD34+ cells from UCB. To provide a similar environment in vitro, we coated DBM scaffold with USSC cells as the matrix for support UCB-CD34+ cells growth. Human placenta USSC was isolated and characterized by morphologic and immunophenotypical analysis. UCB CD34+ cells were expanded by coculture with placental USSC in 2D and 3D environment. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture [LTC-IC] output. Ex vivo expansion of UCB hematopoietic cells, when cultured in different 2D conditions and 3D condition for 3 weeks, was significantly enhanced, the total cell count increased within the 28-day period. For total CFC, the highest CFC expansion was observed at day 14.Flow cytometry analysis of the percentage of CD34+ cells showed a decline in USSC cocultures in 2D and 3D condition at 3 weeks. These results strongly suggest that human USSC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro and USSC-coated DBM can therefore provide an ex vivo mimicry of bone marrow by enhancing of surface/ volume ratio and feeder layers, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation

Development of a novel three-dimensional biocompatible nanofibrous scaffold for the expansion and hepatogenic differentiation of human bone marrow mesenchymal stem cells

In this present study, we examined the differentiation potential of human bone marrow derived mesenchymal stem cells [hBMSCs] into hepatocytes on a three-dimentional [3D] nanofibrous scaffold formed by Poly [e-caprolactone] [PCL], collagen and polyethersulfone [PES]. The nanofiber was prepared by the electrospining technique. HBMSCs were isolated using combining gradient density centrifugation with plastic adherence. Flow cytometric analysis was used to identify the isolated MSCs. The performance of the cells on the scaffold was evaluated by scanning electron microscopy [SEM] and MTT assay. The hBMSCs were then cultured in a hepatic differentiation medium containing hepatocyte growth factor [HGF], oncostatin M [OSM] and dexamethasone [DEX] for up to 21 days. The results showed that the isolated hBMSCs expressed specific markers such as CD44, CD166, CD105 and CD13. The integrity of the MSCs was further confirmed by their differentiation potential to osteogenic and adipogenic lineages. Scanning electron micrographs and MTT analysis revealed that the cells adhered and proliferated well on the nanofibrous hybrid scaffolds. Immunocytochemical analysis of albumin and a-fetoprotein [AFP] showed the accumulation of these markers in the differentiated cells on the scaffold. Hepatocyte differentiation was further confirmed by showing expression of albumin, AFP and cytokeratin-19 [CK-19] at mRNA levels in differentiated cells. In conclusion, the evidences presented in this study show that the engineered scaffold is promising for maintenance of hepatocyte-like cells suitable for transplantation

Analysis of HLA-G gene expression in b-lymphocytes from chronic lymphocytic leukemia patients

The human leukocyte antigen G [HLA-G] molecule exhibits limited tissue distribution, low polymorphism and alternative splicings that generate seven HLA-G isoforms. HLA-G exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells that may favor their escape from anti-tumor immune responses. This study it is an effort to clarify the presence of HLA-G in B-cell chronic lymphocytic leukemia [B-CLL] patients. HLA-G mRNA expression was studied in a pilot study in circulating B-CLL and also healthy controls by reverse transcription [RT]-PCR using a set of pan-HLA-G primers. RT-PCR was performed on B-cells from 74 B-CLL patients and 12 healthy controls. The data showed HLA-G gene expression in 20% of the B-CLL patients. No expression of HLA-G could be detected in the healthy control group. These data suggest that HLA-G is expressed at the gene level in B cells from B-CLL patients but not in B cells from healthy controls. Further study is required to clarify the role of HLA-G as a regulatory factor that could affect immune response in B-CLL patients

Detection of 22q 11.2 microdeletions by Semiquantitaive multiplex PCR [SQMPCR]

22q11.2 chromosomal region is a hot spot for many cytogenetic rearrangements especially micro deletions which are responsible for DiGeorge and VeloCaridoFacial syndromes. The most characteristic sign in these patients is congenital cardiac conotruncal anomalies. The gold standard diagnostic test for these micro deletions is FISH [Fluorescent In Situ Hybridization]. However this diagnostic technique has some drawbacks such a high final cost and low sensitivity in smaller and uncommon micro deletions found in this region. The aim of this study was to introduce a less expensive and a priori more sensitive molecular method to help small and peripheral laboratories to find genetic causes of congenital heart diseases and DiGeorge syndrome. 10 patients with congenital conotruncal anomalies and symptoms of DiGeorge syndrome were included in this study. These patients had been analyzed by FISH probe TUPLE1 before the inclusion. 3 normal persons were included as normal controls for micro deletion region. Semi Quantitative Multiplex PCRs were designed based on known markers in and out of the region of interest. Results were analyzed by Total Lab software. 4 patients showed a decrease in gene dosage more than 60% compared to normal persons. FISH analysis found only one patient with micro deletion. The designed method based on semi quantitative PCR was able to find 4 patients [40%] with micro deletion in a population of 10 patients with congenital cardiac anomalies. This techniques was also able to find micro deletions in three FISH negative patients. Molecular diagnosis of micro deletions is supposed to be more sensitive than FISH in small micro deletions. This study confirms the presence of a typical deletions in Iranian patients and shows that the applied technique can detect some FISH negative patients. However further studies are needed to determine the sensitivity and specificity of the mentioned molecular diagnosis. It seems that this can be used at least for the patients with typical phenotypic features of 22q11Ds and negative FISH results