Chromosomal Abnormalities in Myelodysplastic Syndrome Patients in Upper Northern Thailand.

OBJECTIVE: Chromosome detection is important in the diagnosis and prognosis of Myelodysplastic syndrome (MDS) patients. About 50% of MDS patients have chromosomal abnormalities. Moreover, chromosome 5 and 7 are common genetic abnormalities in MDS patients and use to identify prognosis risk group and the proper treatment in MDS patients. The objective of this study was to evaluate chromosomal abnormalities and clinical features of MDS patients in upper northern Thailand. METHODS: Fifty bone marrow (BM) specimens were examined by conventional cytogenetic (CC) technique and fluorescence in situ hybridization (FISH) technique for detected chromosome 5 and 7 abnormalities. The clinical features were comparison between MDS patients with chromosomal abnormalities and those with normal karyotype. RESULTS: Chromosomal abnormalities were detected in 8/50 MDS patients by CC and 17/50 cases by FISH technique. When the CC and FISH techniques were combined, chromosomal abnormalities increased to 21/50 cases.  Abnormalities of isolated chromosome 5 were found in 13 cases and were associated with lower level of percentage blast of BM (p = 0.003) and higher level of hemoglobin (p = 0.019). Moreover, abnormalities of chromosome 7 were found in 3 cases, 1 case of isolated del(7q) and 2 cases of -7 and del(7q) with complex abnormalities. These three cases were associated with higher level of percentage blast of BM (p = 0.010). CONCLUSION: This study showed the frequency and pattern of chromosomal abnormalities of MDS patients in upper northern Thailand were different from other populations. MDS with isolated chromosome 5 abnormalities had clinical characteristics corresponding with patients in good prognosis risk group. However, MDS patients with chromosome 7 and complex abnormalities showed higher percentage blast of BM which high risk to progression to acute myeloid leukemia (AML). Combined CC and FISH techniques detect chromosomal abnormalities with greater frequency than when either technique is used alone.
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Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells.

The aim of this study was to evaluate the efficiency of ascorbic acid (AA) on cell viability, cytotoxicity and the effects on cardiomyogenic differentiation of the human amniotic fluid mesenchymal stem cells (hAF-MSCs). The results of methylthiazole tetrazolium (MTT) assay and cell apoptosis assay indicated that after 24, 48 and 72 h of treatment, AA had no effect on cells viability and cytotoxicity. After treating the hAF-MSCs with 5-azacytidine (5-aza) and a combination of AA and 5-aza, the alamar blue cells proliferation assay showed the normal growth characteristic similar to control group. Especially, the morphological changes were observed between day 0 and day 21, and it was revealed that the hAF-MSCs exhibited myotube-like morphology after 7 days of cell culturing. Moreover, the treatment with a combination of AA and 5-aza was able to up-regulate the cardiomyogenic specific gene levels, which are known to play an important role in cardiomyogenesis. This was specifically notable with the results of immunofluorescence and immunoenzymatic staining in the AA combined with 5-aza treatment group, the highest expression of cardiomyogenic specific proteins was revealed including for GATA4, cTnT, Cx43 and Nkx2.5. It could be concluded that AA may be a good alternative cardiomyogenic inducing factor for hAF-MSCs and may open new insights into future biomedical applications for a clinically treatment.

Human platelet lysate as an alternative to fetal bovine serum for culture and endothelial differentiation of human amniotic fluid mesenchymal stem cells.

Human amniotic fluid (hAF) mesenchymal stem cells (MSCs) are commonly cultured in medium containing FBS. However, there are concerns about using animal serum in therapeutic applications due to the potential for immunogenic reactions and the risk of transmission of pathogens. For safety reasons, human platelet lysate (hPL) has been suggested as a replacement for FBS because it appears to be a natural source of growth factors. In this present study, it was investigated whether FBS could be substituted with hPL in hAF­MSCs culture without affecting their properties. Pooled hPL was generated by the freeze­thaw method. The concentration of hPL was selected after evaluation by MTT assay. The hAF­MSCs were cultured in FBS­ or hPL­supplemented conditions and shared a fibroblast­like morphology. Cell proliferation assays showed that the growth characteristic of hAF­MSCs cultured in 10% hPL­supplemented media was similar to those cultured in 10% FBS­supplemented media. The expression of MSC markers did not differ between the cells cultured in the different conditions. The endothelial differentiation potential was also investigated. Reverse transcription­quantitative (RT­q)PCR revealed that induced cells supplemented with hPL showed an increase level of endothelial specific gene expression compared to the FBS­supplemented cells. Immunofluorescence analysis showed specific protein localization in both induced cell groups. Additionally, induced cells supplemented with hPL had the potential to form networks on Matrigel. This present study indicated that hPL could be used to culture and enhance the endothelial differentiation potential of hAF­MSCs.

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions.

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.