Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study.

Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.

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.

Amniotic fluid: Source of valuable mesenchymal stem cells and alternatively used as cryopreserved solution.

Mesenchymal stem cells (MSCs), which possess remarkable capabilities, are found in amniotic fluid (AF). The findings of several studies have shown the potential benefits of these cells in applications of regenerative medicine. In clinical applications, an over-period of time is required in a preparation process that makes cell collection become more necessary. Herein, the aim of this study was to preserve and characterize the cell's properties after cell cryopreservation into an appropriate cryogenic medium. The results illustrated that the highest hAF-MSCs viability was found when the cells were conserved in a solution of 5% DMSO + 10% FBS in AF. However, no statistical differences were identified in a chromosomal aberration of the post-thawed cells when compared to the non-frozen cells. These cells could also maintain their MSC features through the ability to express cell prolific quality, illustrating the typical MSC markers and immune privilege properties of CD44, CD73, CD90 and HLA-ABC. Additionally, post-thawed cells were able to differentiate into chondrogenic lineage by exhibiting chondrogenic related genes (SOX9, AGC, COL2A1) and proteins (transcription factor SOX9 protein (SOX9), cartilage oligomeric matrix protein (COMP) and aggrecan core protein (AGC)), as well as to present sGAGs accumulation. Interestingly, the use of a transmission electron microscope (TEM) uncovered the enrichment of the rough endoplasmic reticulum (rER) that coincided with euchromatin and the prominent nucleolus in the chondrogenic-induced cells that are normally found in the cells of natural cartilage. All in all, this study manifested that AF can be a major consideration and applied for use as a co-mixture of cryogenic medium.

FLT3-ITD, NPM1, and DNMT3A Gene Mutations and Risk Factors in Normal Karyotype Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients in Upper Northern Thailand

Objective: Approximately 40-45% of AML and MDS patients have a cytogenetically normal karyotype (CN-AML and CN-MDS). The frequency and types of gene mutations in these cases may differ among various populations. The objective of this study was to identify frequencies and types of FLT3-ITD, NPM1, and DNMT3A mutations, and associations of them with clinical data and risk factors in CN-AML and CN-MDS cases in upper Northern Thailand. Methods: Bone marrow samples of 40 CN-AML and 60 CN-MDS patients were analyzed for gene mutations by direct sequencing. In addition, data for potential risk factors were obtained for comparison. Results: Frequencies of FLT3-ITD, NPM1, and DNMT3A mutations were 25.0%, 17.5%, and 10.0%, respectively in CN-AML, but all zero in CN-MDS cases. NPM1 mutations were found at a median age older than the wild type (58 vs 47 years) while DNMT3A mutations were associated with an increase in the white blood cell count. In all patients, factors for the mutations of these three genes included age ≤ 60 years, and a history of hypertension. Conclusion: When considering mutations in only normal karyotype patients, the frequency of FLT3-ITD, NPM1, DNMT3A mutations in CN-AML patients in upper Northern Thailand were found to occur at lower rates than in Western patients and to differ from other Asian populations including parts of Thailand. No mutations were observed in CN-MDS cases. Some types of gene mutations differed from previous studies, possibly attributable to differences in geography, lifestyle and genetic backgrounds. Links with age ≤ 60 years and history of hypertension were found. Investigation of these three genes in an intermediate risk group with a normal karyotype is useful for a better understanding of molecular leukemogenetic steps in CN-AML and CN-MDS patients and may be beneficial for planning treatment and prevention in the population of upper Northern Thailand.

Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte­like cells.

Ischemic heart disease (IHD) is a major factor influencing worldwide mortality rates. Furthermore, IHD has become a significant health problem among the Thai population. Stem cell therapy using mesenchymal stem cells (MSCs) is an alternative therapeutic method that has been applied to improve the quality of life of patients. Amniotic fluid (AF) contains a heterogeneous cell population, including MSCs, which are multipotent stem cells that have the capability to differentiate into mesenchymal lineages. The purpose of the present study was to evaluate the MSC characteristics of human (h)AF and determine its potency regarding cardiogenic differentiation. MSC characterization following flow cytometric analysis revealed that the cells expressed MSC markers, cluster of differentiation (CD)44, CD90, human leukocyte antigen­ABC and CD73. The results of the alamar blue assay demonstrated that cell proliferation rate continuously increased from the early cultivation phase up to 5­fold during days 1 to 5 of cell culturing. The highest rate of cell proliferation was observed on day 17 with a 30­fold increase compared with that on day 1. During the cardiogenic induction stage, morphological changes were observed between day 0 and day 21, and it was revealed that the hAF derived­MSCs in the cardiogenic­induced group exhibited myotube­like morphology after 7 days of cell culturing. Following cardiogenic induction, immunohistochemistry staining was performed on day 21, and reverse transcription­quantitative polymerase chain reaction on day 7 and 21. These steps were performed to detect the protein and gene expression levels of cardiac specific proteins (GATA4, cardiac troponin T, Nkx2.5 and Connexin43). The results of the present study indicated that hAF­MSCs possess the potential to differentiate into cardiomyocyte­like cells. Thus, it was concluded that hAF may be a suitable source of MSCs for stem cell therapy and tissue engineering.