Figure S3. Characterization of hESC lines derived from MTP. (A) Total transcripts OCT4, SOX2, and NANOG of each hESC line extracted and amplified by PCR using specific primer. PCR product of each transcript from different hESC lines subjected to same agarose gel and same exposure. RT-PCR analysis shows that hESC lines derived from MTP expressed core pluripotent genes, OCT4, NANOG, and SOX2. (B) Immunofluorescent staining shows hESCs derived from MTP expressed typical hESC marker proteins. (C) Spontaneous differentiation of seven hESC cell line-generated derivatives of all three primitive germ layers demonstrated by expression of NESTIN, SMA, and AFP. (D) Hematoxylin and eosin staining of teratoma generated from Si1â Si4 and SiAtra1 showed tissues from three primitive germ layers including endoderm, mesoderm, and ectoderm. Scale bar: 50Â Îźm. (ZIP 7514 kb)
Summary Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, preparation of a unit of concentrated platelet for transfusion requires at least 4–6 units of whole blood. At present, a platelet unit from a single donor can be prepared using apheresis, but lack of donors is still a major problem. Several approaches to produce platelets from other sources, such as haematopoietic stem cells and pluripotent stem cells, have been attempted but the system is extremely complicated, time-consuming and expensive. We now report a novel and simpler technology to obtain platelets using transdifferentiation of human bone marrow erythroblasts to megakaryocytes with overexpression of the FLI1 and ERG genes. The obtained transdifferentiated erythroblasts (both from CD71+ and GPA+ erythroblast subpopulations) exhibit typical features of megakaryocytes including morphology, expression of specific genes (cMPL and TUBB1) and a marker protein (CD41). They also have the ability to generate megakaryocytic CFU in culture and produce functional platelets, which aggregate with normal human platelets to form a normal-looking clot. Overexpression of FLI1 and ERG genes is sufficient to transdifferentiate erythroblasts to megakaryocytes that can produce functional platelets.
Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.
Abstract Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype, IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI), a specific LATS inhibitor, or shRNA targeting LATS1/2 significantly reduced clonogenicity, increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore, depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together, our results suggest that LATS could be one of the key factors contributing to the rapid proliferation, reduced apoptosis, and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM.
Abstract Mesenchymal stem cells (MSCs) are important in regenerative medicine because of their potential for multi-differentiation. Bone marrow, chorion and placenta have all been suggested as potential sources for clinical application. However, the osteogenic differentiation potential of MSCs derived from chorion or placenta is not very efficient. Bone morphogenetic protein-2 (BMP-2) plays an important role in bone development. Its effect on osteogenic augmentation has been addressed in several studies. Recent studies have also shown a relationship between miRNAs and osteogenesis. We hypothesized that miRNAs targeted to Runt-related transcription factor 2 (Runx-2), a major transcription factor of osteogenesis, are responsible for regulating the differentiation of MSCs into osteoblasts. This study examines the effect of BMP-2 on the osteogenic differentiation of MSCs isolated from chorion and placenta in comparison to bone marrow-derived MSCs and investigates the role of miRNAs in the osteogenic differentiation of MSCs from these sources. MSCs were isolated from human bone marrow, chorion and placenta. The osteogenic differentiation potential after BMP-2 treatment was examined using ALP staining, ALP activity assay, and osteogenic gene expression. Candidate miRNAs were selected and their expression levels during osteoblastic differentiation were examined using real-time RT-PCR. The role of these miRNAs in osteogenesis was investigated by transfection with specific miRNA inhibitors. The level of osteogenic differentiation was monitored after anti-miRNA treatment. MSCs isolated from chorion and placenta exhibited self-renewal capacity and multi-lineage differentiation potential similar to MSCs isolated from bone marrow. BMP-2 treated MSCs showed higher ALP levels and osteogenic gene expression compared to untreated MSCs. All investigated miRNAs (miR-31, miR-106a and miR148) were consistently downregulated during the process of osteogenic differentiation. After treatment with miRNA inhibitors, ALP activity and osteogenic gene expression increased over the time of osteogenic differentiation. BMP-2 has a positive effect on osteogenic differentiation of chorion- and placenta-derived MSCs. The inhibition of specific miRNAs enhanced the osteogenic differentiation capacity of various MSCs in culture and this strategy might be used to promote bone regeneration. However, further in vivo experiments are required to assess the validity of this approach.
Mesenchymal stem cells (MSCs) derived from amnion are considered to be adult stem cells that can be easily obtained in large quantities by a less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However; the biological properties and the differentiation capacity of amnion-derived MSCs (AM-MSCs) are still poorly characterized. The objectives of this study were to isolate, characterize and explore the potential of AM-MSCs in differentiating toward neural lineage in comparison to those of BM-MSCs. To isolate AM-MSCs, amnion was digested with trypsin-EDTA and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. The expression profiles of several MSC markers were examined by flow cytometry. AM-MSCs from passage 3-5 were used for adipogenic, osteogenic and neural differentiation assays by culturing in appropriate induction media. The expression of several neural marker genes, including MAP-2, GFAP and beta-tubulin III in AM-MSCs was determined by quantitative real time-PCR. The expression of neural-specific markers, MAP-2 and beta-tubulin III, was subsequently confirmed by immunocytochemistry using confocal laser microscope. The results demonstrated that AM-MSCs could be easily expanded to 18-20 passages while maintaining the undifferentiated state and exhibiting MSC markers (CD73, CD90, and CD105) but do not express the hematopoietic markers (CD34 and CD45). Similar to BM-MSCs, AM-MSCs were able to differentiate to several mesodermal-lineages including adipocytes and osteoblasts. Moreover; these cells could be induced to differentiate to neuron-like cells as characterized by cell morphology and the expression of several neural markers including MAP-2, GFAP and beta-tubulin III. The present study demonstrated that AM-MSCs can be easily obtained and expanded in culture. These cells also have transdifferentiation capacity as evidenced by their neural differentiation potential. According to the results, amnion can be used as an alternative source of MSCs for stem cell therapy in neurodegenerative diseases.
During the last decade, stem cells emerge as a potential alternative treatment for several degenerativediseases. Despite their ability to generate most specific cell types for cell replacement therapy and tissue engineering,the uses of embryonic stem cells, induced pluripotent stem (iPS) cells and adult stem cells in clinical applicationsare still limited. There are several problems that need to be addressed before stem cell treatment can be consideredfor practical applications. According to that, the carefully designed basic and clinical studies have to be performedto improve our understanding on basic biology and therapeutic potentials of stem cells, as well as the safety andlong-term benefits of stem cell therapy. Key words: Stem cell, Regenerative medicine, Therapeutic cloning
Objective: To study the accuracy of self vaginal douching and collection for HPV types 16, 18, 31 and 33 in women visiting Thammasat Hospital for the explicit purpose of cervical screening. Methods: A pelvic examination and Pap smear were performed for all women who came for cervical screening. Specimens were also collected by self vaginal douching before cervical screening and sent to the cell and molecular biology laboratory for analysis of human papillomavirus (HPV) types 16, 18, 31 and 33 using the polymerase chain reaction (PCR). Results: HPV prevalence was 3.6% overall from 250 women in this study. Twenty-four (9.6%) women had an abnormal cytology screening result. No cancer was found. Four women had a high grade squamous intraepithelial lesion (HSIL) and 14 had a low grade squamous intraepithelial lesion (LSIL) from colposcopic biopsy. Self vaginal douching for HPV 16, 18, 31 and 33 was used to predict abnormal Pap smear. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 12.5%, 97.5%, 33.3% and 91.3%, respectively. Conclusion: From our analysis of self vaginal douching for HPV detection using cases from Thammasat university hospital, it cannot replace the Pap smear.
Fisetin and quercetin, polyphenol flavonoids, have been shown to have a wide range of beneficial pharmacological effects including anti-inflammatory, anti-oxidative, and anti-cancer. Our previous work shows that fisetin also affects the specification of the adipogenic-osteogenic lineage of human mesenchymal stem cells (hMSCs) by modulating the Hippo-YAP signaling pathway. Although quercetin has a structure similar to that of fisetin, its effects on the functional properties of hMSCs have not yet been investigated. The objective of this study is to determine the effects of quercetin on the various properties of hMSCs, including proliferation, migration, and differentiation capacity toward adipogenic and osteogenic lineages. The results show that while fisetin increases hMSC adipogenic differentiation, quercetin inhibited adipogenic differentiation of hMSCs. The inhibition is mediated, at least in part, by the activation of hippo signaling and up-regulation of miR-27b, which inhibits the expression of genes involved in all critical steps of lipid droplet biogenesis, resulting in a decrease in the number of lipid droplets in hMSCs. It is possible that the lack of hydroxylation of the 5 position on the A ring of quercetin could be responsible for its different effect on the adipogenic-osteogenic lineage specification of hMSCs compared to fisetin. Molecular docking and molecular dynamics simulation suggested that fisetin and quercetin possibly bind to serine / threonine protein kinases 4 (STK4/MST1), which is an upstream kinase responsible for LATS phosphorylation. Taken together, our results demonstrate more insight into the mechanism underlying the role of flavonoid fisetin and quercetin in the regulation of adipogenesis.
