Aungkura Supokawej

Immunosuppressive properties of mesenchymal stromal cells derived from amnion, placenta, Wharton's jelly and umbilical cord

The role of bone marrow‐derived mesenchymal stromal cells (BM‐MSC) in preventing the incidence and ameliorating the severity of graft‐versus‐host disease (GvHD) has recently been reported. However, as the collection of BM‐MSC is an invasive procedure, more accessible sources of MSC are desirable.

Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment

Objective: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs). Materials and Methods: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 μM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidine–treated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers. Results: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofluorescence analysis examining the expression of the cardiac-specific markers GATA4 and troponin T and the skeletal muscle-specific markers MyoD and myogenin revealed that cells treated with 15 μM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifications indicated that there were higher expression levels of cardiac- and skeletal muscle-specific mRNAs in MSCs treated with 15 μm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated. Conclusion: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 μM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types. Conflict of interest:None declared.

Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells.

Human mesenchymal stem cell (hMSC) is a potential source for cell therapy due to its property to promote tissue repair. Although, it has been known that hMSCs promote tissue repair via angiogenic cytokines, the interaction between hMSC-derived cytokines and the endothelial progenitor cells (EPCs), which play an important role in tissue neovascularization, is poorly characterized. We investigate the effect of cytokine released from different sources of hMSCs including bone marrow and gestational tissues on the EPC functions in vitro. The migration, extracellular matrix invasion and vessel formation of EPCs were studied in the presence or absence of cytokines released from various sources of hMSCs using transwell culture system. The migration of EPCs was highest when co-culture with secretory factors from placenta-derived hMSCs (PL-hMSCs) compared to those co-culture with other sources of hMSCs. For invasion and vessel formation, secretory factors from bone marrow-derived hMSCs (BM-hMSCs) could produce the maximal enhancement compared to other sources. We further identified the secreted cytokines and found that the migratory-enhancing cytokine from PL-hMSCs was PDGF-BB while the enhancing cytokine from BM-hMSCs on invasion was IGF-1. For vessel formation, the cytokines released from BM-hMSCs were IGF1 and SDF-1. In conclusion, hMSCs can release angiogenic cytokines which increase the migration, invasion and vessel forming capacity of EPCs. We can then use hMSCs as a source of angiogenic cytokines to induce neovascularization in injured/ischemic tissues.

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues.

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.

Cell type of origin influences iPSC generation and differentiation to cells of the hematoendothelial lineage

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types, namely human umbilical cord vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs), to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However, only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance.

Osteoporosis: the current status of mesenchymal stem cell-based therapy

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.

Exploring the epigenetic drug discovery landscape

ABSTRACT Introduction: Epigenetic modification has been implicated in a wide range of diseases and the ability to modulate such systems is a lucrative therapeutic strategy in drug discovery. Areas covered: This article focuses on the concepts and drug discovery aspects of epigenomics. This is achieved by providing a survey of the following concepts: (i) factors influencing epigenetics, (ii) diseases arising from epigenetics, (iii) epigenetic enzymes as druggable targets along with coverage of existing FDA-approved drugs and pharmacological agents, and (iv) drug repurposing/repositioning as a means for rapid discovery of pharmacological agents targeting epigenetics. Expert opinion: Despite significant interests in targeting epigenetic modifiers as a therapeutic route, certain classes of target proteins are heavily studied while some are less characterized. Thus, such orphan target proteins are not yet druggable with limited report of active modulators. Current research points towards a great future with novel drugs directed to the many complex multifactorial diseases of humans, which are still often poorly understood and difficult to treat.

Targeting Netrin-1 in glioblastoma stem-like cells inhibits growth, invasion, and angiogenesis.

Glioblastoma (GBM) is an aggressive malignant brain tumor that still lacks effective therapy. Glioblastoma stem cells (GBM-SCs) were identified to contribute to aggressive phenotypes and poor clinical outcomes for GBM. Netrin-1, an axon guidance molecule, has been found in several tumors in adults. However, the role of Netrin-1 in GBM-SCs remains largely unknown. In this study, CD133-positive U251 GBM cells were used as a putative GBM-SC population to identify the functions of Netrin-1. Using lentiviral transduction, Netrin-1 miR RNAi vectors were transduced into CD133-positive U251 cells. We demonstrated that cell proliferation and survival were decreased following targeted deletion of Netrin-1. Cell invasion was dramatically diminished in Netrin-1 knockdown GBM-SCs. Moreover, Netrin-1 knockdown GBM-SCs exhibited less proangiogenic activity. In conclusion, Netrin-1 may represent a therapeutic target in glioblastoma.

CD14−/CD34+ is the founding population of umbilical cord blood-derived endothelial progenitor cells and angiogenin1 is an important factor promoting the colony formation

The origin of endothelial progenitor cells (EPCs) in umbilical cord blood (UCB) is unknown. In this study, we explored the origin of UCB-derived EPCs by culturing CD14+ or CD14− subpopulation separately and co-culturing these two subpopulations either with or without transwells. We found no colony formation with CD14+ or CD14− subpopulation alone, but there were EPC colonies observed in direct co-cultures of both subpopulations. Transwell culture system was used to further study the effect of cytokines on EPC colony formation. We observed the presence of EPC colonies derived from CD14− subpopulation in the presence of CD14+ subpopulation in the upper compartment whereas there was no colony generated from CD14+ subpopulation with CD14− subpopulation in the upper compartment. Therefore, CD14− subpopulation is likely to be the origin of EPCs and EPC colony derivation requires cytokines released from CD14+ subpopulation. We further characterized the founding population of UCB-derived EPCs by separating CD14− subpopulation into CD14−/CD34+ and CD14−/CD34− subpopulations. There were colonies observed only in co-cultures of CD14+ with CD14−/CD34+ subpopulation but not with CD14−/CD34− subpopulation either with or without transwells. We screened 42 cytokines involving in angiogenesis using an ELISA array in the supernatant collected from CD14+ compared to CD14− subpopulations. We found consistently the presence of angiogenin1 in the supernatant of CD14+ subpopulation but not in that of CD14− subpopulation. The addition of angiogenin1 in culture of CD14− subpopulation yielded EPC colonies. We conclude that UCB-derived EPCs are confined to CD14−/CD34+ subpopulation and angiogenin1 released from CD14+ subpopulation may be an important factor promoting the EPC colony formation.

Computational identification of miRNAs that modulate the differentiation of mesenchymal stem cells to osteoblasts

MicroRNAs (miRNAs) are small endogenous noncoding RNAs that play an instrumental role in post-transcriptional modulation of gene expression. Genes related to osteogenesis (i.e., RUNX2, COL1A1 and OSX) is important in controlling the differentiation of mesenchymal stem cells (MSCs) to bone tissues. The regulated expression level of miRNAs is critically important for the differentiation of MSCs to preosteoblasts. The understanding of miRNA regulation in osteogenesis could be applied for future applications in bone defects. Therefore, this study aims to shed light on the mechanistic pathway underlying osteogenesis by predicting miRNAs that may modulate this pathway. This study investigates RUNX2, which is a major transcription factor for osteogenesis that drives MSCs into preosteoblasts. Three different prediction tools were employed for identifying miRNAs related to osteogenesis using the 3’UTR of RUNX2 as the target gene. Of the 1,023 miRNAs, 70 miRNAs were found by at least two of the tools. Candidate miRNAs were then selected based on their free energy values, followed by assessing the probability of target accessibility. The results showed that miRNAs 23b, 23a, 30b, 143, 203, 217, and 221 could regulate the RUNX2 gene during the differentiation of MSCs to preosteoblasts.