PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-ß and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-ß and suppressing Wnt/ß-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

The Human Placental Amniotic Membrane Mesenchymal-Stromal-Cell-Derived Conditioned Medium Inhibits Growth and Promotes Apoptosis of Human Cholangiocarcinoma Cells In Vitro and In Vivo by Suppressing IL-6/JAK2/STAT3 Signaling.

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.

Fucoxanthin diminishes oxidative stress damage in human placenta-derived mesenchymal stem cells through the PI3K/Akt/Nrf-2 pathway.

Placenta-derived mesenchymal stem cells (PL-MSCs) have therapeutic potential in various clinical contexts due to their regenerative and immunomodulatory properties. However, with increasing age or extensive in vitro culture, their viability and function are gradually lost, thus restricting their therapeutic application. The primary cause of this deterioration is oxidative injury from free radicals. Therefore, enhancing cell viability and restoring cellular repair mechanisms of PL-MSCs in an oxidative stress environment are crucial in this context. Fucoxanthin, a carotenoid derived from brown seaweed, demonstrates antioxidant activity by increasing the production of antioxidant enzymes and lowering the levels of reactive oxygen species (ROS). This study aimed to determine whether fucoxanthin protects PL-MSCs from hydrogen peroxide (H2O2)-induced oxidative stress. After characterization, PL-MSCs were co-treated with fucoxanthin and H2O2 for 24 h (co-treatment) or pre-treated with fucoxanthin for 24 h followed by H2O2 for 24 h (pre-treatment). The effects of fucoxanthin on cell viability and proliferation were examined using an MTT assay. The expression of antioxidant enzymes, PI3K/Akt/Nrf-2 and intracellular ROS production were investigated in fucoxanthin-treated PL-MSCs compared to the untreated group. The gene expression and involvement of specific pathways in the cytoprotective effect of fucoxanthin were investigated by high-throughput NanoString nCounter analysis. The results demonstrated that co-treatment and pre-treatment with fucoxanthin restored the viability and proliferative capacity of PL-MSCs. Fucoxanthin treatment increased the expression of antioxidant enzymes in PL-MSCs cultured under oxidative stress conditions and decreased intracellular ROS accumulation. Markedly, fucoxanthin treatment could restore PI3K/Akt/Nrf-2 expression in H2O2-treated PL-MSCs. High-throughput analysis revealed up-regulation of genes involved in cell survival pathways, including cell cycle and proliferation, DNA damage repair pathways, and down-regulation of genes in apoptosis and autophagy pathways. This study demonstrated that fucoxanthin protects and rescues PL-MSCs from oxidative stress damage through the PI3K/Akt/Nrf-2 pathway. Our data provide the supporting evidence for the use of fucoxanthin as an antioxidant cytoprotective agent to improve the viability and proliferation capacity of PL-MSCs both in vitro and in vivo required to increase the effectiveness of MSC expansion for therapeutic applications.

Intron Regions as Genetic Markers for Population Genetic Investigations of Opisthorchis viverrini sensu lato and Clonorchis sinensis.

Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts.

Distribution of Blastocystis subtypes isolated from various animal hosts in Thailand.

Blastocystis is a parasitic protist of a variety of hosts, including humans. Mapping the distribution of Blastocystis and its genetic variants across different host species can help us understand the epidemiology of this organism and its role in health and disease. This study aimed to identify subtypes of Blastocystis detected in different animal hosts in Thailand. A total of 825 fecal samples belonging to 18 vertebrate orders, 36 families, 68 genera, and 80 species were collected. Of these, 111 specimens were Blastocystis-positive by culture. Seventy-nine samples were subjected to small subunit (SSU) ribosomal DNA amplification by PCR, and reliable subtype data were obtained for 61 specimens. At least 14 subtypes (ST), namely ST1 to ST10, ST14/ST24/ST25 complex, ST23, ST26, and ST29 were detected. In addition, Blastocystis was found in tortoises. ST1 (3.2%) and ST5 (11.5%) were found in pigs, ST2 (1.6%) and ST3 (3.2%) in non-human primates, ST4 (14.7%) in rodents and ruminants, ST6 (4.9%), ST7 (30%), ST9 (1.6%), and ST29 (1.6%) in birds, ST8 (6.6%) in Green peafowl and East Asian Porcupine, and ST10 (4.9%), ST14/ST24/ST25 (9.8%), ST23 (1.6%) and ST26 (1.6%) in ruminants. The sequence recovered from the elongated tortoises (Indotestudo elongata) (3.2%) was phylogenetically placed within the reptilian cluster of Blastocystis, for which no subtype system is available yet. Of note, we did not obtain Blastocystis sequences from any of the many canids and felids sampled in the study, and our data are in support of host specificity of Blastocystis, according to both colonization and subtype distribution.

Osteogenic differentiation and proliferation potentials of human bone marrow and umbilical cord-derived mesenchymal stem cells on the 3D-printed hydroxyapatite scaffolds.

Mesenchymal stem cells (MSCs) are a promising candidate for bone repair. However, the maintenance of MSCs injected into the bone injury site remains inefficient. A potential approach is to develop a bone-liked platform that incorporates MSCs into a biocompatible 3D scaffold to facilitate bone grafting into the desired location. Bone tissue engineering is a multistep process that requires optimizing several variables, including the source of cells, osteogenic stimulation factors, and scaffold properties. This study aims to evaluate the proliferation and osteogenic differentiation potentials of MSCs cultured on 2 types of 3D-printed hydroxyapatite, including a 3D-printed HA and biomimetic calcium phosphate-coated 3D-printed HA. MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) were cultured on the 3D-printed HA and coated 3D-printed HA. Scanning electron microscopy and immunofluorescence staining were used to examine the characteristics and the attachment of MSCs to the scaffolds. Additionally, the cell proliferation was monitored, and the ability of cells to differentiate into osteoblast was assessed using alkaline phosphatase (ALP) activity and osteogenic gene expression. The BM-MSCs and UC-MSCs attached to a plastic culture plate with a spindle-shaped morphology exhibited an immunophenotype consistent with the characteristics of MSCs. Both MSC types could attach and survive on the 3D-printed HA and coated 3D-printed HA scaffolds. The MSCs cultured on these scaffolds displayed sufficient osteoblastic differentiation capacity, as evidenced by increased ALP activity and the expression of osteogenic genes and proteins compared to the control. Interestingly, MSCs grown on coated 3D-printed HA exhibited a higher ALP activity and osteogenic gene expression than those cultured on the 3D-printed HA. The finding indicated that BM-MSCs and UC-MSCs cultured on the 3D-printed HA and coated 3D-printed HA scaffolds could proliferate and differentiate into osteoblasts. Thus, the HA scaffolds could provide a suitable and favorable environment for the 3D culture of MSCs in bone tissue engineering. Additionally, biomimetic coating with octacalcium phosphate may improve the biocompatibility of the bone regeneration scaffold.

Human Mesenchymal Stem Cells Derived from the Placenta and Chorion Suppress the Proliferation while Enhancing the Migration of Human Breast Cancer Cells.

Background: Breast cancer is the most frequently diagnosed malignancy among women, resulting from abnormal proliferation of mammary epithelial cells. The highly vascularized nature of breast tissue leads to a high incidence of breast cancer metastases, resulting in a poor survival rate. Previous studies suggest that human mesenchymal stem cells (hMSCs) play essential roles in the growth, metastasis, and drug responses of many cancers, including breast cancer. However, hMSCs from different sources may release different combinations of cytokines that affect breast cancer differently. Methods: In this study, we have isolated hMSCs from the placenta (PL-hMSCs) and the chorion (CH-hMSCs) and determined how these hMSCs affect the proliferation, migration, invasion, and gene expression of two human breast cancer cells, MCF-7 and MDA-MB-231, as well as the possible mechanisms underlying those effects. Results: The results showed that the soluble factors derived from PL-hMSCs and CH-hMSCs inhibited the proliferation of MCF-7 and MDA-MB-231 cells but increased the migration of MDA-MB-231 cells. The study of gene expression showed that PL-hMSCs and CH-hMSCs downregulated the expression levels of the protooncogene CyclinD1 while upregulating the expression levels of tumor suppressor genes, P16 and P21 in MCF-7 and MDA-MB-231 cells. Furthermore, hMSCs from both sources also increased the expression levels of MYC, SNAI1, and TWIST, which promote the epithelial-mesenchymal transition and migration of breast cancer cells in both cell lines. The functional study suggests that the suppressive effect of CH-hMSCs and PL-hMSCs on MCF-7 and MDA-MB231 cell proliferation was mediated, at least in part, through IFN-γ. Conclusions: Our study suggests that CH-hMSCs and PL-hMSCs inhibited breast cancer cell proliferation by negatively regulating CYCLIND1 expression and upregulating the expression of the P16 and P21 genes. In contrast, hMSCs from both sources enhanced breast cancer cell migration, possibly by increasing the expression of MYC, SNAI1, and TWIST genes in those cells.

Human chorion-derived mesenchymal stem cells suppress JAK2/STAT3 signaling and induce apoptosis of cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment.

Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of Rhipicephalus microplus clade C and the rarely detected R. haemaphysaloides.

In total, 160 ticks infesting cattle in the northeast region of Thailand were collected and used for molecular investigation. Three tick species-Rhipicephalus microplus Canestrini, Rhipicephalus haemaphysaloides Supino and Haemaphysalis bispinosa Neumann-were identified based on morphology and DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S rRNA). In total, 26 and seven unique haplotypes of the CO1 and 16S rRNA genes, respectively, were recovered. Phylogenetic analysis using the CO1 sequence revealed that the R. microplus from northeastern Thailand were grouped into the previously described clades A and C, whereas the 16S rRNA phylogenetic tree assigned all isolates of R. microplus from Northeast Thailand into the previously described clade B. Clade C of the CO1 phylogenetic tree is a new genetic assemblage recently discovered from India and Malaysia, which has now been detected in our study. The haplotype network also demonstrated that R. microplus is divided into two haplogroups corresponding to the assemblage of the CO1 phylogenetic tree. Our findings strongly support the previous genetic assemblage classification and evidence that R. microplus from Northeast Thailand is a species complex comprising at least two genetic assemblages, i.e., clades A and C. However, further investigation is needed and should involve more comprehensive genetic and morphological analyses and cover a larger part of their distributional range throughout Southeast Asia.

Andrographolide Reduces Lipid Droplet Accumulation in Adipocytes Derived from Human Bone Marrow Mesenchymal Stem Cells by Suppressing Regulators of Adipogenesis.

Obesity has become a major public health concern; so, a strategy to prevent or reduce obesity is a priority. The inhibition of lipid droplet accumulation and adipogenesis process provides a target for the treatment of obesity. Herein, the effect of andrographolide (AP) on lipid accumulation in adipocytes derived from human bone marrow mesenchymal stem cells (hBM-MSCs) was examined. AP at concentrations of 1, 2.5, 5, and 10 µM reduced lipid droplet accumulation in the adipocytes by suppressing the adipogenic differentiation of hBM-MSCs. Concurrently, the expressions of adipogenic marker genes and the level of adipokines secreted by adipocytes were suppressed. Gene screening analysis showed a negative regulation of genes involved in the adipogenesis process. In conclusion, we demonstrated for the first time an antilipid accumulation in adipocytes from hBM-MSCs by AP. The compound may potentially be a novel therapeutic agent for the treatment of obesity as well as obesity-related diseases.

Genetic structure and evidence for coexistence of three taxa of Bithynia (Gastropoda: Bithyniidae), the intermediate host of Opisthorchis viverrini sensu lato (Digenea: Opisthorchiidae) in Thailand examined by mitochondrial DNA sequences analyses.

The freshwater snails, Bithynia are the first intermediate hosts of the liver fluke, Opisthorchis viverrini, the causative agent of cholangiocarcinoma (CCA) in Southeast Asia. In Thailand, there are three traditionally recognized taxa of Bithynia: Bithynia funiculata; B. siamensis siamensis; B. s. goniomphalos. This study examines the geographical distribution and genetic structure of Bithynia species from five previously reported water catchments and six new catchments in Thailand. Of these, three new catchments Kok, Wang, and Nan are from the north and the remaining three new catchments are Phetchaburi, Prachuap Khiri Khan Coast, Mae Klong from the west of Thailand. We sampled 291 Bithynia snails from 52 localities in 11 catchment systems in the northern, western and central regions of Thailand. Mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal DNA (16S rDNA) sequences were used to examine genetic diversity of Bithynia snails which revealed 200 and 27 haplotypes of COI and 16S rDNA, respectively. However, as 16S rDNA is a conserved gene, it is not suitable to distinguish Bithynia at the species and sub-species levels in our study. The phylogenetic tree and haplotype network analyses included sequences of COI from GenBank. B. funiculata was found only in the north of Thailand and the genetic structure did not differ among populations. Genetic differentiation (ΦST) analyses showed that B. s. goniomphalos contained three distinct lineages. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR. Lineage II contained all B. s. goniomphalos from the Prachin Buri and Bang Pakong catchment systems in eastern and central Thailand, including samples from all catchment systems in Cambodia. While lineage III contained B. s. goniomphalos from the Songkram and Nam Kam catchment systems in Thailand and the Nam Ngum and Huai Som Pak catchment systems in Lao PDR. Furthermore, results showed that all samples of B. s. siamensis were classified into one lineage and placed phylogenetically between B. s. goniomphalos lineages I and II. Thus, the taxonomic status of B. s. goniomphalos and B. s. siamensis requires reassessment, and they should be reclassified as belonging to the species complex "Bithynia siamensis sensu lato".

Andrographolide promotes proliferative and osteogenic potentials of human placenta-derived mesenchymal stem cells through the activation of Wnt/ß-catenin signaling.

INTRODUCTION: The in vitro expansion and differentiation of mesenchymal stem cells derived from bone marrow (BM-hMSCs) are considered as potential therapeutic tools for clinical applications in bone tissue engineering and regenerative medicine. However, invasive sampling and reduction in number and proliferative capacity with age are the major limitations of BM-hMSCs. Recently, human placenta-derived MSCs (PL-hMSCs) obtained by a non-invasive procedure have attracted much interest. Attempts to increase the potential of PL-hMSCs would be an important paradigm in regenerative medicine. Herein, we examined the proliferative and osteogenic effect of andrographolide (AP) on PL-hMSCs. METHODS: Mesenchymal stem cells were isolated from full-term normal human placentas and were characterized before using. Cell cytotoxicity and proliferative effect of AP were examined by MTT and BrdU assay, respectively. The non-toxicity concentrations of AP were further assessed for osteogenic effect determined by alkaline phosphatase (ALP) expression and activity, alizarin red staining, and osteoblast-specific gene expressions. Screening of genes involved in osteogenic differentiation-related pathways modulated by AP was explored by a NanoString nCounter analysis. RESULTS: PL-hMSCs generated in this study met the MSC criteria set by the International Society of Cellular Therapy. The non-cytotoxic concentrations of AP on PL-hMSCs are up to 10 µM. The compound increased PL-hMSC proliferation concomitant with increases in Wnt/ß-catenin level and activity. It also enhanced osteogenic differentiation in association with osteoblast-specific mRNA expression. Further, AP promoted bone formation and increased bone structural protein level, osteocalcin, in osteoblastic cells. Gene screening analysis showed the upregulation of genes related to Wnt/ß-catenin, TGFß/BMP, SMAD, and FGF signaling pathways. CONCLUSION: We demonstrated, for the first time, the potential role of AP in promoting proliferation, osteogenic differentiation, and osteoblast bone formation of PL-hMSCs. This study suggests that AP may be an effective novel agent for the improvement of PL-hMSCs and stem cell-based therapy for bone regeneration.

MicroRNA treatment modulates osteogenic differentiation potential of mesenchymal stem cells derived from human chorion and placenta.

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.

Genetic differentiation of Southeast Asian Paragonimus Braun, 1899 (Digenea: Paragonimidae) and genetic variation in the Paragonimus heterotremus complex examined by nuclear DNA sequences.

Southeast Asian lung flukes, the causative agents of human and animal paragonimiasis, comprise at least 14 species. Of these, seven species; Paragonimus bangkokensis, P. harinasutai, P. macrorchis, P. siamensis, P. westermani, P. heterotremus and P. pseudoheterotremus were studied. Two regions of domain 1 of taurocyamine kinase; TkD1 (exon) and TkD1Int2 (intron 2), were used as genetic markers for elucidating their genetic differentiation, genetic variation, and heterozygosity. The TkD1 region was conserved between these species but can potentially be used to differentiate all seven species. However, the TkD1Int2 region had a high level of polymorphism, which is suitable for investigation of genetic variation within or between closely related species, especially P. heterotremus and P. pseudoheterotremus as well as for a phylogenetic analyses of the genus Paragonimus. Heterozygosity was mostly observed in DNA samples extracted from adult P. heterotremus including samples taken from sputum of paragonimiasis patients, whereas DNA extracted from metacercariae was not, except in the samples from Myanmar. Our findings provide evidence of DNA recombination and incomplete lineage sorting of P. heterotremus and P. pseudoheterotremus in TkD1Int2, which suggesting gene flow between these two species.

Phylogeographic genetic variation of Indoplanorbis exustus (Deshayes, 1834) (Gastropoda: Planorbidae) in South and Southeast Asia.

The freshwater snail Indoplanorbis exustus play an important role as the sole intermediate host of several medically- and economically-important trematodes, especially zoonotic schistosomes and echinostomes, which can infect and cause diseases in livestock and people. This study aims to explore the mitochondrial cytochrome c oxidase subunit 1 sequence variation of I. exustus collected from new geographical areas; 459 specimens of I. exustus were collected from 43 localities in South and Southeast Asia. The 42 haplotypes (Ie1 - Ie42) we detected were classified into haplogroups I - V. Phylogenetic analyses revealed five major clades, A - E, in concordance with all previous studies. Clade E contained two subclades, E1 (haplogroup I) and E2 (haplogroup II). The most widespread genetic group was subclade E1. Clade A, clade B (haplogroup V), and clade C (haplogroup IV) were found only in South Asia, whereas clade D (haplogroup III) was specifically found in Southeast Asia. In Thailand, I. exustus showed high genetic divergence with 21 haplotypes. Several isolates showed significant genetic differences from others with unique haplotype(s). Hence, we confidently conclude our findings support all previous studies that I. exustus is a species complex with at least four major lineages and five haplogroups. Our additional analyses of 35 samples from Sri Lanka showed these were indeed an independent genetic group as previously found, but they can now be classified as a unique group forming subclade E2 (haplogroup II) of I. exustus sensu lato.

Intron sequence variation of the echinostomes (Trematoda; Echinostomatidae): implications for genetic investigations of the 37 collar-spined, Echinostoma miyagawai Ischii, 1932 and E. revolutum (Fröelich, 1802).

Echinostomes are a diverse group of digenetic trematodes that are difficult to classify by predominantly traditional techniques and contain many cryptic species. Application of contemporary genetic/molecular markers can provide an alternative choice for comprehensive classification or systematic analysis. In this study, we successfully characterized the intron 5 of domain 1 of the taurocyamine kinase gene (TkD1Int5) of Artyfechinostomum malayanum and the other two species of the 37 collar-spined group, Echinostoma revolutum and Echinostoma miyagawai, whereas TkD1Int5 of Hypoderaeum conoideum cannot be amplified. High levels of nucleotide polymorphism were detected in TkD1Int5 within E. revolutum and E. miyagawai, but not in A. malayanum. Thus, TkD1Int5 can be potentially used as genetic marker for genetic investigation of E. miyagawai and E. revolutum. We therefore used TkD1Int5 to explore genetic variation within and genetic differentiation between 58 samples of E. miyagawai and five samples of E. revolutum. Heterozygosity was observed in 17 and two samples with 16 and three insertion/deletion (indel) patterns in E. miyagawai and E. revolutum, respectively. Heterozygous samples were then cloned and nucleotide sequence was performed revealing the combined haplotypes in a particular sample. Based on nucleotide variable sites (excluding indels), the 72 E. miyagawai and seven E. revolutum haplotypes were subsequently classified. The haplotype network revealed clear genetic differentiation between E. miyagawai and E. revolutum haplogroups, but no genetic structure correlated with geographical localities was detected. High polymorphism and heterogeneity of the TkD1Int5 sequence found in our study suggest that it can be used in subsequent studies as an alternate independent potential genetic marker to investigate the population genetics, genetic structure, and possible hybridization of the other echinostomes, especially the 37 collar-spined group distributed worldwide.

Multiplex PCR development for the differential detection of four medically important echinostomes (Trematoda: Echinostomatidae) in Thailand.

Four species of echinostomes, Echinostoma revolutum (Froelich, 1802), Echinostoma ilocanum (Garrison, 1908), Hypoderaeum conoideum (Bloch, 1872) Dietz, 1909, and Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 commonly infect humans in Thailand, but their eggs present similar morphologies resulting in difficult differentiation for diagnosis. Present molecular methods have a great potential to provide superior detection/diagnosis. DNA sequences, especially the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene, have already been used to differentiate among echinostomes; thus, we aimed to develop species-specific primers for the differential detection of four medically important echinostomes by multiplex PCR. The species-specific reverse primers and a forward primer were based on variable regions and conserved regions of the ND1 gene, respectively. Four reverse primers and a forward primer were combined in a multiplex PCR reaction to amplify the ND1 fragment. Different ND1 fragment sizes were amplified: 108, 209, 384 and 419 bp of E. revolutum H. conoideum, E. ilocanum and A. malayanum, respectively. Specificity was tested with other medically important parasite DNA; no cross-reaction occurred. Sensitivity ranged between 0.1 and 0.05 ng. The species-specific primers developed in this study could be of further use in differential diagnosis for these medically important echinostomes infection in human and animal hosts.

Genetic structure and geographical variation of Bithynia siamensis goniomphalos sensu lato (Gastropoda: Bithyniidae), the snail intermediate host of Opisthorchis viverrini sensu lato (Digenea: Opisthorchiidae) in the Lower Mekong Basin revealed by mitochondrial DNA sequences.

The freshwater snail Bithynia siamensis goniomphalos sensu lato is widely distributed in the Lower Mekong Basin where it acts as the first intermediate host of the liver fluke Opisthorchis viverrini, a group 1 carcinogen causing cholangiocarcinoma. This study explores the genetic structure and geographical variation of B. s. goniomphalos from eight previously studied catchments and eight new catchments. These catchments belong to five previously studied catchment systems and one new catchment system (Tonlesap) in the Lower Mekong Basin. Two new catchment systems, Prachin Buri and Bang Pakong from eastern and central Thailand, respectively, were also examined. We collected 289 specimens of B. s. goniomphalos from 15 previously studied localities and 18 new localities in Thailand, Lao PDR (People's Democratic Republic), and Cambodia. The mitochondrial cytochrome c oxidase subunit 1 and 16S ribosomal DNA sequences were used to determine genetic variation. Classification of haplotypes specified 100 at the cox1 locus and 15 at the rrnL locus. Comparison between 16 catchment populations found significant genetic differences (ФST) between all populations. The phylogenetic tree and haplotype network analyses classified B. s. goniomphalos into three evolutionary lineages (lineage I-III). Lineage I contained B. s. goniomphalos from the Mekong, Chi, Mun, Prachin Buri and Bang Pakong catchments in Thailand, including the Nam Ngum catchment in Lao PDR. Lineage II contained all specimens from the Tonlesap catchment, whereas lineage III contained specimens from the Mekong and Sea Bang Heang catchments in Thailand and Lao PDR, respectively. Interestingly, Bithynia siamensis siamensis was placed between lineages I and II of B. s. goniomphalos. This study supports the hypothesis that B. s. goniomphalos is a species complex containing at least three distinct evolutionary lineages in the Lower Mekong Basin, and that comprehensive molecular genetic analyses need to be conducted to further our understanding of the evolutionary and systematic relationships of these Bithynia snail taxa.

Gestational Tissue-Derived Human Mesenchymal Stem Cells Use Distinct Combinations of Bioactive Molecules to Suppress the Proliferation of Human Hepatoblastoma and Colorectal Cancer Cells.

BACKGROUND: Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy, treatments of advance stage cancers are mostly ineffective resulting in poor survival of patients. Recent evidences suggest that multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However, the effect of hMSCs on cancer cells is still controversial because there are also evidences demonstrating that hMSCs inhibited growth and metastasis of some cancers. METHODS: In this study, we investigated the effects of bioactive molecules released from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cells, including C3A, HT29, A549, Saos-2, and U251. We also characterized the hMSC-derived factors that inhibit cancer cell proliferation by protein fractionation and mass spectrometry analysis. RESULTS: We herein make a direct comparison and show that the effects of hMSCs on cancer cell proliferation and migration depend on both hMSC sources and cancer cell types and cancer-derived bioactive molecules did not affect the cancer suppressive capacity of hMSCs. Moreover, hMSCs use distinct combination of bioactive molecules to suppress the proliferation of human hepatoblastoma and colorectal cancer cells. Using protein fractionation and mass spectrometry analysis, we have identified several novel hMSC-derived factors that might be able to suppress cancer cell proliferation. CONCLUSION: We believe that the procedure developed in this study could be used to discover other therapeutically useful molecules released by various hMSC sources for a future in vivo study.

Human serum enhances the proliferative capacity and immunomodulatory property of MSCs derived from human placenta and umbilical cord.

BACKGROUND: Mesenchymal stromal cells (MSCs) are considered potential candidates that hold great promise in the treatment of immune-related diseases. For therapeutic applications, it is necessary to isolate and expand MSCs with procedures complying with good manufacturing practice (GMP). Recent studies reported the use of human serum (HS) instead of fetal bovine serum (FBS) for the expansion of bone marrow-derived MSCs. Nevertheless, there are only limited data on HS as an alternative to FBS for the isolation and expansion of umbilical (UC-MSCs) and placenta-derived MSCs (PL-MSCs). In this study, we evaluate the effect of HS compared to FBS on the proliferative and immunosuppressive capacities of these MSCs. METHODS: PL-MSCs and UC-MSCs were isolated and cultured in HS- or FBS-supplemented media. The MSC characteristics, including morphology, immunophenotype, and differentiation ability, were verified. The proliferative and immunosuppressive capacities were also examined. In addition, the proliferative-enhancing factors in both sera were explored using proteomic analysis. RESULTS: PL-MSCs and UC-MSCs proliferated faster in HS-supplemented medium than in equivalent levels of FBS-supplemented medium. Adipogenic and osteogenic differentiations occurred at nearly identical levels in HS- and FBS-supplemented media. Interestingly, MSCs cultured in HS-supplemented medium had a similar immunosuppressive effect as MSCs cultured in FBS-supplemented medium. Proteomic analysis revealed that Con-A binding glycoproteins with a molecular weight > 100 kDa in FBS could significantly enhance MSC proliferation. In contrast, the proliferative enhancing factors in HS were found in the Con-A non-binding fraction and WGA binding fraction with a molecular weight > 100 kDa. CONCLUSIONS: Taken together, our results suggest applications for the use of HS instead of FBS for the isolation and expansion of PL-MSCs and UC-MSCs for cell therapy in the future. Furthermore, this study identifies factors in HS that are responsible for its proliferative and immunosuppressive effects and might thus lead to the establishment of GMPs for the therapeutic use of MSCs.