Kalliopi I. Pappa

Novel sources of fetal stem cells: where do they fit on the developmental continuum?

The recent isolation of fetal stem cells from several sources either at the early stages of development or during the later trimesters of gestation, sharing similar growth kinetics and expressing pluripotency markers, provides strong support to the notion that these cells may be biologically closer to embryonic stem cells, actually representing intermediates between embryonic stem cells and adult mesenchymal stem cells, regarding proliferation rates and plasticity features, and thus able to confer an advantage over postnatal mesenchymal stem cells derived from conventional adult sources such as bone marrow. This conclusion has been strengthened by the different pattern of growth potential between the two stage-specific types of sources, as assessed by transcriptomic and proteomic analysis. A series of recent studies regarding the numerous novel features of fetal stem cells has reignited our interest in the field of stem-cell biology and in the possibilities for the eventual repair of damaged organs and the generation of in vitro tissues on biomimetic scaffolds for transplantation. These studies, employing elegant approaches and novel technologies, have provided new insights regarding the nature and the potential of fetal stem cells derived from placenta, amniotic fluid, amnion or umbilical cord. In this update, we highlight the major progression that has occurred in fetal stem-cell biology and discuss the most important areas for future investigation in the field of regenerative medicine.

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure.

Background There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. Methods AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. Results Both HPL cells and AF-MSCs were incorporated into CCl4-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. Conclusions Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver.

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF‐MPCs consisted of two morphologically distinct adherent cell types, termed as spindle‐shaped (SS) and round‐shaped (RS). A detailed analysis of these two populations showed that SS‐AF‐MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS‐AF‐MPCs and RS‐AF‐MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS‐AF‐MPCs. Furthermore, SS‐AF‐MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS‐AF‐MPCs and thus we further evaluated SS‐AF‐MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP‐lentiviral transduced SS‐AF‐MPCs retained their stem cell identity, proliferation and differentiation potential. GFP‐SS‐AF‐MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF‐MPCs consisted of at least two different MPC populations. In addition, SS‐AF‐MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.

Spindle Shaped Human Mesenchymal Stem/Stromal Cells from Amniotic Fluid Promote Neovascularization

Human amniotic fluid obtained at amniocentesis, when cultured, generates at least two morphologically distinct mesenchymal stem/stromal cell (MSC) subsets. Of these, the spindle shaped amniotic fluid MSCs (SS-AF-MSCs) contain multipotent cells with enhanced adipogenic, osteogenic and chondrogenic capacity. Here, we demonstrate, for the first time, the capacity of these SS-AF-MSCs to support neovascularization by umbilical cord blood (UCB) endothelial colony forming cell (ECFC) derived cells in both in vitro and in vivo models. Interestingly, although the kinetics of vascular tubule formation in vitro were similar when the supporting SS-AF-MSCs were compared with the best vasculogenic supportive batches of bone marrow MSCs (BMSCs) or human dermal fibroblasts (hDFs), SS-AF-MSCs supported vascular tubule formation in vivo more effectively than BMSCs. In NOD/SCID mice, the human vessels inosculated with murine vessels demonstrating their functionality. Proteome profiler array analyses revealed both common and distinct secretion profiles of angiogenic factors by the SS-AF-MSCs as opposed to the hDFs and BMSCs. Thus, SS-AF-MSCs, which are considered to be less mature developmentally than adult BMSCs, and intermediate between adult and embryonic stem cells in their potentiality, have the additional and very interesting potential of supporting increased neovascularisation, further enhancing their promise as vehicles for tissue repair and regeneration.

Impact of Maternal Diabetes on Epigenetic Modifications Leading to Diseases in the Offspring

Gestational diabetes, occurring during the hyperglycemic period of pregnancy in maternal life, is a pathologic state that increases the incidence of complications in both mother and fetus. Offspring thus exposed to an adverse fetal and early postnatal environment may manifest increased susceptibility to a number of chronic diseases later in life. Compelling evidence for the role of epigenetic transmission in these complications has come from comparison of siblings born before and after the development of maternal diabetes, exposure to this intrauterine diabetic environment being shown to cause alterations in fetal growth patterns which predispose these infants to developing overweight and obesity later in life. Diabetes of the offspring is also mainly the consequence of exposure to the diabetic intrauterine environment, in addition to genetic susceptibility. Since obesity and diabetes are known to increase the risk of cardiovascular disease, cardiovascular sequelae in the offspring of diabetic mothers are virtually inevitable. Research data also suggest that exposure to a diabetic intrauterine environment during pregnancy is associated with an increase in dyslipidemia, subclinical vascular inflammation, and endothelial dysfunction processes in the offspring, all of which are linked with development of cardiovascular disease later in life. The main underlying mechanisms involve persistent hyperglycemia hyperinsulinemia and leptin resistance.

Gestational diabetes mellitus shares polymorphisms of genes associated with insulin resistance and type 2 diabetes in the Greek population

Gestational diabetes mellitus (GDM) and type 2 diabetes (T2D) share common pathophysiological features, including β-cell dysfunction and insulin resistance. In this study, we investigated the association between GDM and five recently identified T2D susceptibility loci, in a Greek population. We studied 148 women with GDM and 107 non-diabetic unrelated pregnant Greek women, for polymorphisms in the TCF7L2 gene (rs7903146 C/T), the PPARG gene (Pro12Ala), the KCNJ11 gene (E23K), the IRS1 gene (G972R) and in the FOXC2 gene (-512C>T). The T-allele of the TCF7L2 rs7903146 (C/T) polymorphism was found to be significantly associated with an increased risk of GDM [p = 0.0003; odds ratio (OR) 2.04 (95%CI 1.38–3.00)]. Additionally, CT and TT genotypes were significantly overrepresented in women with GDM compared to controls (p = 0.0003 and p = 0.0148, respectively). Analysis of the IRS1 G972R polymorphism showed that the R-allele frequency was increased in women with GDM [(p = 0.009; OR 1.67 (95%CI 1.14–2.47)]. The genotypes and allele frequencies of the other polymorphisms studied did not statistically differ between the GDM and the control women. Thus, our data suggest that the common T2D susceptibility polymorphism of TCF7L2 (rs7903146 C/T) gene, and the G972R polymorphism of the IRS1 gene, seem to predispose to GDM in Greek women.

OCT4 spliced variant OCT4B1 is expressed in human colorectal cancer

OCT4, a POU‐domain transcription factor is considered to be a key factor in maintaining the pluripotency of stem cells. Several OCT4 isoforms are differentially expressed in human pluripotent and non‐pluripotent cells. Reactivation of OCT4 expression is postulated to occur in differentiated cells that have undergone tumorigenesis. To examine OCT4 expression in colorectal cancer (CRC) tissues, and to assess the efficacy of OCT4 as a potential biomarker for CRC, in this study, we investigated its expression in CRC tissues, evaluated its relationship to various clinicopathological parameters and defined the isoform of OCT4 that was found to be expressed in CRC cases. Primary tumor tissues and matching adjacent non‐cancerous tissues were obtained from 84 CRC patients. OCT4 expression and isoform determination were documented by reverse transcription‐PCR and real‐time PCR. OCT4, Sox‐2, and NANOG localization were performed using immunohistochemistry. The isoforms expressed in the studied cases were confirmed by sequencing. Twenty biopsy specimens representing healthy tissues, retrieved from colonoscopy were studied in parallel as controls. OCT4 expression levels were higher in CRC tissues compared to matching, adjacent non‐cancerous tissues, and healthy controls. Additionally, the levels of OCT4 expression in CRC tissues correlated with tumor stage. OCT4 and Sox‐2 were localized in the nuclei and the cytoplasm of CRC cells. In all CRC cases, we found that the OCT4B1 isoform is expressed. Over‐expression of OCT4B1 was found in poorly and moderately differentiated CRC tissues. In conclusion, the data imply that OCT4B1 isoform may represent a potential biomarker for the initiation, progression, and differentiation of CRC. Mol. Carcinog.

Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application.

BackgroundmicroRNAs (miRNAs) are single-stranded RNA molecules of about 20–23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance.ResultsGOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application.ConclusionGOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA.

Sox2 Suppression by miR‐21 Governs Human Mesenchymal Stem Cell Properties

MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle‐shaped (SS) and round‐shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR‐21 was expressed at higher levels in RS‐AF‐MSCs and BM‐MSCs compared with SS‐AF‐MSCs. We further demonstrated for the first time a direct interaction between miR‐21 and the pluripotency marker Sox2. The induction of miR‐21 strongly inhibited Sox2 expression in SS‐AF‐MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR‐21 inhibition in RS‐AF‐MSCs and BM‐MSCs, which led to an enhanced proliferation rate. Finally, miR‐21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS‐AF‐MSCs. Therefore, these findings suggest that miR‐21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

Role of Adipokines and Other Inflammatory Mediators in Gestational Diabetes Mellitus and Previous Gestational Diabetes Mellitus

Previous Gestational Diabetes Mellitus (pGDM) is a common condition and has been associated with future development of Type 2 Diabetes Mellitus (T2DM) and Metabolic Syndrome (MS) in women affected. The pathogenesis and risk factors implicated in the development of these conditions later in the lives of women with pGDM are not as yet fully understood. Research has recently focused on a group of substances produced mainly by adipose tissue called adipokines, this group including, among others, adiponectin, leptin, Retinol-Binding Protein-4 (RBP-4), and resistin. These substances as well as other inflammatory mediators (CRP, IL-6, PAI-1, TNF-α) seem to play an important role in glucose tolerance and insulin sensitivity dysregulation in women with pGDM. We summarize the data available on the role of these molecules.