Tissues derived from reprogrammed Wharton's jelly stem cells of the umbilical cord as a platform to study gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) has been strongly associated with an increased risk of type 2 diabetes mellitus (T2DM) in later child and adulthood. The human umbilical cord and its contents are of fetal origin and represent the fetus genetically and physiologically. Since it is not possible to obtain tissues from the fetus and newborn to investigate the association between GDM and later T2DM, we reprogrammed the stem cells from the Wharton's jelly of umbilical cords (hWJSCs) of GDM and non-GDM mothers into induced pluripotent stem cells (iPSCs) and then differentiated the iPSCs into insulin-producing cells (IPCs) to provide pancreatic tissues that represent the fetus of GDM and normal mothers. These tissues are an attractive model to study the effects of glucose on the fetus. Interestingly, GDM-iPSCs had a decreased potential towards differentiation into IPCs. IPCs differentiated from GDM-iPSCs also had lower total insulin content and a lower capacity for insulin secretion to glucose stimulation compared to their normal-iPSC counterparts. This abnormal pathogenesis in GDM-iPSCs pancreatic differentiation recapitulates the pathology that may be observed in the infants of the diabetic mother (IDM) and while indicating adaptive mechanisms for fetal survival, may lead to the development of T2DM later in life. (199 words).

Manufacturing of human Wharton's jelly stem cells for clinical use: selection of serum is important.

BACKGROUND: Human Wharton's jelly-derived mesenchymal stromal cells (hWJSCs) have gained considerable attention for their use in cell therapy. Many of these applications would require manufacturing of millions of hWJSCs. It is, therefore, necessary to develop a Good Manufacturing Practice (GMP)-compliant hWJSC expansion protocol, allowing the generation of a large quantity of cells to meet both clinical and regulatory requirements. Here, we compared human platelet lysate (HPL) and human serum (HS) in supporting clinical-grade hWJSC expansion. METHODS: hWJSCs were successfully isolated from six different umbilical cords using GMP-compliant dissociation enzymes. Freshly isolated hWJSCs were cultured in media supplemented with 10% of one of the following sera: fetal bovine serum (FBS), HPL and HS. Properties of the expanded hWJSCs were analyzed. RESULTS: We showed that GMP-compliant dissociation enzymes were as efficient as research-grade dissociation enzymes in isolating hWJSCs. hWJSC fresh cell yield and cell viability using HPL and HS supplementations were at greater advantages than FBS. Moreover, hWJSCs expanded in HPL and HS supplementations not only preserved classical MSCs phenotypes and differentiation potential to adipocytes, osteocytes and chondrocytes, they also enhanced the migration of skin fibroblasts. However, HS, unlike HPL, did not alter immunogenicity properties of hWJSCs. hWJSCs expanded in HS supplementation also exerted greater immunosuppressive action in inhibiting T-cell proliferation and increased extracellular matrix (ECM) gene expression, making them useful in tissue repair clinical application. CONCLUSION: Our findings indicate that HS can be considered as a promising and safer alternative to FBS, and should be recommended for clinical-grade expansion of hWJSCs.

Changes in Stemness Properties, Differentiation Potential, Oxidative Stress, Senescence and Mitochondrial Function in Wharton's Jelly Stem Cells of Umbilical Cords of Mothers with Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) has been associated with an increased risk of maternal and neonatal morbidity. The Wharton's jelly (WJ) of the umbilical cord (UC) is a useful indicator of the deleterious effects of hyperglycemia on fetal tissues as it represents the fetus embryologically, physiologically and genetically. We studied WJ mesenchymal stem cells (hWJSCs) from UC from mothers without GDM (Normal; n = 3); insulin-controlled GDM mothers (GDMi; n = 3) and diet-controlled GDM mothers (GDMd; n = 3)]. Cell proliferation, stemness markers, telomerase, osteogenic and chondrogenic differentiation, antioxidant enzymes and gene expression for mitochondrial function (ND2, TFAM, PGC1α, and NDUFB9) were significantly lower in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs (P < 0.05). On the other hand, cell cycle inhibitors (p16, p21, p27) and p53 were remarkably up-regulated in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs. The results from this study confirmed that maternal hyperglycemia even though managed with insulin or diet, induced changes in the properties of the WJ and its cells. These changes may also be observed in fetal tissues and if true, prevention of the onset of gestational diabetes should be a priority over management. Generation of tissues that simulate those of the fetus such as pancreatic and cardiovascular cells from GDM-hWJSCs by direct differentiation or via induced pluripotent stem cell reprogramming provide possible platforms to evaluate the effects of glucose on specific fetal organ.

The use of transformed IMR90 cell model to identify the potential extra-telomeric effects of hTERT in cell migration and DNA damage response.

BACKGROUND: Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90. RESULTS: Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR). CONCLUSIONS: Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.

Telomere shortening in human diseases.

The discovery of telomeres dates back to the early 20th century. In humans, telomeres are heterochromatic structures with tandem DNA repeats of 5'-TTAGGG-3' at the chromosomal ends. Telomere length varies greatly among species and ranges from 10 to 15 kb in humans. With each cell division, telomeres shorten progressively because of the 'end-replication problem'. Short or dysfunctional telomeres are often recognized as DNA DSBs, triggering cell-cycle arrest and result in cellular senescence or apoptotic cell death. Therefore, telomere shortening serves as an important tumor-suppressive mechanism by limiting cellular proliferative capacity by regulating senescence checkpoint activation. Although telomeres serve as a mitotic clock to cells, they also confer capping on chromosomes, with help from telomere-associated proteins. Over the past decades, many studies of telomere biology have demonstrated that telomeres and telomere-associated proteins are implicated in human genetic diseases. In addition, it has become more apparent that accelerated telomere erosion is associated with a myriad of metabolic and inflammatory diseases. Moreover, critically short or unprotected telomeres are likely to form telomeric fusions, leading to genomic instability, the cornerstone for carcinogenesis. In light of these, this minireview summarizes studies on telomeres and telomere-associated proteins in human diseases. Elucidating the roles of telomeres involved in the mechanisms underlying pathogenesis of these diseases may open up new possibilities for novel molecular targets as well as provide important diagnostic and therapeutic implications.

Elite model for the generation of induced pluripotent cancer cells (iPCs).

The inefficiency of generating induced pluripotent somatic cells (iPS) engendered two contending models, namely the Stochastic model and Elite model. Although the former is more favorable to explain the inherent inefficiencies, it may be fallible to extrapolate the same working model to reprogramming of cancer cells. Indeed, tumor cells are known to be inherently heterogeneous with respect to distinctive characteristics thus providing a suitable platform to test whether the reprogramming process of cancer cells is biased. Here, we report our observations that all randomly picked induced pluripotent cancer cells (iPCs) established previously do not possess mutations known in the parental population. This unanticipated observation is most parsimoniously explained by the Elite model, whereby putative early tumor progenies were selected during induction to pluripotency.

Reversal of aberrant cancer methylome and transcriptome upon direct reprogramming of lung cancer cells.

Recent reports on direct reprogramming of cancer cells (iPCs) which results in reduced tumorigenic potential has attributed the importance of epigenetics in tumorigenesis, but lacked genome-wide analysis. Here we describe successful generation of iPCs from non-small cell lung cancer (NSCLC) cell lines. Following reprogramming, they resembled embryonic stem and induced pluripotent stem cells in pluripotency markers expression, gene expression patterns and in vitro differentiation ability. Genome-wide methylation analysis revealed that aberrantly methylated promoters which were mostly developmental-associated genes and tumor suppressors; as well as commonly upregulated genes in NSCLC i.e. KRT19 and S100P were reversed in iPCs upon reprogramming. Also, the reversal of oncogenes and tumor suppressors status were partially explainable by DNA methylation. These findings suggest that DNA methylation patterns explain the downstream transcriptional effects, which potentially caused the reduced tumorigenicity in iPCs, thus providing evidence that reprogramming reverses the aberrantly dysregulated genes in NSCLC both epigenetically and transcriptionally.