The human prostate contains two types of stromal cells, peripheral stromal cells (PSCs) and transitional stromal cells (TSCs). Here, we demonstrate the effects of PSCs and TSCs on tumorigenesis in prostate cancer (PCa) and identify the mechanisms underlying these effects. Using microarray analysis, we identified 3,643 differentially expressed genes in cocultures of TSCs, PSCs, and DU145 cells, a human prostate cancer cell line. Expression of cell division cycle 25 homolog A (CDC25A) was lower and that of tumor-associated calcium signal transducer 2 (TACSTD2) was higher in TSCs than in PSCs. Additionally, increased CDC25A expression or decreased TACSTD2 expression modulated the survival, growth, and migration of DU145 cells. These data suggest that PSCs promote and TSCs inhibit tumorigenesis by regulating the expression of CDC25A and TACSTD2.

Ventricular septal defect (VSD) is one of the common congenital heart malformations. Several factors lead to the development of VSD, including familial causes, exposure to certain drugs, infectious agents, and maternal metabolic disturbances. We considered that induced pluripotent stem (iPS) cells derived from VSD patients can be used to study the origin and pathogenesis of the VSD. Here, we show generation and cardiomyocyte differentiation potential of iPS cells from thymic epithelial cells of a patient with VSD (TECs-VSD) by overexpressing the four factors: OCT4, SOX2, NANOG, and LIN28 with lentiviral vectors. The self-renewal and pluripotency of the VSD-iPS cells was verified in iPS cells by in vitro expression of pluripotency markers and formation of teratoma in vivo. iPS cell lines from VSD patients differentiated into functional cardiomyocytes can serve as a model system for studying the pathophysiology and identifying etiology of VSD.

BACKGROUND:Tetralogy of Fallot (ToF) is the most common form of cyanotic congenital heart disease and is a major cause of significant morbidity and mortality. VANGL2 is a critical gene in the planar cell polarity pathway that plays an important role in the development of the heart. This study investigates the methylation status of the promoter region of VANGL2 and the expression pattern of VANGL2 in cardiac tissue.METHODS:The promoter region of VANGL2 was sequenced in 200 ToF patients and 400 control subjects. Methylation levels were measured in four regions of the VANGL2 promoter (B1-1: -282 bp ∼ -117 bp, B1-2: -117 bp ∼ 41 bp, B2: 8 bp ∼ 157 bp, B3: 132 bp ∼ 401 bp) by bisulfite sequencing PCR in the right ventricular outflow tract of the myocardium. Quantitative real-time PCR and immunohistochemistry were used to detect the mRNA and protein expression levels, respectively.RESULTS:No mutations were found in the promoter region, but two SNPs (rs11582932 T>G, rs11265385 T>G) were found in ToF patients and controls with similar frequencies (p>0.05). The overall methylation status of the VANGL2 promoter was significantly higher in ToF patients than in controls (p=0.0234). Specifically, the methylation levels of regions B1-1 and B3 were significantly higher in ToF patients (p=0.0042, p=0.0418). Both the VANGL2 mRNA and protein levels were significantly lower in ToF patients than in controls (p<0.05).CONCLUSION:The aberrant VANGL2 promoter methylation and the decreased gene expression in ToF patients may provide important clues for the development of ToF.

Transforming growth factor β2 (TGFβ2) plays an essential role in cardiac morphogenesis. However, the prevalence of TGFβ2 mutations in congenital heart disease (CHAD) and the correlation between the TGFβ2 genotype and the CHAD phenotype have not been studied extensively. The aim of this study was to examine DNA sequence changes in the TGFβ2 gene in sporadic patients with tetralogy of Fallot (TOF), and to observe whether TGFβ2 is the susceptibility gene for TOF. A cohort of 100 pediatric patients with TOF was recruited to the study; 200 healthy children were used as controls. PCR and genotyping were conducted for the detection of DNA changes in TGFβ2. The exons and the 5' untranslated region (5'UTR) sequences of the TGFβ2 gene were amplified. No mutations were identified in the coding region in any of the TOF patients. However, three single nucleotide changes, including 9126 A>AC, 9353 A>AG and 9040_9043 del CTTC, in the 5'UTR were found. There were no significant differences in allelic frequencies and genotype frequencies of position 9126 and 9353 between the TOF group and the control group. On the contrary, a significant difference was identified in the allelic frequencies (χ(2)=17.469, P<0.001) of position 9040_9043 in the 5'UTR between the TOF group and the control group. Our results suggest that TGFβ2 may be a potential candidate gene of TOF. SNPs at position 9040_9043 del CTTC in the 5'UTR of TGFβ2 may be associated with susceptibility to TOF. The CTTC allele may be the susceptibility allele for TOF. However, the exact effect of these sequence changes requires further study using functional experiments.

Stromal cells play a decisive role in regulating tumor progression. In this study, we assessed the significance of normal prostate‐derived stromal cells (PSCs) in prostate cancer development. An in vivo s.c. tumor model was established as follows: Group 1, DU145 cells alone; Group 2, DU145 + PSCs; Group 3, DU145 cells alone injected into pre‐castrated mice; and Group 4, DU145 + PSCs injected into pre‐castrated mice. Following injection, tumors were only detectable in the first two groups, with more aggressive growth in Group 2 than in Group 1 (P < 0.05). Immunohistochemical analysis revealed significantly higher proliferation (P < 0.05), but not apoptosis or altered expression of androgen receptor in Group 2, as compared with Group 1. In vitro, DU145 cells isolated from Group 1 tumors showed lower viability and migratory capability than those from Group 2. cDNA microarray on isolated DU145 cells from Groups 1 and 2 revealed the differential expression of genes regulating cell cycle progression and cell mobility, including GADD45A, RHOV, KLK11, and PCK1. Our results suggest that stromal cells derived from normal prostate potentiate the development of tumor growth in vivo, which is achieved at least in part through the regulation of cell‐cycle‐ and migration‐related gene expression within the tumor cells. (Cancer Sci 2011; 102: 1630–1635)

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.(1) The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.(2) Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.(3) iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells. Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.