OBJECTIVE: This study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water.
METHODS: To create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results.
RESULTS: Proliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001).
CONCLUSIONS: Intentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.

Lymphovascular invasion (LVI) is a relevant prognostic factor in germ cell tumors of the testis (GCTT) and it has been included in the AJCC staging system. Nevertheless, its histological assessment is challenging, with low/moderate interobserver agreement also among expert uropathologists. Few studies focused on the potential role of immunohistochemistry to solve this critical issue; as result, in current guidelines there is no indication for additional staining to detect this histological feature. In the present study, we investigated the detection of LVI invasion in a small cohort of GCTT with double staining for OCT4/CD34. Although our results need to be validated in larger case series with follow-up data, they suggest as OCT4/CD34 could be a useful tool for the histological assessment of these tumors, helping to identify some histological mimickers of LVI and modifying the pT/stage in a significant percentage of patients.

Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivo iPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.

The cancer stem cells deliver uncontrolled proliferative capacity within the tumor imparting to increasing size while epithelial mesenchymal transition adds to the invasive potential. Studies using specific markers elucidating the role of these phenomena may bring advancement in the targeted therapy of tumor. SOX2 and OCT4 are two among few stem cell markers indicative of proliferative potential and WNT5A is an epithelial mesenchymal transition marker indicative of invasive potential. We aimed to determine the association between expression of SOX2, OCT4 and WNT5A in oral epithelial dysplasia, oral squamous cell carcinoma and normal oral mucosa. 20 cases of oral squamous cell carcinoma, 20 cases of oral epithelial dysplasia (leukoplakia with dysplasia) and 25 normal oral mucosa tissues specimens were immunohistochemically stained to assess SOX2, OCT4 and WNT5A expression. SOX2 expression was higher in oral squamous cell carcinoma than in oral epithelial dysplasia and very low in normal oral mucosa. OCT4 was very low in oral squamous cell carcinoma and oral epithelial dysplasia when compared to SOX2, while negative in normal tissues. Co-expression of SOX2 and OCT4 showed statistically non-significant difference for tumor proliferation. WNT5A expression was found to be increasing from normal oral mucosa to oral epithelial dysplasia and oral squamous cell carcinoma. In conformity with present study, SOX2 itself can act as a potential marker for proliferation in tumor cells while OCT4 has non-significant role in regulation of tumor behavior in oral squamous cell carcinoma as well as in oral epithelial dysplasia. WNT5A can be a putative marker in studying invasive potential of oral squamous cell carcinoma.

OBJECTIVE: To report a series of 11 ovarian and one endometrial neoplasm in elderly patients with mixed clear cell tumour and germ cell tumour (GCT) components, to compare their immunohistochemical profiles and demonstrate a putative stem cell population.
RESULTS: The clear cell tumours included 11 clear cell carcinomas (CCC) and one borderline clear cell tumour, while the GCT always included glandular yolk sac tumour (YST). In four cases, there were also foci of teratoma with immature neuroepithelial and endodermal tissues and undifferentiated areas showing true embryoids. To distinguish between the clear cell and YST components, the following antibodies were used: HNF1-β, napsin-A, cytokeratin 7 (CK7), PAX8, EMA, AFP, SALL4, villin, glypican-3 (GPC-3), GATA3, HepPar-1, OCT4, CDX2, CD30 and SOX2. HNF1-β, CK7, EMA and GPC-3 were often expressed in both components. Other markers had higher specificity for each cellular lineage; napsin-A and PAX8 were expressed only in CCC, while SALL4, villin, AFP and HepPar-1 were positive in the glandular YST component but negative in the clear cell component. OCT4 expression occurred in six of 10 cases and consistently in teratoma (four of four).
CONCLUSIONS: There is considerable immunophenotypical overlap between the two components in these mixed neoplasms, and a panel of markers should be used to facilitate the distinction. We propose that OCT4-expressing somatic cancer cells differentiate into GCT and represent spontaneously induced pluripotent stem cells, possibly conditioned by age-related epigenetic factors. These neoplasms have features of prepubertal type GCT showing lack of 12p gain, preponderance of YST and coexistence with immature neuroectoderm. However, there may also be undifferentiated stem cell areas with embryoid bodies, of the type seen in postpubertal testicular GCT, but lacking a complete embryonal carcinoma immunophenotype.

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient\'s tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.