Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

SummaryReprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.PaperCli

FISH for HER-2/neu in breast cancer: Standardization makes the difference!

CONTEXT: Overexpression of HER-2/neu oncogene in breast cancer patients is correlated with disease free survival (DFS) and overall survival (OS). The most commonly used methods for the detection of HER-2/neu status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, therse is a lot of controversy with regard to the best method. Most of the FISH studies chose arbitrary cut-off levels for positive results (10%) and had no validation. AIM: In order to address these issues, we designed a pilot study of 38 samples with known IHC status representing all 4 categories. SETTINGS AND DESIGN: FISH was performed using Vysis Pathvysion\u99 probe. For validation, 5 cases of reduction mammoplasty were analyzed using same protocols. RESULTS: Our results showed significant discordance between FISH and IHC. The rate of discordance was much higher in the 0, 1+, and 2+ categories compared to published literature. This could be due to the lower cut-off rates for positive amplification established by validation in our study (5.7% vs 10%). Our analysis showed that FISH positive and IHC negative patients have a poor prognosis in terms of DFS and OS compared to FISH negative and IHC negative patients. Further, our results also showed that IHC in comparison to FISH has a comparable specificity (98%), but has a very low sensitivity (46%). CONCLUSION: Based on these results, we consider FISH to be the gold standard for detecting HER-2/neu status in breast cancer

Original Article - FISH for HER-2/neu in breast cancer: Standardization makes the difference!

CONTEXT: Overexpression of HER-2/neu oncogene in breast cancer patients is correlated with disease free survival (DFS) and overall survival (OS). The most commonly used methods for the detection of HER-2/neu status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, therse is a lot of controversy with regard to the best method. Most of the FISH studies chose arbitrary cut-off levels for positive results (10%) and had no validation. AIM: In order to address these issues, we designed a pilot study of 38 samples with known IHC status representing all 4 categories. SETTINGS AND DESIGN: FISH was performed using Vysis Pathvysion™ probe. For validation, 5 cases of reduction mammoplasty were analyzed using same protocols. RESULTS: Our results showed significant discordance between FISH and IHC. The rate of discordance was much higher in the 0, 1+, and 2+ categories compared to published literature. This could be due to the lower cut-off rates for positive amplification established by validation in our study (5.7% vs 10%). Our analysis showed that FISH positive and IHC negative patients have a poor prognosis in terms of DFS and OS compared to FISH negative and IHC negative patients. Further, our results also showed that IHC in comparison to FISH has a comparable specificity (98%), but has a very low sensitivity (46%). CONCLUSION: Based on these results, we consider FISH to be the gold standard for detecting HER-2/neu status in breast cancer

Oct4 Expression Is Not Required for Mouse Somatic Stem Cell Self-Renewal

SummaryThe Pou domain containing transcription factor Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in embryonic stem cells. While it has been shown that the Oct4 gene is inactivated through a series of epigenetic modifications following implantation, recent studies have detected Oct4 activity in a variety of somatic stem cells and tumor cells. Based on these observations it has been suggested that Oct4 may also function in maintaining self-renewal of somatic stem cells and, in addition, may promote tumor formation. We employed a genetic approach to determine whether Oct4 is important for maintaining pluripotency in the stem cell compartments of several somatic tissues, including the intestinal epithelium, bone marrow (hematopoietic and mesenchymal lineages), hair follicle, brain, and liver. Oct4 gene ablation in these tissues revealed no abnormalities in homeostasis or regenerative capacity. We conclude that Oct4 is dispensable for both self-renewal and maintenance of somatic stem cells in the adult mammal

Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing

Increased methylation of CpG islands and silencing of affected target genes is frequently found in human cancer; however, in vivo the question of causality has only been addressed by loss-of-function studies. To directly evaluate the role and mechanism of de novo methylation in tumor development, we overexpressed the de novo DNA methyltransferases Dnmt3a1 and Dnmt3b1 in ApcMin/+ mice. We found that Dnmt3b1 enhanced the number of colon tumors in ApcMin/+ mice approximately twofold and increased the average size of colonic microadenomas, whereas Dnmt3a1 had no effect. The overexpression of Dnmt3b1 caused loss of imprinting and increased expression of Igf2 as well as methylation and transcriptional silencing of the tumor suppressor genes Sfrp2, Sfrp4, and Sfrp5. Importantly, we found that Dnmt3b1 but not Dnmt3a1 efficiently methylates the same set of genes in tumors and in nontumor tissues, demonstrating that de novo methyltransferases can initiate methylation and silencing of specific genes in phenotypically normal cells. This suggests that DNA methylation patterns in cancer are the result of specific targeting of at least some tumor suppressor genes rather than of random, stochastic methylation followed by clonal selection due to a proliferative advantage caused by tumor suppressor gene silencing

Generation of iPSCs from cultured human malignant cells

Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients