Homozygosity for a null mutation in the scl gene causes mid‐gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl−/−) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl−/− embryonic stem cells make a substantial contribution to all non‐hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl−/− embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

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BackgroundAcute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML.MethodsWe interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p <0.01 and Δβ >0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons.ResultsWe identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis.ConclusionsThe DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology.

Differentiation induction in murine Ml leukemia cells by interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) is postulated to occur via a common receptor chain, gpl3O. In this study, growth factorinduced differentiation of Ml cells was accompanied by a late and persistent decrease in levels of mRNA and protein for a helix-oop-helix transcription factor, the SCL gene product. (18,(21)(22)(23). Despite differences in these receptor complexes, receptor signaling for IL-6, LIF, and OSM is postulated to occur via a common receptor chain (gpl3O) (24)(25)(26)(27).In the work reported here, we studied the behavior of SCL mRNA and protein during growth factor-induced differentiation of Ml leukemia cells. We then established M1/SCL cell The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.lines in which overexpression of SCL mRNA and protein was enforced. Using these M1/SCL cell lines, we examined the effects of SCL overexpression on terminal differentiation induced by LIF, IL-6, and OSM. MATERIALS AND METHODSEnforced Expression of SCL in Ml Cells. An SCL retrovirus was constructed by introducing the entire murine SCL coding region (28) into the MPZen/Neo retrovirus (29) so that SCL expression was under control of the long terminal repeat promoter of myeloproliferative sarcoma virus. Ml cells were then infected by cocultivation with an SCL-retroviral "packaging" cell line (30). After 3 days of cocultivation, cells were cloned in agar by selection with the neomycin analogue G418 (400 ,ug/ml). On day 7, individual G418-resistant colonies were resuspended to establish four M1/SCL clonal cell lines. M1/SCL clonal cell lines were confrmed to express the retroviral (exogenous) SCL mRNA (see below). Three control cell lines were obtained by using the vector alone; clonal cell lines derived by G418 selection in agar (M1/neo cell lines) were examined in addition to parental Ml cells. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 bovine calf serum. At various time points cells were prepared for morphological examination (using Cytospin preparations stained with May-Grunwald and Giemsa reagents and examining a minimum of 200 cells) and Northern analysis, with medium being changed every 3 days during the culture period.Agar Cultures. Cells (300 per ml) were cultured with serial dilutions of purified recombinant mouse LIF, IL-6, or OSM in 35-mm Petri dishes using DMEM with a final concentration of 20% preselected bovine calf serum and 0.3% agar in a final volume of 1 ml. Cultures were incubated at 37°C in a fully humidified atmosphere of 10% CO2 in air. Colonies (clones of >40 cells) were scored at x 35 magnifications with a dissection microscope after 7 days of incubation. Differentiated colonies were identified by their characteristic dispersed morphology (31).Recloning experiments were performed by select...

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.

The role of telomerase and telomeres within the hematopoietic compartment needs further clarification. Advances in our knowledge in this field may improve clinical outcomes for the treatment of hematologic disease.