induced pluripotent stem cells ͉ epigenetic regulation ͉ Oct4 ͉ Nanog ͉ Sox2

Increasing studies suggest that SALL4 may play vital roles in leukemogenesis and stem cell phenotypes. We have mapped the global gene targets of SALL4 using chromatin immunoprecipitation followed by microarray hybridization and identified more than 2000 highconfidence, SALL4-binding genes in the human acute promyelocytic leukemic cell line, NB4. Analysis of SALL4-binding sites reveals that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (P < .05).These genes include 38 important apoptosis-inducing genes (TNF, TP53, PTEN, CARD9, CARD11, CYCS, LTA) and apoptosis-inhibiting genes (Bmi-1, BCL2, XIAP, DAD1, TEGT). Real-time polymerase chain reaction has shown that expression levels of these genes changed significantly after SALL4 knockdown, which ubiquitously led to cell apoptosis.

BackgroundInfantile hemangiomas (IH) are the most common benign tumors of infancy. The typical clinical course consists of rapid growth during the first year of life, followed by natural and gradual involution over a multi-year time span through unknown cellular mechanisms. Some tumors respond to medical treatment with corticosteroids or beta-blockers, however, when this therapy fails or is incomplete, surgical extirpation may be necessary. Noninvasive therapies to debulk or eliminate these tumors would be an important advance. The development of an in vitro cell culture system and an animal model would allow new insights into the biological processes involved in the development and pathogenesis of IH.ResultsWe observed that proliferative stage IH specimens contain significantly more SALL4+ and CD133+ cells than involuting tumors, suggesting a possible stem cell origin. A tumor sphere formation assay was adapted to culture IH cells in vitro. Cells in IH tumor spheres express GLUT1, indicative of an IH cell of origin, elevated levels of VEGF, and various stem/progenitor cell markers such as SALL4, KDR, Oct4, Nanog and CD133. These cells were able to self-renew and differentiate to endothelial lineages, both hallmarks of tumor stem cells. Treatment with Rapamycin, a potent mTOR/VEGF inhibitor, dramatically suppressed IH cell growth in vitro. Subcutaneous injection of cells from IH tumor spheres into immunodeficient NOD-SCID mice produced GLUT1 and CD31 positive tumors with the same cellular proliferation, differentiation and involution patterns as human hemangiomas.ConclusionsThe ability to propagate large numbers of IH stem cells in vitro and the generation of an in vivo mouse model provides novel avenues for testing IH therapeutic agents in the future.

Our group and others have recently shown that a murine transcriptional factor, SALL4 plays an essential role in maintaining the properties of embryonic stem cells and in governing the fate of the primitive inner cell mass through regulation of Oct4. Over-expression of SALL4 in mice exhibit myelodysplastic syndrome (MDS) which transforms to acute myeloid leukemia (AML) associated with expansion of leukemic stem cells. Aberrant expression of SALL4 in leukemic blasts is commonly seen in a variety of AML and MDS cases. However, the transcriptional programs downstream of the SALL4 stem cell gene in leukemia are unknown. We mapped its global gene targets using chromatin-immunoprecipitation followed by microarray hybridization (ChIP-on-ChIP). We identified over two thousands high-confidence SALL4 binding sites in the acute leukemic cell line, NB4. Analysis of the SALL4 binding sites revealed that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (p&lt;0.05) when functionally classified. This is consistent with promoter binding of over 280 apoptosis-related genes including important apoptosis and cell-cycle arrest induction genes (DAP3, CARD9, TP53, DAXX, CASP6, CASP9, CYCS, TNF, TAL1, and PTEN) and apoptosis inhibition genes (Bmi-1, BCL2, TEGT and XIAP). This indicates that SALL4 may play a role in leukemogenesis. Reduction of wild type SALL4 mRNA levels by approximately 50 percent resulted in a 21-fold increase in caspase-3 activity from 4.6 percent to 98.3 percent. In addition, suppression of SALL4 expression caused a significant decrease in NB4 cell growth rate. To further study the role of the SALL4 in cell growth, we monitored cell-cycle changes and cellular DNA synthesis in SALL4-suppressed and wild type NB4 cells through flow cytometry analysis and the BrdU incorporation assay. SALL4-reduced NB4 showed about a four-fold decrease in S phase cells and a significant increase in the G1 and G2 phases, which paralleled the drop in DNA synthesis as measurement from the level of BrdU incorporation. Similar results were observed in other cancer cell lines. In addition, SALL4-reduced NB4 cells showed significantly decreased tumorigenicity in immunodeficient mice. Our studies provide a foundation for developing a leukemia stem cell-specific therapy targeting SALL4.