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A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 [CV2, also known as BMP endothelial cell precursor derived regulator (BMPER) ] is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.

miRNAs (miR) play a critical role in human cancers, including hepatocellular carcinoma. Although miR-302b has been suggested to function as a tumor repressor in other cancers, its role in hepatocellular carcinoma is unknown. This study investigated the expression and functional role of miR-302b in human hepatocellular carcinoma. The expression level of miR-302b is dramatically decreased in clinical hepatocellular carcinoma specimens, as compared with their respective nonneoplastic counterparts, and in hepatocellular carcinoma cell lines. Overexpression of miR-302b suppressed hepatocellular carcinoma cell proliferation and G1–S transition in vitro, whereas inhibition of miR-302b promoted hepatocellular carcinoma cell proliferation and G1–S transition. Using a luciferase reporter assay, AKT2 was determined to be a direct target of miR-302b. Subsequent investigation revealed that miR-302b expression was inversely correlated with AKT2 expression in hepatocellular carcinoma tissue samples. Importantly, silencing AKT2 recapitulated the cellular and molecular effects seen upon miR-302b overexpression, which included inhibiting hepatocellular carcinoma cell proliferation, suppressing G1 regulators (Cyclin A, Cyclin D1, CDK2) and increasing p27Kip1 phosphorylation at Ser10. Restoration of AKT2 counteracted the effects of miR-302b expression. Moreover, miR-302b was able to repress tumor growth of hepatocellular carcinoma cells in vivo. Implications: Taken together, miR-302b inhibits HCC cell proliferation and growth in vitro and in vivo by targeting AKT2. Mol Cancer Res; 12(2); 190–202. ©2013 AACR.

Rationale: Vascular calcification is a regulated process that involves osteoprogenitor cells and frequently complicates common vascular disease, such as atherosclerosis and diabetic vasculopathy. However, it is not clear whether the vascular endothelium has a role in contributing osteoprogenitor cells to the calcific lesions. Objective: To determine whether the vascular endothelium contributes osteoprogenitor cells to vascular calcification. Methods and Results: In this study, we use 2 mouse models of vascular calcification, mice with gene deletion of matrix Gla protein, a bone morphogenetic protein (BMP)-inhibitor, and Ins2 Akita/+ mice, a diabetes model. We show that enhanced BMP signaling in both types of mice stimulates the vascular endothelium to contribute osteoprogenitor cells to the vascular calcification. The enhanced BMP signaling results in endothelial–mesenchymal transitions and the emergence of multipotent cells, followed by osteoinduction. Endothelial markers colocalize with multipotent and osteogenic markers in calcified arteries by immunostaining and fluorescence-activated cell sorting. Lineage tracing using Tie2 - Gfp transgenic mice supports an endothelial origin of the osteogenic cells. Enhancement of matrix Gla protein expression in Ins2 Akita/+ mice, as mediated by an Mgp transgene, limits the generation of multipotent cells. Moreover, matrix Gla protein–depleted human aortic endothelial cells in vitro acquire multipotency rendering the cells susceptible to osteoinduction by BMP and high glucose. Conclusions: Our data suggest that the endothelium is a source of osteoprogenitor cells in vascular calcification that occurs in disorders with high BMP activation, such as deficiency of BMP-inhibitors and diabetes mellitus.

White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5–7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.