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Purpose: Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects., Procedures: To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter., Results: In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers., Conclusions: We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.

Motivation: Primary purpose of modeling gene regulatory networks for developmental process is to reveal pathways governing the cellular differentiation to specific phenotypes. Knowledge of differentiation network will enable generation of desired cell fates by careful alteration of the governing network by adequate manipulation of cellular environment., Results: We have developed a novel integer programming-based approach to reconstruct the underlying regulatory architecture of differentiating embryonic stem cells from discrete temporal gene expression data. The network reconstruction problem is formulated using inherent features of biological networks: (i) that of cascade architecture which enables treatment of the entire complex network as a set of interconnected modules and (ii) that of sparsity of interconnection between the transcription factors. The developed framework is applied to the system of embryonic stem cells differentiating towards pancreatic lineage. Experimentally determined expression profile dynamics of relevant transcription factors serve as the input to the network identification algorithm. The developed formulation accurately captures many of the known regulatory modes involved in pancreatic differentiation. The predictive capacity of the model is tested by simulating an in silico potential pathway of subsequent differentiation. The predicted pathway is experimentally verified by concurrent differentiation experiments. Experimental results agree well with model predictions, thereby illustrating the predictive accuracy of the proposed algorithm., Contact: ipb1@pitt.edu, Supplementary Information: Supplementary data are available at Bioinformatics online.

One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.

Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.