Integral to the development of embryonic stem cell therapeutic strategies for hepatic disorders is the identification and establishment of a controllable hepatic differentiation strategy. In order to address this issue we have established an alginate microencapsulation approach which provides a means to modulate the differentiation process through changes in key encapsulation parameters. We report that a wide array of hepatocyte specific markers is expressed by cells differentiated during a 23-day period within an alginate bead microenvironment. These include urea and albumin secretion, glycogen storage, and cytochrome P450 transcription factor activity. In addition, we demonstrate that cellular aggregation is integral to the control of differentiation within the bead environment and this process is mediated by the E-cadherin protein. The temporal expression of surface E-cadherin and hepatocyte functional expression occur concomitantly and both cellular aggregation and albumin synthesis are blocked in the presence of anti E-cadherin immunoglobulin. Furthermore, by establishing a compartmental model of differentiation, which incorporates this aggregation phenomenon, we can optimize key encapsulation parameters.

The temporal patterns of adverse event reports are more complex than those described in Weber's classic report. The number of reports does not reliably rise and then fall after launch and the reporting rate does not reliably decrease with time from launch.

Control of genetic expression is a critical issue in the field of stem cell biology, where determining a cell fate or reprogramming adult somatic cells into pluripotent cells has become a common experimental practice. In turn, for these cells to have therapeutic clinical potential, techniques for controlling gene expression are needed that minimizes or eliminates the risk of oncogenesis and mutagenesis. Possible routes for achieving this outcome could come in the form of a transient nonviral gene delivery system. In this study, we improved the efficiency of transient gene delivery to differentiating murine embryonic stem (ES) cells via serum starvation for 3 days before transfection. The transient expression of a constitutively-controlled plasmid increased from ~50% (replated control) to ~83% when transfected after 3 days of serum starvation but decreased to ~28% when transfected after 3 days in normal high serum-containing media. When probed with a liver-specific reporter, Cyp7A1, expression increased from ~1.4% (replated control) to ~3.7% when transfected after 3 days of serum starvation but decreased to ~0.7% when transfected after 3 days in high serum-containing media. Cy3-tagged oligonucleotides were used to rapidly quantify DNA uptake and predict ultimate transfection efficiency. This study suggests that modifications in media serum levels before transfection can have a profound effect on improving nonviral gene delivery.

There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. We have reported previously a variety of methods for differentiating murine embryonic stem (ES) cells into hepatocyte-like cells. One major challenge of our work and others in the field has been the ability to selectively purify and enrich these cells from a heterogeneous population. Traditional approaches for inserting new genes (e.g., stable transfection, knock-in, retroviral transduction) involve permanent alterations in the genome. These approaches can lead to mutations and involve the extra costs and time of developing, validating and maintaining new cell lines. We have developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells are purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis is performed. In an effort to test the ability of the reporters to work in a transient environment for our differentiation system, we engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection efficiency of delivering these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. Development of gene reporter systems that allow us to identify, purify and assess homogeneous populations of cells is important in better understanding stem cell differentiation pathways. And engineering cellular systems without making permanent gene changes will be critical for the generation of clinically acceptable cellular material in the future.