Transient gene delivery for functional enrichment of differentiating embryonic stem cells

Abstract

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.

Serum starvation improves transient transfection efficiency in differentiating embryonic stem cells

“…2,[6][7][8][9] Recently, we developed a transient gene delivery system to deliver two fluorescent liver-specific reporter plasmids into differentiating, semi-mature murine embryonic stem (ES) cells for enriching a subpopulation of hepatocyte-like cells. 10 The benefit of the transient expression of the plasmids fulfilled the system's needs, as the activation of the fluorescent reporters was only necessary up to and through the completion of the cell sorting. Nonetheless, the scale-up of this system is limited by inherent low plasmid transfection efficiency.…”

“…In earlier work, we found that purifying differentiating ES cells expressing the Cyp7A1driven reporter yields greater hepato-specific functional enrichment than cells sorted with a more ubiquitous reporter driven by the albumin enhancer/promoter. 10 In the current study, we first assessed the role of serum starvation in modulating transfection efficiency using a general constitutive CMV-driven plasmid. We then specifically targeted liver-specific cells using the Cyp7A1 reporter in an effort to purify the number of cells in this subpopulation expressing the reporter.…”

Engineering Embryonic Stem-Cell Aggregation Allows an Enhanced Osteogenic Differentiation In Vitro

“…[5][6][7][8] It is hypothesized that by controlling EB formation, it may be possible to control or enrich specified ES cell differentiation. [9][10][11] Previous studies have demonstrated selective differentiation of osteoblasts from ES cells via EB culture with factors including ascorbic acid, b-glycerophosphate (BGP), and the osteoinductive glucocorticoid dexamethasone (Dex). [12][13][14] In vitro cultured osteoblasts hold great promise for tissue repair and regeneration of bone, damaged as a result of disease and trauma.…”

Establishment of Novel Detection System for Embryonic Stem Cell-Derived Hepatocyte-Like Cells Based on Nongenetic Manipulation with Indocyanine Green

“…[5][6][7][8] Thus, several studies have used ESC lines transfected with the green fluorescent protein reporter gene controlled by hepatocytespecific promoters to select and/or detect ESC-derived hepatocytes. 9,10 However, this technique is relatively time consuming and leads to significant cell injury. Further, genetic modification carries risks such as possible tumor formation.…”