Retrospective assessment of clonality of a legacy cell line by analytical subcloning of the master cell bank.

In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

Serum starvation improves transient transfection efficiency in differentiating embryonic stem cells.

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.

Enrichment of hepatocyte-like cells with upregulated metabolic and differentiated function derived from embryonic stem cells using S-NitrosoAcetylPenicillamine.

The generation of a large number of fully functional hepatocytes from a renewable cell source can provide an unlimited resource for bioartificial liver devices and cell replacement therapies. We have established a directed differentiation system using sodium butyrate treatment to generate an enriched population of hepatocyte-like cells from embryonic stem cells. A metabolic analysis of the hepatocyte populations revealed glycolytic and mitochondrial phenotypes similar to mouse hepatoma cells, implying that these cells represent an immature hepatocyte phenotype. To mediate further differentiation, S-NitrosoAcetylPenicillamine (SNAP), a nitric oxide donor, was utilized to induce mitochondrial development in the precursor populations. A comparative analysis of the different treated populations showed that 500microM SNAP treatment resulted in the generation of an enriched population of metabolically mature hepatocyte-like cells with increased differentiated function. Specifically, 500microM SNAP treatment increased glucose consumption, lactate production rates, mitochondrial mass, and potential as compared to untreated populations. In addition, functional analysis revealed that intracellular albumin content, urea secretion rates, and cytochrome P450 7a1 promoter activity were increased in the treated population. The methodology described here to generate an enriched population of metabolically and functionally mature hepatocyte-like cells may have potential implications in drug discovery and regenerative medicine.

Transient gene delivery for functional enrichment of differentiating embryonic stem cells.

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.

Augmentation of EB-directed hepatocyte-specific function via collagen sandwich and SNAP.

The development of implantable engineered liver tissue constructs and ex vivo hepatocyte-based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)-mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin-18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin-M (OSM) or S-nitroso-N-acetylpenicillamine (SNAP) supplementation of EB-derived hepatocyte-lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long-term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver-specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte-like function. Maintenance and augmentation of hepatocyte-like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.

Control of hepatic differentiation via cellular aggregation in an alginate microenvironment.

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.