Michael P. Storm

Three-Dimensional Culture Systems for the Expansion of Pluripotent Embryonic Stem Cells

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two‐dimensional (2D) static culturing techniques are inadequate for large‐scale production. The culture of mammalian cells in three‐dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher‐S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher‐S out‐performed the Solohill microcarriers. When cultured for successive passages on Cultispher‐S microcarriers, mESCs maintained their pluripotency, demonstrated by self‐renewal, expression of pluripotency markers and the ability to undergo multi‐lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher‐S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra‐1–60, NANOG, and OCT‐4. Our study highlights the importance of optimization of initial seeding parameters and provides proof‐of‐concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695.

Characterization of the Phosphoinositide 3‐Kinase‐Dependent Transcriptome in Murine Embryonic Stem Cells: Identification of Novel Regulators of Pluripotency

Phosphoinositide 3‐kinase (PI3K)‐dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K‐dependent signaling. Using filtering of greater than 1.5‐fold change in expression and an analysis of variance significance level of p < .05, we have defined a dataset comprising 646 probe sets that detect changes in transcript expression (469 down and 177 up) on inhibition of PI3Ks. Changes in expression of selected genes have been validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analyses reveal significant over‐representation of transcriptional regulators within our dataset. In addition, several known regulators of ES cell pluripotency, for example, Nanog, Esrrb, Tbx3, and Tcl‐1, are among the downregulated genes. To evaluate the functional involvement of selected genes in regulation of ES cell self‐renewal, we have used short interfering RNA‐mediated knockdown. These studies identify genes not previously associated with control of ES cell fate that are involved in regulating ES cell pluripotency, including the protein tyrosine phosphatase Shp‐1 and the Zscan4 family of zinc finger proteins. Further gain‐of‐function analyses demonstrate the importance of Zscan4c in regulation of ES cell pluripotency. STEM CELLS 2009;27:764–775

High Basal γH2AX Levels Sustain Self‐Renewal of Mouse Embryonic and Induced Pluripotent Stem Cells

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double‐strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self‐renewal of these cells. Here, we report that not only mESCs but also mouse‐induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self‐renewal‐enhancing small molecules. We observe that self‐renewal activity is highly compromised in H2AX−/− cells and that it can be restored in these cells through reconstitution with a wild‐type, but not a phospho‐mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells. STEM CELLS2012;30:1414–1423

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells.

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.

MicroRNA-122: A Novel Hepatocyte-Enriched in vitro Marker of Drug-Induced Cellular Toxicity

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Next generation in vitro liver model design: Combining a permeable polystyrene membrane with a transdifferentiated cell line

Herein we describe the manufacture and characterisation of biocompatible, porous polystyrene membranes, suitable for cell culture. Though widely used in traditional cell culture, polystyrene has not been used as a hollow fibre membrane due to its hydrophobicity and non-porous structure. Here, we use microcrystalline sodium chloride (4.7 ± 1.3 µm) to control the porosity of polystyrene membranes and oxygen plasma surface treatment to reduce hydrophobicity. Increased porogen concentration correlates to increased surface pore density, macrovoid formation, gas permeability and mean pore size, but a decrease in mechanical strength. For tissue engineering applications, membranes spun from casting solutions containing 40% (w/w) sodium chloride represent a compromise between strength and permeability, having surface pore density of 208.2 ± 29.7 pores/mm2, mean surface pore size of 2.3 ± 0.7 µm, and Youngs modulus of 115.0 ± 8.2 MPa. We demonstrate the biocompatibility of the material with an exciting cell line-media combination: transdifferentiation of the AR42J-B13 pancreatic cell line to hepatocyte-like cells. Treatment of AR42J-B13 with dexamethasone/oncostatin-M over 14 days induces transdifferentiation towards a hepatic phenotype. There was a distinct loss of the pancreatic phenotype, shown through loss of expression of the pancreatic marker amylase, and gain of the hepatic phenotype, shown through induction of expression of the hepatic markers transferrin, carbamoylphosphate synthetase and glutamine synthetase. The combination of this membrane fabrication method and demonstration of biocompatibility of the transdifferentiated hepatocytes provides a novel, superior, alternative design for in vitro liver models and bioartificial liver devices.