hESC Culture Research Articles

Hepatic differentiation of human embryonic stem cells on microcarriers

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells, especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system, high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1×106cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers, both in static culture and stirred bioreactors, induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea, and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment.

Physiological Oxygen Prevents Frequent Silencing of the DLK1-DIO3 Cluster during Human Embryonic Stem Cells Culture

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs.

The nature of floating cells in human embryonic stem cell culture

A consistent presence of floating cells is a common phenomenon in cultures of human embryonic stem cells (hESCs). However, little attention has been paid to their existence. It is currently believed that unavoidable imperfections in culture conditions lead the cells to undergo senescence and apoptosis resulting in unattached cells floating in the culture medium. Inspired by recent studies on mitotic activities in human embryonic stem cell colonies, we believe the existence of floating cells is not simply the result of unfavorable growth conditions but an intrinsic phenomenon resulted from maintaining the pluripotency of hESCs under the culture conditions. We tested this hypothesis with a set of systematic experiments and discovered: 1) the ratio of floating cells to attached cells was significantly increased with culture time; 2) the number of floating cells could be manipulated. For example, we were able to reduce the number of floating cells by providing the colonies with more horizontal or vertical cultural spaces and maintaining the cells’ pluripotency. The results open a new avenue to increase the stem cell culture efficiencies by rescuing the floating cells. On the other hand, by placing a physical barrier on the top of colonies, the number of floating cells was decreased, at the same time, hESCs also showed signs of differentiation. In addition, when inducing cells to differentiate with retinoic acid, the number of floating cells no longer increased with prolonged culture time. Taken together, these results suggested that continuous cell division across the colonies is responsible for the emergence of floating cells during hESC culture. This is quite different from the bacterial colony growth where the cells in the center of colonies are quiescent. Our results indicated that continuous cell division, even at the cost of floating cells formation, is essential for human embryonic stem cell proliferation.

Derivation of Chondrogenic Cells from Human Embryonic Stem Cells for Cartilage Tissue Engineering.

Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative applications in dentistry and medicine. We previously described a micromass culture system as a model system to induce and study the chondrogenic commitment of hESCs. Using this system, chondrogenic cells can be further isolated and expanded under specific growth factor conditions. When encapsulated in hyaluronic acid (HA)-based hydrogels and cultured under appropriate growth factor and medium conditions, these chondrogenic cells synthesized and deposited extracellular matrix (ECM) characteristic of neocartilage. Here, we describe the micromass culture of hESCs, the isolation and expansion of hESC-derived chondrogenic cells, and the three-dimensional (3-D) culture of the chondrogenic cells in hydrogels for cartilage tissue engineering. We will also describe the various tools and techniques used for characterizing the tissue-engineered cartilage.

High‐Density Polymer Microarrays: Identifying Synthetic Polymers that Control Human Embryonic Stem Cell Growth

The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications.

A Synthetic Peptide-Acrylate Surface for Production of Insulin-Producing Cells from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

An In Vitro Mechanism Study on the Proliferation and Pluripotency of Human Embryonic Stems Cells in Response to Magnesium Degradation

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials, we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg, cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence, their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose, the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4–40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages, the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4, SSEA3, and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM, however, hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications, providing their degradation rate is moderate. Additionally, the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro, and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds.

MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells

IntroductionTissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells.MethodshESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis.ResultsWe first found that TF was expressed only in trophoblasts and granulocyte–monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway.ConclusionsThe miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.

Genomic Analysis of hESC Pedigrees Identifies De Novo Mutations and Enables Determination of the Timing and Origin of Mutational Events

SummaryGiven the association between mutational load and cancer, the observation that genetic aberrations are frequently found in human pluripotent stem cells (hPSCs) is of concern. Prior studies in human induced pluripotent stem cells (hiPSCs) have shown that deletions and regions of loss of heterozygosity (LOH) tend to arise during reprogramming and early culture, whereas duplications more frequently occur during long-term culture. For the corresponding experiments in human embryonic stem cells (hESCs), we studied two sets of hESC lines: one including the corresponding parental DNA and the other generated from single blastomeres from four sibling embryos. Here, we show that genetic aberrations observed in hESCs can originate during preimplantation embryo development and/or early derivation. These early aberrations are mainly deletions and LOH, whereas aberrations arising during long-term culture of hESCs are more frequently duplications. Our results highlight the importance of close monitoring of genomic integrity and the development of improved methods for derivation and culture of hPSCs.

Identification of Unsafe Human Induced Pluripotent Stem Cell Lines Using a Robust Surrogate Assay for Pluripotency

Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.

Spontaneously DifferentiatedGATA6-Positive Human Embryonic Stem Cells Represent an Important Cellular Step in Human Embryonic Development; They Are Not Just an Artifact of In Vitro Culture

In this study, we isolated and characterized spontaneously differentiated human embryonic stem cells (SD-hESCs) found in hESC colonies in comparison to the morphologically premature ESCs in the colonies to investigate the potential role of SD-hESCs in embryogenesis. SD-hESCs were distinguished from undifferentiated hESCs by their higher expression of GATA6, a marker for primitive endoderm and transthyretin, a marker visceral endoderm in embryoid bodies (EBs). SD-hESCs expressed OCT4 and NANOG, markers for pluripotent stem cells, at significantly lower levels than undifferentiated hESCs. EBs derived from isolated SD-hESCs were morphologically distinct from cells directly derived from the undifferentiated hESCs; they contained higher number of cysts compared to EBs from undifferentiated hESC-derived EBs (42% vs. 20%). Furthermore, the extracellular signal molecule, BMP2/4, induced a higher GATA4/6 expression and cystic EB formation than control and noggin-treated EBs. Since cystic formation in EBs play a role in primitive endoderm formation during embryogenesis, the SD-hESC may be a relevant cell type equipped to differentiate into primitive endoderm. Our results suggest that SD-ESCs generated during routine hESC culture are not just an artifact of in vitro culture and these cells could serve as a useful model to study the process of embryogenesis.

Derivation of human embryonic stem cell lines from single blastomeres of low-quality embryos by direct plating

To explore a simple method of establishing pluripotent human embryonic stem cell (hESC) lines from single blastomeres of low-quality (LQ) embryos. Blastomeres were isolated from normally fertilized, day-3 pre-implantation LQ embryos by dissolving of the zona pellucida and were then plated directly onto inactivated human foreskin fibroblasts. The subsequent culture was identical to that used to derive a hESC line from the inner cell mass of a blastocyst. The established hESC lines were passaged and characterized. Two hESC lines were produced by culturing the blastomeres individually in a hESC culture system (hESC-CS). Both of the hESC lines maintained a normal 46-chromosome XY karyotype, expressed stemness markers, and showed a pluripotent phenotype, including the ability to differentiate into all three germ layers in vitro and in vivo. The blastomeres of LQ embryos have a developmental capacity that necessitates prolonged culture. Plating of blastomeres from LQ embryos directly into the hESC-CS is a feasible method for deriving hESC lines.

Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Abstract 3833: A novel 3D remodelable hydrogel for bioengineering tumor .

Abstract The need for new tools and technologies to decrease the cost of drug discovery and reduce animal testing has long been recognized. Three-dimensional (3-D) cell cultures have great potential for reducing costly late-stage drug failures by providing an in vivo-like early stage evaluation of efficacy and toxicity. We have developed a novel 3D culture system, which will serve as a simple and rapid method to examine cell proliferation, migration, apoptosis, and tissue formation in a physiologically relevant 3-D environment, similar to the in vivo setting, than standard 2-D arrays. This transglutaminase crosslinked gelatin (T-gel) system provides a native ECM scaffold for cell-cell and cell-matrix interaction; its transparency property enables routine observation with ease; the proteolysis sensitivity scaffold supports cell migration and invasion; the controllable rigidity mimics physiological and pathological environments. Importantly, 3D gel models have great potential as drug evaluation tools as they exhibit in vivo-like cell-cell and cell-ECM interaction, possess metabolic, proliferative and diffusion gradients. It is simple to use on a large scale. Compare to Matrigel, Tgel is with well defined ingredients, wide range of handling properties, easy to prepare at room temperature, and little batch to batch variation. It supports both anchorage dependent and independent cell growth due to hydrogel and natural ECM network properties. We make 3D cultures more relevant by including two or more cell types in different configurations co-cultures. Cells can be tracked on their formation of aggregates, migration (individual or collective), invasion direction (random or directional), speed, and responses to hypoxic and nutritional gradients. At the end of test, viable cells can be easily removed from the 3D T-gel for further experimentation. T-gel can also be used as a carrier for xenograft tumor formation in nude mice models. Other applications include cell growth and differentiation studies, metabolism/toxicology Studies, in vitro/vivo angiogenesis assay, hESC and iPSC culture and Differentiation. Citation Format: Bo Han, Josephine Fang, Zhi Yang, Marcel Nimni, Charisse Tayag. A novel 3D remodelable hydrogel for bioengineering tumor . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3833. doi:10.1158/1538-7445.AM2013-3833

Passaging Techniques and ROCK Inhibitor Exert Reversible Effects on Morphology and Pluripotency Marker Gene Expression of Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) are known for their potential usage in regenerative medicine, but also for handling sensitivity. Much effort has been put into optimizing the culture methods of hESCs. It has been shown that the use of Rho-associated coiled-coil kinase inhibitor (ROCKi) decreases the cellular stress response and the apoptotic cell death in hESC cultures that have been passaged enzymatically. These observations sparked a wide use of ROCKi in hESC cultures. We and others, however, noted that cells passaged enzymatically with the use of ROCKi had a different morphology compared to cells passaged mechanically. Here we show that hESCs that were enzymatically passaged displayed alterations in the nuclear size compared to cultures that were mechanically passaged. Notably, a dramatically decreased expression of the genes encoding common pluripotency markers, such as OCT4/POU5F1 and NANOG were revealed in enzymatically passaged hESCs compared to mechanically passaged, while such differences were not significant when assessing protein levels. The differences in gene expression did not correlate strongly with commonly analyzed histone modifications (H3K4me3, H3K9me3, H3K27me3, and H4K16ac) on the promoters of these genes. Surprisingly, the effects of enzymatic passaging were at least in part reversible as the gene expression profile of enzymatically passaged hESCs that were transferred back to mechanical passaging, showed no significant difference compared to those hESCs that were continuously passaged mechanically. Our results suggest that enzymatic passaging influences parameters associated with hESC characteristics, and emphasizes the importance of using cells handled in the same manner when comparing results both within and between projects.

318 DIFFERENTIATION OF ES-DERIVED HEPATIC PROGENITORS AND HepaRG CELL LINE INTO CHOLANGIOCYTE-LIKE CELLS

Background and Aims: Primary hepatocytes are difficult to culture ex vivo and their utility is limited by the lack of available human hepatocytes. These problems provide incentive into finding sources of hepatocyte-like cells from stem cell populations. The hepatic differentiation of human embryonic stem cells (hESC) has been extensively described for 2D cultures, however differentiated cells exhibit poor hepatic function and no typical hepatic architecture is observed. Our aims were to investigate whether 3D culture of hESC in bioreactor devices can enhance the hepatic function and whether a more physiological architecture can be promoted. Methods and Results: Human ESC (WA01, WiCell, James A. Thomson, University of Wisconsin, Madison, USA) were initially subjected comparatively to five established protocols [1–5] for promoting hepatic differentiation. In two of these approaches [1,2] we observed successful differentiation of hESC into hepatocyte-like cells, which were obtained for further analysis and culture. The culture and differentiation of hESC was carried out in a miniaturised bioreactor system with a cell compartment volume of 0.4ml and 2D cultures were performed in parallel as controls. The differentiation in the bioreactors was performed by adaptation of the protocols published by Hay et al [1,2]. Medium samples were taken daily and analysed for hepatic differentiation markers. At the end of culture immunohistochemistry for hepatic, biliary and pluripotency markers was performed for both bioreactor and 2D cultures. RT-PCR analysis for hepatocyte-specific genes including albumin and various cytochrome P450 isotypes showed a significant increase in expression in 3D cultures. Furthermore observations of the cellular architecture from bioreactor cultures demonstrated 3D structures that were closer to primary liver cell structures than those observed from 2D cultures. Conclusions: These data show that hESC can be successfully differentiated in 3D bioreactors where they express a number of hepatocyte-specific genes and form 3D structures. Thus, the bioreactor system provides a new instrument for generating functional hepatocyte-like cells from hESC, which has great potential for clinical and pharmacological studies.

317 HEPATIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS IN 3D BIOREACTOR CULTURES

Background and Aims: Primary hepatocytes are difficult to culture ex vivo and their utility is limited by the lack of available human hepatocytes. These problems provide incentive into finding sources of hepatocyte-like cells from stem cell populations. The hepatic differentiation of human embryonic stem cells (hESC) has been extensively described for 2D cultures, however differentiated cells exhibit poor hepatic function and no typical hepatic architecture is observed. Our aims were to investigate whether 3D culture of hESC in bioreactor devices can enhance the hepatic function and whether a more physiological architecture can be promoted. Methods and Results: Human ESC (WA01, WiCell, James A. Thomson, University of Wisconsin, Madison, USA) were initially subjected comparatively to five established protocols [1–5] for promoting hepatic differentiation. In two of these approaches [1,2] we observed successful differentiation of hESC into hepatocyte-like cells, which were obtained for further analysis and culture. The culture and differentiation of hESC was carried out in a miniaturised bioreactor system with a cell compartment volume of 0.4ml and 2D cultures were performed in parallel as controls. The differentiation in the bioreactors was performed by adaptation of the protocols published by Hay et al [1,2]. Medium samples were taken daily and analysed for hepatic differentiation markers. At the end of culture immunohistochemistry for hepatic, biliary and pluripotency markers was performed for both bioreactor and 2D cultures. RT-PCR analysis for hepatocyte-specific genes including albumin and various cytochrome P450 isotypes showed a significant increase in expression in 3D cultures. Furthermore observations of the cellular architecture from bioreactor cultures demonstrated 3D structures that were closer to primary liver cell structures than those observed from 2D cultures. Conclusions: These data show that hESC can be successfully differentiated in 3D bioreactors where they express a number of hepatocyte-specific genes and form 3D structures. Thus, the bioreactor system provides a new instrument for generating functional hepatocyte-like cells from hESC, which has great potential for clinical and pharmacological studies.

Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.