Generation of a novel three-dimensional scaffold-based model of the bovine endometrium.

Bovine in vitro endometrial models that resemble tissue function in vivo are needed to study infertility, long-term uterine alterations induced by pathogens and impact of endocrine disruptor chemicals on reproductive function and other reproductive system complications that cause high economic losses in livestock species. The present study aimed to generate an innovative, reproducible, and functional 3D scaffold-based model of the bovine endometrium structurally robust for long term-culture. We developed a multicellular model containing both endometrial epithelial and stromal cells. Epithelial cells organized to form a luminal-like epithelial layer on the surface of the scaffold. Stromal cells produced their own extracellular matrix forming a stable subepithelial compartment that physiologically resembles the normal endometrium. Both cell types released prostaglandin E2 and prostaglandin F2α following a treatment with oxytocin and arachidonic acid. Additionally signal pathways mediating oxytocin and arachidonic acid stimulation of prostaglandin synthesis were analyzed by real time PCR (RT-PCR). Oxytocin receptor (OXTR), prostaglandin E2 receptor 2 (EP2), prostaglandin E2 receptor 4 (EP4), prostaglandin F receptor (PTGFR), prostaglandin E synthase (PTGES), PGF-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (COX-2) expression was detected in both control and treatment groups, however, only significant changes in abundance of OXTR mRNA transcripts were found. The results obtained by this study are a step forward in bovine in vitro culture technology. This 3D scaffold-based model provides a platform to study regulatory mechanisms involved in endometrial physiology and can set the basis for a broader tool for designing and testing novel therapeutic strategies for recurrent uterine pathologies.

Application of biomimetic surfaces and 3D culture technology to study the role of extracellular matrix interactions in neurite outgrowth and inhibition.

The microenvironment that cells experience during in vitro culture can often be far removed from the native environment they are exposed to in vivo. To recreate the physiological environment that developing neurites experience in vivo, we combine a well-established model of human neurite development with, functionalisation of both 2D and 3D growth substrates with specific extracellular matrix (ECM) derived motifs displayed on engineered scaffold proteins. Functionalisation of growth substrates provides biochemical signals more reminiscent of the in vivo environment and the combination of this technology with 3D cell culture techniques, further recapitulates the native cellular environment by providing a more physiologically relevant geometry for neurites to develop. This biomaterials approach was used to study interactions between the ECM and developing neurites, along with the identification of specific motifs able to enhance neuritogenesis within this model. Furthermore, this technology was employed to study the process of neurite inhibition that has a detrimental effect on neuronal connectivity following injury to the central nervous system (CNS). Growth substrates were functionalised with inhibitory peptides released from damaged myelin within the injured spinal cord (Nogo & OMgp). This model was then utilised to study the underlying molecular mechanisms that govern neurite inhibition in addition to potential mechanisms of recovery.

Design considerations of benchtop fluid flow bioreactors for bio-engineered tissue equivalents in vitro.

One of the major aims of bio-engineering tissue equivalents in vitro is to create physiologically relevant culture conditions to accurately recreate the cellular microenvironment. This often includes incorporation of factors such as the extracellular matrix, co-culture of multiple cell types and three-dimensional culture techniques. These advanced techniques can recapitulate some of the properties of tissue in vivo, however fluid flow is a key aspect that is often absent. Fluid flow can be introduced into cell and tissue culture using bioreactors, which are becoming increasingly common as we seek to produce increasingly accurate tissue models. Bespoke technology is continuously being developed to tailor systems for specific applications and to allow compatibility with a range of culture techniques. For effective perfusion of a tissue culture many parameters can be controlled, ranging from impacts of the fluid flow such as increased shear stress and mass transport, to potentially unwanted side effects such as temperature fluctuations. A thorough understanding of these properties and their implications on the culture model can aid with a more accurate interpretation of results. Improved and more complete characterisation of bioreactor properties will also lead to greater accuracy when reporting culture conditions in protocols, aiding experimental reproducibility, and allowing more precise comparison of results between different systems. In this review we provide an analysis of the different factors involved in the development of benchtop flow bioreactors and their potential biological impacts across a range of applications.

Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts.

Various methods are currently used to investigate human tissue differentiation, including human embryo culture and studies utilising pluripotent stem cells (PSCs) such as in vitro embryoid body formation and in vivo teratoma assays. Each method has its own distinct advantages, yet many are limited due to being unable to achieve the complexity and maturity of tissue structures observed in the developed human. The teratoma xenograft assay allows maturation of more complex tissue derivatives, but this method has ethical issues surrounding animal usage and significant protocol variation. In this study, we have combined three-dimensional (3D) in vitro cell technologies including the common technique of embryoid body (EB) formation with a novel porous scaffold membrane, in order to prolong cell viability and extend the differentiation of PSC derived EBs. This approach enables the formation of more complex morphologically identifiable 3D tissue structures representative of all three primary germ layers. Preliminary in vitro work with the human embryonal carcinoma line TERA2.SP12 demonstrated improved EB viability and enhanced tissue structure formation, comparable to teratocarcinoma xenografts derived in vivo from the same cell line. This is thought to be due to reduced diffusion distances as the shape of the spherical EB transforms and flattens, allowing for improved nutritional/oxygen support to the developing structures over extended periods. Further work with EBs derived from murine embryonic stem cells demonstrated that the formation of a wide range of complex, recognisable tissue structures could be achieved within 2-3 weeks of culture. Rudimentary tissue structures from all three germ layers were present, including epidermal, cartilage and epithelial tissues, again, strongly resembling tissue structure of teratoma xenografts of the same cell line. Proof of concept work with EBs derived from the human embryonic stem cell line H9 also showed the ability to form complex tissue structures within this system. This novel yet simple model offers a controllable, reproducible method to achieve complex tissue formation in vitro. It has the potential to be used to study human developmental processes, as well as offering an animal free alternative method to the teratoma assay to assess the developmental potential of novel stem cell lines.

Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues.

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

Application of synthetic photostable retinoids induces novel limb and facial phenotypes during chick embryogenesis in vivo.

We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal-distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.

A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

Mesenchymal differentiation propensity of a human embryonic stem cell line.

OBJECTIVES: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL-002. MATERIALS AND METHODS: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real-time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. RESULTS: Undifferentiated KCL-002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox-2, Oct-4 and TRA 1-60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR-2, α-actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. CONCLUSIONS: The data presented suggest that the KCL-002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.

Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins.

The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination.

Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins.

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.

The role of retinoids in the adult nervous system and their therapeutic potential.

The mode of action of retinoids in relation to their activity in the adult central nervous system and the potential of synthetic retinoid analogues is reviewed. Investigation into the activity of such molecules will further our understanding of the retinoid pathway during nervous system development and in various neurological disease states.

Following the differentiation of human pluripotent stem cells by proteomic identification of biomarkers.

Following the differentiation of cultured stem cells is often reliant on the expression of genes and proteins that provide information on the developmental status of the cell or culture system. There are few molecules, however, that show definitive expression exclusively in a specific cell type. Moreover, the reliance on a small number of molecules that are not entirely accurate biomarkers of particular tissues can lead to misinterpretation in the characterization of the direction of cell differentiation. Here we describe the use of technology that examines the mass spectrum of proteins expressed in cultured cells as a means to identify the developmental status of stem cells and their derivatives in vitro. This approach is rapid and reproducible and it examines the expression of several different biomarkers simultaneously, providing a profile of protein expression that more accurately corresponds to a particular type of cell differentiation.

Growth of teratomas derived from human pluripotent stem cells is influenced by the graft site.

Pluripotent stem cells transplanted into immune-deficient mice form complex teratomas. Although such tumors are generally haphazard in their organization, they do contain some structures that resemble tissues normally seen in the embryo. As a consequence, the teratoma model is useful for exploring the developmental potential of stem cells and studying certain aspects of tissue development. To further our understanding of this process, we examined whether the anatomical location into which human pluripotent stem cells were grafted influenced their growth in situ. Here we report that cells grafted into the liver rapidly produced large tumors containing predominantly immature cells. In contrast, subcutaneous implants were significantly slower growing and eventually formed tumors composed of differentiated tissues. The alternative growth patterns recorded between these two graft sites indicates how environmental cues affect stem cell behavior. This approach may lead to the identification of new ways to control stem cell growth and differentiation.

Rewritable DNA microarrays.

Thiol-terminated single-stranded deoxyribonucleic acids (ssDNA) can be immobilized onto pulsed plasma deposited poly(allylmercaptan) surfaces via disulfide bridge chemistry and are found to readily undergo nucleic acid hybridization. Unlike other methods for oligonucleotide attachment to solid surfaces, this approach is shown to be independent of substrate material or geometry, and amenable to highly efficient rewriting.

Neural development by transplanted human embryonal carcinoma stem cells expressing green fluorescent protein.

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

Growth of human stem cell-derived neurons on solid three-dimensional polymers.

Understanding neural differentiation and the development of complex neurite networks in three-dimensional matrices is critical for neural tissue engineering in vitro. In this study we describe for the first time the growth of human stem cell-derived neurons on solid polystyrene matrices coated with bioactive molecules. Highly porous foams were prepared from poly(styrene/divinylbenzene) using a high internal phase emulsion (HIPE) as a template to create the porous structure. The resulting polyHIPE matrices were readily coated with aqueous-based solutions including poly-d-lysine and laminin. Human neurons adhered well to poly-d-lysine coated surfaces and extended neural processes, however, neurite outgrowth was particularly enhanced when polymers also received a coating of laminin. These data clearly demonstrate the potential use of solid polystyrene scaffolds to create three-dimensional environments for cell growth and differentiation. We propose that these robust and stable matrices can be conveniently and routinely used in the tissue culture laboratory to study the behaviour of cells grown in three-dimensions.

Human embryonal carcinoma stem cells: models of embryonic development in humans.

There are few reliable experimental systems available to study the molecular mechanisms that govern human embryonic development. Embryonal carcinoma (EC) cells are pluripotent stem cells derived from teratocarcinomas and are considered the malignant counterparts of human embryonic stem (ES) cells. Several of the existing human EC stem cell lines provide robust and simple culture systems to study certain aspects of cellular differentiation in a manner pertinent to human embryogenesis. Here we review the strategies used to derive and characterize the established and recognized human EC stem cell line TERA2.cl.SP12. Furthermore, we demonstrate the value of human EC stem cells as a model of early development and focus on cell fate determination in the embryonic ectoderm.

Generation of neuroprogenitor-like cells from adult mammalian bone marrow stromal cells in vitro.

Recently, it has been proposed that bone marrow stromal cells (BMSCs) have a broader capacity for differentiation than previously contemplated. In vitro studies have indicated that BMSCs may have the capacity to differentiate into neuroectodermal-like cells in response to various growth conditions, including those commonly used to maintain and differentiate cultures of primary neural stem cells (NSCs). Interpreting the wealth of data on this subject has been difficult because of variation in the starting cell population and the differences between the methods used to induce their differentiation. Here we evaluate how cultures of expanded BMSCs with a consistent immunophenotype respond to a variety of growth conditions and induction agents and review their ability to form neural-like derivatives. In addition, we report on some modifications to previously published techniques for the generation of neural-like cells from BMSCs in vitro.

Proteomic identification of biomarkers expressed by human pluripotent stem cells.

The ability to effectively monitor the behaviour of pluripotent stem cells and their differentiation is key to their use in basic and clinical research. Molecules expressed in particular cell types can be used to report the status of cell differentiation and is a recognised means of assessing the behaviour of cell cultures. There are currently few useful markers of stem cells and there is no rapid way to accurately determine their level of expression. In this study, we describe for the first time the potential of surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) to identify novel biomarkers of human pluripotent embryonal carcinoma stem cells and their differentiated derivatives. This approach allows the rapid and sensitive screening of cell samples without the need to purify the specimen prior to analysis. The identification of proteins expressed in specific cell populations will provide valuable tools for monitoring cellular development.

Enhanced neurite outgrowth by human neurons grown on solid three-dimensional scaffolds.

Growing and differentiating human stem cells in vitro can provide access to study the molecular mechanisms that control cellular development in a manner pertinent to human embryogenesis. To fully understand such processes, however, it is important to recreate culture conditions that most closely relate to those in living tissues. As step in this direction, we have developed a robust three-dimensional cell culture system using inert highly porous solid matrices manufactured from polystyrene that can be routinely used to study the differentiation of human pluripotent stem cell-derived neurons in vitro. Neurite outgrowth was significantly enhanced when neurons were grown in a three-dimensional environment compared to traditional flat surfaces and resulted in the formation of extensive neural networks. These data suggest that the topography within the culture environment can significantly alter cell development and will therefore be an important feature when investigating the potential of human stem cells.