A chimeric vitronectin : IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells

This paper was retracted by the Journal of Stem Cells and Development on February 15, 2013.

Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.

A note on a reaction-diffusion model describing the bone morphogen protein gradient in Drosophila embryonic patterning

In this article, we consider the Eldar model [3] from embryology in which a bone morphogenic protein, a short gastrulation protein, and their compound react and diffuse. We carry out a perturbation analysis in the limit of small diffusivity of the bone morphogenic protein. This analysis establishes conditions under which some elementary results of [3] are valid.

Stage-specific embryonic antigen-4 is not a marker for chondrogenic and osteogenic potential in cultured chondrocytes and mesenchymal progenitor cells

One important challenge for regenerative medicine is to produce a clinically relevant number of cells with consistent tissue-forming potential. Isolation and expansion of cells from skeletal tissues results in a heterogeneous population of cells with variable regenerative potential. A more consistent tissue formation could be achieved by identification and selection of potent progenitors based on cell surface molecules. In this study, we assessed the expression of stage-specific embryonic antigen-4 (SSEA-4), a classic marker of undifferentiated stem cells, and other surface markers in human articular chondrocytes (hACs), osteoblasts, and bone marrow-derived mesenchymal stromal cells (bmMSCs) and characterized their differentiation potential. Further, we sorted SSEA-4-expressing hACs and followed their potential to proliferate and to form cartilage in vitro. Cells isolated from cartilage and bone exhibited remarkably heterogeneous SSEA-4 expression profiles in expansion cultures. SSEA-4 expression levels increased up to approximately 5 population doublings, but decreased following further expansion and differentiation cultures; levels were not related to the proliferation state of the cells. Although SSEA-4-sorted chondrocytes showed a slightly better chondrogenic potential than their SSEA-4-negative counterparts, differences were insufficient to establish a link between SSEA-4 expression and chondrogenic potential. SSEA-4 levels in bmMSCs also did not correlate to the cells' chondrogenic and osteogenic potential in vitro. SSEA-4 is clearly expressed by subpopulations of proliferating somatic cells with a MSC-like phenotype. However, the predictive value of SSEA-4 as a specific marker of superior differentiation capacity in progenitor cell populations from adult human tissue and even its usefulness as a stem cell marker appears questionable.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Regulation of human stem cell research in Australia

Australia is currently well placed to contribute to the global growth of human stem cell research. However, as the science has progressed, authorities have had to deal with the ongoing challenges of regulating such a fast moving field of scientific endeavour. Australia’s past and current approach to regulating the use of embryos in human embryonic stem cell research provides an insight into how Australia may continue to adapt to future regulatory challenges presented by human stem cell research. In the broader context, a number of issues have been identified that may impact upon the success of future human stem cell research in Australia.

Stem cell technologies : regulation, patents and problems

Human embryonic stem cell research promises to deliver in the future a whole range of therapeutic treatments, but currently governments in different jurisdictions must try to regulate this burgeoning area. Part of the problem has been, and continues to be, polarised community opinion on the use of human embryonic stem cells for research. This article compares the approaches of the Australian, United Kingdom and United States governments in regulating human embryonic stem cell research. To date, these governments have approached the issue through implementing legislation or policy to control research. Similarly, the three jurisdictions have viewed the patentability of human embryonic stem cell technologies in their own ways with different policies being adopted by the three patent offices. This article examines these different approaches and discusses the inevitable concerns that have been raised due to the lack of a universal approach in relation to the regulation of research; the patenting of stem cell technologies; and the effects patents granted are having on further human embryonic stem cell research.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Characterisation and culturing of adipose-derived precursor cells

This book focuses on practical applications for using adult and embryonic stem cells in the pharmaceutical development process. It emphasizes new technologies to help overcome the bottlenecks in developing stem cells as therapeutic agents. A key reference for professionals working in stem cell science, it presents the general principles and methodologies in stem cell research and covers topics such as derivitization and characterization of stem cells, stem cell culture and maintenance, stem cell engineering, applications of high-throughput screening, and stem cell genetic modification with their use for drug delivery.