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.

How to extract and expand randomness : a summary and explanation of existing results

We examine the use of randomness extraction and expansion in key agreement (KA) pro- tocols to generate uniformly random keys in the standard model. Although existing works provide the basic theorems necessary, they lack details or examples of appropriate cryptographic primitives and/or parameter sizes. This has lead to the large amount of min-entropy needed in the (non-uniform) shared secret being overlooked in proposals and efficiency comparisons of KA protocols. We therefore summa- rize existing work in the area and examine the security levels achieved with the use of various extractors and expanders for particular parameter sizes. The tables presented herein show that the shared secret needs a min-entropy of at least 292 bits (and even more with more realistic assumptions) to achieve an overall security level of 80 bits using the extractors and expanders we consider. The tables may be used to �nd the min-entropy required for various security levels and assumptions. We also �nd that when using the short exponent theorems of Gennaro et al., the short exponents may need to be much longer than they suggested.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds

Insufficient availability of osteogenic cells limits bone regeneration through cell-based therapies. This study investigated the potential of amniotic fluid–derived stem (AFS) cells to synthesize mineralized extracellular matrix within porous medical-grade poly-e-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in two-dimensional (2D) culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6-mm diameter9-mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive micro computed tomography (microCT) analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of predifferentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced seven times more mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.

Kava Anxiety Depression Spectrum Study (KADSS): a mixed methods RCT using an aqueous extract of Piper methysticum

Objectives: To report on the design, significance and potential impacts of the first documented human clinical trial assessing the anxiolytic and thymoleptic efficacy of an aqueous monoextract of Piper methysticum (kava). The significance of the qualitative element of our clinical trial is also explored. The Kava Anxiety Depression Spectrum Study (KADSS) is a 3-week placebocontrolled, double-blind, cross-over trial involving 60 adult participants (18—65) with elevated stable anxiety and varying levels of depressive symptoms. Aims: The aims of KADSS are: (1) to determine whether an aqueous standardised extract of kava is effective for the treatment of anxiety; (2) to assess the effects of kava on differing levels of depression; and (3) to explore participants’ experience of taking kava via qualitative research. The study also provides preliminary assessment of the safety of an aqueous extract of kava in humans. Conclusion: If results reveal that the aqueous kava preparation exerts significant anxiolytic effects and appears safe, potentially beneficial impacts may occur. Data supporting a safe and effective kava extract may encourage a re-introduction of kava to Europe, UK and Canada. This may provide a major socioeconomic benefit to Pacific Island nations, and to sufferers of anxiety disorders.

The Kava Anxiety Depression Spectrum Study (KADSS): A randomized, placebo-controlled, cross-over trial using an aqueous extract of Piper methysticum.

Rationale: Piper methysticum (Kava) has been withdrawn in European, British, and Canadian markets due to concerns over hepatotoxic reactions. The WHO recently recommended research into “aqueous” extracts of Kava. Objective: The objective of this study was to conduct the first documented human clinical trial assessing the anxiolytic and antidepressant efficacy of an aqueous extract of Kava. Design and participants: The Kava Anxiety Depression Spectrum Study was a 3-week placebo-controlled, double-blind crossover trial that recruited 60 adult participants with 1 month or more of elevated generalized anxiety. Five Kava tablets per day were prescribed containing 250 mg of kavalactones/day. Results: The aqueous extract of Kava reduced participants' Hamilton Anxiety Scale score in the first controlled phase by −9.9 (CI = 7.1, 12.7) vs. −0.8 (CI = −2.7, 4.3) for placebo and in the second controlled phase by −10.3 (CI = 5.8, 14.7) vs. +3.3 (CI = −6.8, 0.2). The pooled effect of Kava vs. placebo across phases was highly significant (p < 0.0001), with a substantial effect size (d = 2.24, η² [sub]p[sub] = 0.428). Pooled analyses also revealed highly significant relative reductions in Beck Anxiety Inventory and Montgomery–Asberg Depression Rating Scale scores. The aqueous extract was found to be safe, with no serious adverse effects and no clinical hepatotoxicity. Conclusions: The aqueous Kava preparation produced significant anxiolytic and antidepressant activity and raised no safety concerns at the dose and duration studied. Kava appears equally effective in cases where anxiety is accompanied by depression. This should encourage further study and consideration of globally reintroducing aqueous rootstock extracts of Kava for the management of anxiety.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells

The growth and differentiation of mesenchymal stem cells is controlled by various growth factors, the activities of which can be modulated by heparan sulfates. We have previously underscored the necessity of sulfated glycosaminoglycans for the FGF-2-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of heparan sulfate to cultures of primary rat MSCs stimulates their proliferation leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation and osteogenic differentiation of rMSCs when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. Furthermore, we show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth resulting in the cells being unable to reach the critical density necessary to induce differentiation. Interestingly, blocking FGFR1 signaling in post-confluent osteogenic cultures significantly increased calcium deposition. Taken together our data clearly suggests that FGFR1 signaling plays an important role during osteogenic differentiation, firstly by stimulating cell growth that is closely followed by an inhibitory affect once the cells have reached confluence. It also underlines the importance of HS as a co-receptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

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.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.