Nanotopography mediated osteogenic differentiation of human dental pulp derived stem cells

Advanced medical devices, treatments and therapies demand an understanding of the role of interfacial properties on the cellular response. This is particularly important in the emerging fields of cell therapies and tissue regeneration. In this study, we evaluate the role of surface nanotopography on the fate of human dental pulp derived stem cells (hDPSC). These stem cells have attracted interest because of their capacity to differentiate to a range of useful lineages but are relatively easy to isolate. We generated and utilized density gradients of gold nanoparticles which allowed us to examine, on a single substrate, the influence of nanofeature density and size on stem cell behavior. We found that hDPSC adhered in greater numbers and proliferated faster on the sections of the gradients with higher density of nanotopography features. Furthermore, greater surface nanotopography density directed the differentiation of hDPSC to osteogenic lineages. This study demonstrates that carefully tuned surface nanotopography can be used to manipulate and guide the proliferation and differentiation of these cells. The outcomes of this study can be important in the rational design of culture substrates and vehicles for cell therapies, tissue engineering constructs and the next generation of biomedical devices where control over the growth of different tissues is required

Surface engineering for long-term culturing of mesenchymal stem cell microarrays

The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks.

Gradients of Al/Al2O3 nanostructures for screening mesenchymal stem cell proliferation and differentiation

By decomposing a molecular precursor we fabricated a novel surface based on an aluminium/aluminiumoxide composite incorporating nanotopography gradient to address high-throughput and fast analysis method for studying stem cell differentiation by nanostructures. Depending on the topography of the nanostructures, mesenchymal stem cells exhibit a diverse proliferation and differentiation behavior

Exploring the mesenchymal stem cell niche using high throughput screening

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment – or niche – encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed.

Evaluation of Morpholino Antisense Oligos’ Role on BCR-ABL Gene Silencing in the K562 Cell Line

Objective: Chronic myeloid leukemia (CML) develops when a hematopoietic stem cellacquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cellsto have a greater than normal proliferation rate. Scientists attempt to improve the CMLtreatment process by silencing the BCR/ABL oncogene. In this work, we used morpholinoantisense oligos to silence the BCR/ABL oncogene.Materials and Methods: In this study, the K562 was used as a BCR/ABL fusion-genepositive cell line and the Jurkat cell line as a control. We explored the inhibiting capacityof morpholino antisense oligos in the the expression of the BCR/ABL oncogene andstudied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation ofapoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos.Results: Prolonged exposure of the K562 cell line to the morpholino antisense oligostargeted against the BCR-ABL gene showed proliferation inhibition as its main feature.After western blotting, we found that complete silencing of BCR/ABL was achieved, butflow cytometric analysis showed no broad apoptosis.Conclusion: The results indicate that the Morpholino antisense oligo is able to inhibitp210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR

Identification and In Vitro Expansion of Buccal Epithelial Cells

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME progenitor cells in ex vivo cultures may improve the outcome of BME cell transplantation. However, the parameter of cell seeding density in this context has largely been neglected. This study investigates how varying cell seeding density influences BME cell proliferation and differentiation on tissue culture polystyrene (TCPS). The highest cell proliferation activity was seen when cells were seeded at 5×104 cells/cm2. Both below and above this density, the cell proliferation rate decreased sharply. Differential immunofluorescence analysis of surface markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell seeding density also significantly affected the progenitor cell marker expression profile. Hence, this study demonstrates that seeding density has a profound effect on the proliferation and differentiation of BME stem cells in vitro, and this is relevant to downstream cell therapy applications.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by ‘NHMRC Australia Project grant’, grant agreement No. 595901

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response

Targeted drug delivery using genetically engineered diatom biosilica

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites