Screening and improving the safe provision of mesenchymal stem cells in regenerative medicine: an in vitro study

A number of studies have suggested that mesenchymal stem cells (MSCs) may undergo genetic alterations and spontaneous malignant transformation to form tumour cells, or at least can become contaminated with other cell types following extended periods in culture. Possible transformation or contamination of MSCs during cell culture expansion prior to their use in transplantation therapies is a risk, which should be taken into account. There is a continued need for the development of improved tools for monitoring safety and release criteria of cells intended for cell-based therapies. In order to help address these risks, this thesis aimed to develop improved safety measures in MSC-based cell therapies. Initially, this was investigated through the use of microscopic imaging and image analysis platforms to screen, characterise and distinguish between cultures of non-transformed MSCs and MSC-derived tumour cells, i.e. the osteosarcoma cell lines, SAOS2 and MG63, as well as cells derived from a chondrosarcoma. High content screening (HCS) and live-cell imaging and analysis platforms were used to enable these experiments. Phenotypic features that distinguished the normal versus malignant cell types were identified, including immunoreactivity for the proliferation-associated Ki67 antigen and pluripotency marker Oct4, as well as significant differences in nuclear morphology. These findings help inform release criteria for therapeutic MSCs. To further potentially improve the safety of MSC-based therapies, research was also performed to address the possibility that tumour cells in MSC cultures might remain undetected, thereby still providing a risk in MSC transplantations. Novel combinatorial regimes of potential anti-tumour drugs, i.e. bezafibrate, medroxyprogesterone, and valproic acid (termed V-BAP) were tested in vitro for their effects on MSCs versus SAOS2 and MG63 cells. At determined concentrations, these drugs were shown to significantly inhibit the growth of the osteosarcoma cells, but had little effect on MSCs. Thus, this thesis has made inroads into improved safety of MSC-based therapies by (i) demonstrating the application of imaging-based cell screening platforms to help characterise MSC cultures intended for cell transplantation, and (ii) identifying a novel drug regime that selectively targets osteosarcoma cells whilst having little effect on MSCs. The findings on V-BAP also have application in anti-tumour treatments for osteosarcoma

Automation of 3D cell culture using cellulose-based scaffolds

Introduction: It is recognized that screening of drugs on 2D models is unable to precisely select clinically active medicaments; therefore, 3D culture systems are emerging and show potential for better simulating the in vivo tumour microenvironment and for eliminating the species differences to allow drug testing directly in human systems before drugs move into clinical trials. The purpose of this study was to automate the production and cultivation of human primary osteogenic sarcoma cells line, SaOS-2, in scaffold-based, multiple spheroids in GrowDex® (GDS) and scaffold-free systems, single spheroids (SS) for high-throughput screening. Methods: For scaffold-based models, SAOS-2 were embedded in nanofibrillar cellulose hydrogel, GrowDex®, in flat ultra-low attachment (ULA) 96-well plates while for the scaffold-free system cells were let to form spheroids in U-bottom ULA 96-well plates. Experiments were conducted on the Fluent® 780 automation workstation. The scripts for automated model production and media exchange were established in the FluentControlTM. Technical parameters such as aspiration and dispensing speed as well as XYZ dispensing positions were empirically defined and optimized i) to allow production of stable models, ii) to avoid disturbing during the medium exchange neither the GDS nor the GrowDex structure/shape, iii) to avoid SS displacement and aspiration. GDS and SS models were cultivated up to 11 days and were characterized for their viability (ATP assay), morphology and size (MTT assay). Dose-response tests with taxol and doxorubicin were carried out for both system types. Drugs were automated dispensed on the 3D models at day 4 for a treatment time of 72h. Cells were analysed for cell viability, measuring ATP levels, morphology, and size. Results: The established scripts allowed the formation of multiple spheroids in GrowDex as well as the aggregation of SAOS-2 in single spheroids in the U-bottom ULA plates. 70% of medium could be successfully automated exchanged maintaining unaltered the shape and the position of the models in the wells. GDS and SS remained stable for up to 11 days and increased in size over time, showing a similar growth rate. Viable GDS populated the entire model at different Z values with a compact morphology, parameter that characterized the SS too. GDS showed, as expected, a wide size distribution while SS were bigger and more homogenous in size in comparison to GDS (SS area 10-fold bigger than GDS area). SAOS-2 responded in both systems to taxol and doxorubicin, showing higher IC50 values for GDS compared to SS. Taxol (fig.1) and doxorubicin were 3.5- and 4.5-fold more potent in SS than in GDS. Collapsed morphology with a viable and compact core and a loose cell layer around border at high drugs concentrations characterized exclusively SS. Discussion & Conclusions: The automation protocols were successfully established allowing the reproducible production and maintenance of GrowDex multiple spheroids and scaffold-free models. Although its viscosity, GrowDex is automation compatible, and the results obtained in this project shows its high potential for high-throughput drug screening. Acknowledgements: The ZHAW authors would like to thank the Tecan for the technical support and the consumables, and the UPM Biomedicals for its contribution

Combined bezafibrate, medroxyprogesterone acetate and valproic acid treatment inhibits osteosarcoma cell growth without adversely affecting normal mesenchymal stem cells.

This document is the Accepted Manuscript version of a published work that appeared in final form in Bioscience Reports. To access the final edited and published work see http://dx.doi.org/10.1042/BSR20202505Drug repurposing is a cost effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts

Electrical stimulation of adipose-derived stem cells in 3D nanofibrillar cellulose increases their osteogenic potential

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ACSs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASC. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures

Optically Transparent Anionic Nanofibrillar Cellulose Is Cytocompatible with Human Adipose Tissue-Derived Stem Cells and Allows Simple Imaging in 3D.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.Peer Reviewe

Biomimetic and electroactive 3D scaffolds for human neural crest-derived stem cell expansion and osteogenic differentiation

Abstract: Osteoporosis is a skeletal disease characterized by bone loss and bone microarchitectural deterioration. The combination of smart materials and stem cells represents a new therapeutic approach. In the present study, highly porous scaffolds are prepared by combining the conducting polymer PEDOT:PSS with collagen type I, the most abundant protein in bone. The inclusion of collagen proves to be an effective way to modulate their mechanical properties and it induces an increase in scaffolds’ electrochemical impedance. The biomimetic scaffolds support neural crest-derived stem cell osteogenic differentiation, with no need for scaffold pre-conditioning contrarily to other reports

Introductory programming: a systematic literature review

As computing becomes a mainstream discipline embedded in the school curriculum and acts as an enabler for an increasing range of academic disciplines in higher education, the literature on introductory programming is growing. Although there have been several reviews that focus on specific aspects of introductory programming, there has been no broad overview of the literature exploring recent trends across the breadth of introductory programming. This paper is the report of an ITiCSE working group that conducted a systematic review in order to gain an overview of the introductory programming literature. Partitioning the literature into papers addressing the student, teaching, the curriculum, and assessment, we explore trends, highlight advances in knowledge over the past 15 years, and indicate possible directions for future research

Development and characterisation of a novel, megakaryocyte NF-κB reporter cell line for investigating inflammatory responses

Background Due to the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterise a novel megakaryocyte NF-κB reporter cell line in order to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-κB-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-κB-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-κB to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics including the expression of both GPIb and integrin β3. Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as TNFα and Pam3CSK4 increased NF-κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to LPS (non-ultrapure) although it was not able to react to ultrapure LPS due to the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterisation of a novel megakaryocyte NF-κB reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-κB activity