Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons.

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Highly active nisin coated polycaprolactone electrospun fibers against both Staphylococcus aureus and Pseudomonas aeruginosa.

In this study, a wound dressing of electrospun polycaprolactone (PCL) fibers incorporating the antimicrobial peptide (AMP) nisin was fabricated. Nisin was physically adsorbed to the PCL fibers and tested for antibacterial activity against both Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). The PCL fibers had an average diameter of 1.16 µm ± 0.42 µm and no significant change in diameter occurred after nisin adsorption. X-ray photoelectron spectroscopy (XPS) analysis of the fibers detected nitrogen indicative of adsorbed nisin and the signal was used to quantify the levels of coverage on the fiber surfaces. In vitro nisin release studies showed a burst release profile with 80 % of the nisin being released from the fibers within 30 min. Air plasma pre-treatment of the PCL fibers to render them hydrophilic improved nisin loading and release. Antibacterial testing was performed using minimum inhibitory concentration (MIC) and surface attachment assays. The released nisin remained active against both Gram positive S. aureus and Gram negative P. aeruginosa, which has previously been difficult to achieve with single polymer fiber systems. Mammalian cell culture of the nisin coated fibers with L-929 mouse fibroblasts and human epidermal keratinocytes (HEKa) showed that the nisin did not have a significant effect on the biocompatibility of the PCL fibers. The results presented here demonstrate that the physical adsorption, which is a post-treatment, overcomes the potential limitations of harsh chemicals and fabrication conditions of electrospinning from organic solvents and provides a drug loading system having effective antibacterial properties in wound dressings.

Efficient Generation of Stable Cell Lines with Inducible Neuronal Transgene Expression Using the piggyBac Transposon System.

The piggyBac transposon system has been adapted to be a highly efficient genome engineering tool for transgenesis of eukaryotic cells and organisms. As with other methods of transgenesis, incorporation of an inducible promoter, such as a tetracycline-responsive element, enables inducible transgene expression. Here, we describe an efficient method of using the piggyBac system to create stably transfected mammalian cell lines, including inducible transgene expression. Gibson assembly is used to construct the required vectors as it enables multiple DNA fragments to be seamlessly assembled in a single isothermal reaction. We demonstrate an application of this approach to generate a stably transfected pluripotent stem cell line that can be induced to express a transcription factor transgene and rapidly differentiate into neurons in a single step.

Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.

CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.

Tools for Efficient Genome Editing; ZFN, TALEN, and CRISPR.

The last two decades have marked significant advancement in the genome editing field. Three generations of programmable nucleases (ZFNs, TALENs, and CRISPR-Cas system) have been adopted to introduce targeted DNA double-strand breaks (DSBs) in eukaryotic cells. DNA repair machinery of the cells has been exploited to introduce insertion and deletions (indels) at the targeted DSBs to study function of any gene-of-interest. The resulting indels were generally assumed to be "random" events produced by "error-prone" DNA repair pathways. However, recent advances in computational tools developed to study the Cas9-induced mutations have changed the consensus and implied the "non-randomness" nature of these mutations. Furthermore, CRISPR-centric tools are evolving at an unprecedented pace, for example, base- and prime-editors are the newest developments that have been added to the genome editing toolbox. Altogether, genome editing tools have revolutionized our way of conducting research in life sciences. Here, we present a concise overview of genome editing tools and describe the DNA repair pathways underlying the generation of genome editing outcome.

Alloreactivity of Allogeneic Mesenchymal Stem/Stromal Cells and Other Cellular Therapies: A Concise Review.

Cellular therapies, deemed live medicine, have brought a wave of new generation biological therapies to treat previously untreatable diseases such as cancers and degenerative diseases like osteoarthritis. These cellular therapies have gained significant recognition in clinical research. The area has been further strengthened with the approval of Chimeric Antigen Receptor added on T cells (CAR-T) therapies by the regulatory authorities USA's Food and Drugs Administration (FDA), European Medical Agency (EMA), the Australian Therapeutic Goods Administration (TGA), and in many countries in 2017 to treat hematological cancers. Another milestone was achieved when allogeneic Mesenchymal Stem Cell- (MSC-) based therapy was approved by the EMA to treat Chrohn's disease in 2018. Allogeneic donor-derived MSC therapies in particular hold great promise and real hope because of their 'off-the shelf' availability and accessibility for patients in need of urgent treatment. So far, thousands of clinical trials have explored the safety and efficacy of both autologous and allogeneic cell therapies, deeming them safe, however with varying degrees of efficacy. In the current pandemic, clinical trials have begun in many parts of the world to treat severe cases of COVID with MSCs. However, the risk of tissue rejection and the development of undesirable effects due to alloreactivity of allogeneic cells are currently not adequately addressed. Therefore, this warrants careful investigation and detailed reporting of such events by clinical researchers. This review aims at discussing the current landscape of approved allogeneic MSCs along with a few other cellular therapies. We explore any possible reactivity reported to inform the readers of any safety concern and on the efficacy of such therapies.

The Mechanisms of Restenosis and Relevance to Next Generation Stent Design.

Stents are lifesaving mechanical devices that re-establish essential blood flow to the coronary circulation after significant vessel occlusion due to coronary vessel disease or thrombolytic blockade. Improvements in stent surface engineering over the last 20 years have seen significant reductions in complications arising due to restenosis and thrombosis. However, under certain conditions such as diabetes mellitus (DM), the incidence of stent-mediated complications remains 2-4-fold higher than seen in non-diabetic patients. The stents with the largest market share are designed to target the mechanisms behind neointimal hyperplasia (NIH) through anti-proliferative drugs that prevent the formation of a neointima by halting the cell cycle of vascular smooth muscle cells (VSMCs). Thrombosis is treated through dual anti-platelet therapy (DAPT), which is the continual use of aspirin and a P2Y12 inhibitor for 6-12 months. While the most common stents currently in use are reasonably effective at treating these complications, there is still significant room for improvement. Recently, inflammation and redox stress have been identified as major contributing factors that increase the risk of stent-related complications following percutaneous coronary intervention (PCI). The aim of this review is to examine the mechanisms behind inflammation and redox stress through the lens of PCI and its complications and to establish whether tailored targeting of these key mechanistic pathways offers improved outcomes for patients, particularly those where stent placement remains vulnerable to complications. In summary, our review highlights the most recent and promising research being undertaken in understanding the mechanisms of redox biology and inflammation in the context of stent design. We emphasize the benefits of a targeted mechanistic approach to decrease all-cause mortality, even in patients with diabetes.

Comparative analysis of extracellular vesicles isolated from human mesenchymal stem cells by different isolation methods and visualisation of their uptake.

Various types of cells secrete extracellular vesicle (EVs) which contain proteins, lipids and nucleic acids and play important roles in inter-cellular signalling and pathological processes to impact the recipient cells. EVs have demonstrated their potential as biomarkers for disease and as therapeutic agents in regenerative medicine. In recent times, EVs derived from mesenchymal stem cells (MSCs), which are widely used as a promising medicinal product in many clinical applications, are being tested in many preclinical trials. However, the lack of standardization of MSC-derived EV isolation and analysis methods, restricts the utility of MSC-derived EVs in the clinical setting. Here, we focused on optimising the isolation method for EVs derived from MSCs. Four samples of EVs were isolated from human adipose derived MSC culture medium by differential ultracentrifugation with three different ultracentrifuge durations to investigate the influence of ultracentrifuge time on quality and quantity of MSC-derived EVs. Additionally, we used a commercial kit to extract EVs from MSC cultured medium and compared it with the ultracentrifugation method. The EV samples were then characterised for particle concentration, protein concentration, size distribution and the presence of known EV protein markers, by western blot and flow cytometry. A comparison of these results for the five samples demonstrated that 1 h of differential ultracentrifugation was optimal to isolate high quality and quantity of MSC-derived EVs from MSC cultured medium. Additionally, fluorescence imaging of the freshly isolated vs frozen EVs showed that freshly isolated EVs are taken up by cells more efficiently than frozen EVs. These finding establish a simple and reliable method of EV isolation from MSCs.

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model.

BACKGROUND: Mesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment. AIM: In light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device. METHODS: The conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium. RESULTS: The A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups. CONCLUSIONS: Hyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.

Forward Programming of Pluripotent Stem Cells to Neurons.

Pluripotent stem cells (PSCs) are powerful tools for studying developmental biology and neuronal diseases. Conventional differentiation protocols require several intermediate states and different culture conditions, inefficiently generating mixed subtypes of neuronal cells with immature characteristics. Direct programming of PSCs by forced expression of neuronal transcription factors has shown rapid cell fate determination with high purity as it can bypass sequential developmental steps that traditional culture requires. In this review, we focus on neuronal differentiation from PSCs to specific subtypes by various transcription factors. Furthermore, the potential applications and limitations of this novel technology are discussed.

Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia.

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

Mesenchymal Stem Cell-Derived Extracellular Vesicles and Their Therapeutic Potential.

Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles, which act as shuttles, delivering a range of biomolecules to diverse target cells. They play an important role in maintenance of biophysiological homeostasis and cellular, physiological, and pathological processes. EVs have significant diagnostic and therapeutic potentials and have been studied both in vitro and in vivo in many fields. Mesenchymal stem cells (MSCs) are multipotent cells with many therapeutic applications and have also gained much attention as prolific producers of EVs. MSC-derived EVs are being explored as a therapeutic alternative to MSCs since they may have similar therapeutic effects but are cell-free. They have applications in regenerative medicine and tissue engineering and, most importantly, confer several advantages over cells such as lower immunogenicity, capacity to cross biological barriers, and less safety concerns. In this review, we introduce the biogenesis of EVs, including exosomes and microvesicles. We then turn more specifically to investigations of MSC-derived EVs. We highlight the great therapeutic potential of MSC-derived EVs and applications in regenerative medicine and tissue engineering.

Binary Colloidal Crystal (BCC) Substrates for Controlling the Fate of Mouse Embryonic Stem Cells.

Understanding the interactions of stem cells with surface topography can give us an invaluable tool in controlling stemness and fate of stem cells for further use in biomedical applications. In this study, we have fabricated topographical features using a class of cell culture substrates called binary colloidal crystals (BCCs), that are made by self-assembly of mixtures of spherical micron sized silica (Si) and nanometer sized polystyrene (PS) or poly (methyl methacrylate) (PMMA) particles. The substrates formed are arrays of ordered, hexagonally packed large Si particles inter-dispersed with the PS particles that are stabilized by gentle heating, which melts the PS or PMMA forming substrates suitable for cell culture. BCC substrates were used for culture of mouse embryonic stem cells (mESCs). Compared to tissue culture plates, COM1 (Si5-PMMA0.4), COM2 (Si5-PS0.4) and COM4 (Si2-PSC0.22) have shown to provide a better support for mESC proliferation in the presence of the cytokine leukemia inhibitory factor (LIF). The behavior of mESCs with the BCCs in presence and absence of LIF, was further explored and it was found that interaction of mESCs with the culture substrate can be controlled by tuning surface topography and roughness, which is determined by the size and type of particles used in making BCCs. Furthermore, it was shown that limiting cell-surface interactions and controlling colony shape can promote stemness maintenance on COM1 and COM2 substrates as indicated by better proliferation and higher expression of pluripotency genes including Nanog both in presence and in absence of LIF. Together with higher expression of GATA6 gene, it can be stated that these surfaces can be used for endodermic priming of mESCs. Therefore, we believe that these surfaces, especially COM1 and COM2 surfaces can be beneficial as stem cell culture systems for further use in biomedical research.

Selective Cytotoxicity of a Novel Trp-Rich Peptide against Lung Tumor Spheroids Encapsulated inside a 3D Microfluidic Device.

There is a globally rising healthcare need to develop new anticancer therapies as well as to test them on biologically relevant in vitro cancer models instead of overly simplistic 2D models. To address both these needs, a 3D lung cancer spheroid model is developed using human A549 cells trapped inside a collagen gel in a compartmentalized microfluidic device and homogenously sized (35-45 µm) multicellular tumor spheroids are obtained in 5 days. The novel tryptophan-rich peptide P1, identified earlier as a potential anticancer peptide (ACP), shows enhanced cytotoxic efficacy against A549 tumor spheroids (>75%) in clinically relevant low concentrations, while it does not affect human amniotic membrane mesenchymal stem cells at the same concentrations (<15%). The peptide also inhibits the formation of tumor spheroids by reducing cell viability as well as lowering the proliferative capacity, which is confirmed by the expression of cell proliferation marker Ki-67. The ACP offers a novel therapeutic strategy against lung cancer cells without affecting healthy cells. The microfluidic device used is likely to be useful in helping develop models for several other cancer types to test new anticancer agents.

On-chip anticancer drug screening - Recent progress in microfluidic platforms to address challenges in chemotherapy.

There is an increasing need for advanced and inexpensive preclinical models to accelerate the development of anticancer drugs. While costly animal models fail to predict human clinical outcomes, in vitro models such as microfluidic chips ('tumor-on-chip') are showing tremendous promise at predicting and providing meaningful preclinical drug screening outcomes. Research on 'tumor-on-chips' has grown enormously worldwide and is being widely accepted by pharmaceutical companies as a drug development tool. In light of this shift in philosophy, it is important to review the recent literature on microfluidic devices to determine how rapidly the technology has progressed as a promising model for drug screening and aiding cancer therapy. We review the past five years of successful developments and capabilities in microdevice technology (cancer models) for use in anticancer drug screening. Microfluidic devices that are being designed to address current challenges in chemotherapy, such as drug resistance, combinatorial drug therapy, personalized medicine, and cancer metastasis are also reviewed in detail. We provide a perspective on how personalized 'tumor-on-chip', as well as high-throughput microfluidic platforms based on patient-specific tumor cells, can potentially replace the more expensive and 'non-human' animal models in preclinical anticancer drug development.

New Approaches to Treat Osteoarthritis with Mesenchymal Stem Cells.

Osteoarthritis is one of the most common chronic health problems in the world that causes disability and chronic pain with reduced mobility and is a progressive degenerative disease in weight-bearing joints such as the knee. The pathology of the joint resulting from OA includes loss of cartilage volume and cartilage lesions leading to inflammation of the articular joint structures; its incidence and progression are associated with a variety of risk factors. Most of the current treatments focus on symptom management such as physical and occupational therapies, pharmacological intervention for pain management, and surgical intervention with limited success and do not address nor halt the progression of the disease. In this review, we will describe the current treatment options for OA and the exciting new translational medical research currently underway utilising mesenchymal stem cells for OA therapy.

Outcome of Allogeneic Adult Stem Cell Therapy in Dogs Suffering from Osteoarthritis and Other Joint Defects.

Osteoarthritis is a common condition that causes joint pain and stiffness that affects both humans and dogs. In Australia, allogeneic canine adipose-derived mesenchymal stem cells for therapy have been commercially available since 2010. In this report, we describe the outcome of the treatment of two hundred and three dogs diagnosed with degenerative arthritis with severe chronic pain in joints causing lameness at walk, reduced mobility, and functional disability. Posttreatment assessment data after 10 weeks revealed significant improvement (p < 0.007) of the symptoms: pain reduction, improvement of mobility, and increased daily activity as measured as quality of life score. Ninety percent of young dogs (<9 years) showed excellent improvement in pain and mobility and were able to run and resume normal activity. Sixty percent of older dogs showed good improvement. However, 12% of dogs did not exhibit any change in symptoms; one dog showed worsening of the symptoms. This report provides the support for the safety and efficacies of allogeneic adipose-derived mesenchymal stem cells in a regenerative therapeutic veterinary model.

Heterogeneity of mesenchymal and pluripotent stem cell populations grown on nanogrooves and nanopillars.

Surface nanotopographies are an important way of mimicking the stem cell niche on biomaterial surfaces. Previous studies have focused on the differentiation of stem cells into a defined lineage using nanotopographies, but they have rarely considered the homogeneity of cell populations produced. We examined the impact of two types of substrates (i.e. nanogrooves and nanopillars made by soft lithography) on the surface-induced differentiation of human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) and mouse embryonic stem cells (mESCs) without the use of additional chemical induction medium components. Cell morphology and proliferation were analysed at day 1 and day 3. Gene expression was analysed at day 14 for hAM-MSCs and at day 7 for mESC-derived embryoid bodies (mEBs) using quantitative real-time polymerase chain reaction (qPCR). The substrates with nanogrooves had a noticeable effect on cell alignment in a depth dependent manner with both cell types showing strong alignment along the deep grooves. On the other hand, the nanopillar substrates showed inhibition of cell spreading for both cell types. The nanogrooves showed inhibition of hAM-MSC growth but enhanced mEB proliferation, especially on the deeper grooves. The nanopillars did not significantly affect hAM-MSC growth, but can modulate mEB growth depending on the pillar density, indicating that mEBs are more sensitive to nanotopographies in terms of proliferation, while hAM-MSCs are only sensitive to specific structures and sizes. Genes associated with bone, cartilage, and fat were investigated for hAM-MSCs, whereas genes of the endoderm, mesoderm, ectoderm, and pluripotency were investigated for mEBs. In general, gene expression for hAM-MSCs was not enhanced significantly by the nanotopographies compared to the flat control. On the other hand, genes of bone, cartilage, skeletal muscle, heart, and liver were up-regulated on both nanopillars and nanogrooves, especially OP65 (ordered pillars with 65% density) and SG40 (shallow grooves with 40 nm depth) in a feature size dependent manner. We found that a small portion of mEBs was composed of cardiac-like beating cells (i.e. GFP-NKX2.5 positive) and a bone cell marker (i.e. OCN) indicating a heterogeneous cell population being generated on those types of surfaces. This work highlights the importance of nanotopographies in stem cell differentiation and how studying multiple properties of the substrate and cells is needed as we strive to generate homogeneous and mature cell populations using biomaterials.