Cellular Outgrowth Research Articles

Functional heterogeneity of meniscal fibrochondrocytes and microtissue models is dependent on modality of fibrochondrocyte isolation.

Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology-related studies. However, the impact of these modalities on study outcomes is unknown. Here, we show that og- and d-isolated MFC have distinct proliferative capacities, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming, and migratory capacities. Our data indicate that microtissue pellet models developed from og-isolated MFC display a contractile phenotype with higher expressions of alpha-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC isolation designated digestion-after-outgrowth (dog). The transcriptomic profile of dog-MFC is distinct from d- and og-MFC, including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue pellet models containing a higher frequency of smaller collagen fibre diameters. Thus, we demonstrate that the modalities of MFC isolation influence the downstream outcomes of bioengineering and mechanobiology-related studies.

A biomimetic approach to modulating the sustained release of fibroblast growth factor 2 from fibrin microthread scaffolds.

The pleiotropic effect of fibroblast growth factor 2 (FGF2) on promoting myogenesis, angiogenesis, and innervation makes it an ideal growth factor for treating volumetric muscle loss (VML) injuries. While an initial delivery of FGF2 has demonstrated enhanced regenerative potential, the sustained delivery of FGF2 from scaffolds with robust structural properties as well as biophysical and biochemical signaling cues has yet to be explored for treating VML. The goal of this study is to develop an instructive fibrin microthread scaffold with intrinsic topographic alignment cues as well as regenerative signaling cues and a physiologically relevant, sustained release of FGF2 to direct myogenesis and ultimately enhance functional muscle regeneration. Heparin was passively adsorbed or carbodiimide-conjugated to microthreads, creating a biomimetic binding strategy, mimicking FGF2 sequestration in the extracellular matrix (ECM). It was also evaluated whether FGF2 incorporated into fibrin microthreads would yield sustained release. It was hypothesized that heparin-conjugated and co-incorporated (co-inc) fibrin microthreads would facilitate sustained release of FGF2 from the scaffold and enhance in vitro myoblast proliferation and outgrowth. Toluidine blue staining and Fourier transform infrared spectroscopy confirmed that carbodiimide-conjugated heparin bound to fibrin microthreads in a dose-dependent manner. Release kinetics revealed that heparin-conjugated fibrin microthreads exhibited sustained release of FGF2 over a period of one week. An in vitro assay demonstrated that FGF2 released from microthreads remained bioactive, stimulating myoblast proliferation over four days. Finally, a cellular outgrowth assay suggests that FGF2 promotes increased outgrowth onto microthreads. It was anticipated that the combined effects of fibrin microthread structural properties, topographic alignment cues, and FGF2 release profiles will facilitate the fabrication of a biomimetic scaffold that enhances the regeneration of functional muscle tissue for the treatment of VML injuries.

Characterisation of the R2R3 Myb subgroup 9 family of transcription factors in tomato.

Tomato (Solanum lycopersicum) has many epidermal cell outgrowths including conical petal cells and multiple types of trichomes. These include the anther-specific trichome mesh which holds the anthers connate. The R2R3 Myb Subgroup 9 family of transcription factors is involved in development of epidermal cell outgrowths throughout the angiosperms. No previous study has examined all members of this transcription factor family in a single species. All 7 R2R3 Myb Subgroup 9 genes were isolated from tomato. They were ectopically expressed in tobacco to assess their ability to induce epidermal cell outgrowth. Endogenous expression patterns were examined by semi-quantitative RT-PCR at different stages of floral development relative to the development of anther trichomes. We report variation in the degree of epidermal cell outgrowth produced in transgenic tobacco by each ectopically expressed gene. Based on expression profile and ectopic activity, SlMIXTA-2 is likely involved in the production of leaf trichomes. SlMIXTA-2 is expressed most strongly in the leaves, and not expressed in the floral tissue. SlMYB17-2 is the best candidate for the regulation of the anther trichome mesh. SlMYB17-2 is expressed strongly in the floral tissue and produces a clear phenotype of epidermal cell outgrowths when ectopically expressed in tobacco. Analysis of the phenotypes of transgenic plants ectopically expressing all 7 genes has revealed the different extent to which members of the same transcription factor subfamily can induce cellular outgrowth.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor β (TGFβ), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

FABP7: a glial integrator of sleep, circadian rhythms, plasticity, and metabolic function.

Sleep and circadian rhythms are observed broadly throughout animal phyla and influence neural plasticity and cognitive function. However, the few phylogenetically conserved cellular and molecular pathways that are implicated in these processes are largely focused on neuronal cells. Research on these topics has traditionally segregated sleep homeostatic behavior from circadian rest-activity rhythms. Here we posit an alternative perspective, whereby mechanisms underlying the integration of sleep and circadian rhythms that affect behavioral state, plasticity, and cognition reside within glial cells. The brain-type fatty acid binding protein, FABP7, is part of a larger family of lipid chaperone proteins that regulate the subcellular trafficking of fatty acids for a wide range of cellular functions, including gene expression, growth, survival, inflammation, and metabolism. FABP7 is enriched in glial cells of the central nervous system and has been shown to be a clock-controlled gene implicated in sleep/wake regulation and cognitive processing. FABP7 is known to affect gene transcription, cellular outgrowth, and its subcellular localization in the fine perisynaptic astrocytic processes (PAPs) varies based on time-of-day. Future studies determining the effects of FABP7 on behavioral state- and circadian-dependent plasticity and cognitive processes, in addition to functional consequences on cellular and molecular mechanisms related to neural-glial interactions, lipid storage, and blood brain barrier integrity will be important for our knowledge of basic sleep function. Given the comorbidity of sleep disturbance with neurological disorders, these studies will also be important for our understanding of the etiology and pathophysiology of how these diseases affect or are affected by sleep.

Neurotoxicity of mycotoxin citrinin: Novel evidence in developing zebrafish and underlying mechanisms in human neuron cells.

Citrinin (CTN) is a mycotoxin that is found as a contaminant in various types of food/feed grains and fermented food supplements. Previous studies have already established the nephrotoxicity and hepatotoxicity of CTN, but the neurotoxicity of CTN has not been clearly examined. In this study, CTN at 2-20μM was first found to interfere with the neural ganglia formation and locomotive behavior of embryonic zebrafish, a vertebrate animal model, at 24 hpf and 6 dpf, respectively. Further exposure of human neuroblastoma SH-SY5Y cells to 10 and 20μM CTN for 72h indicated that pathways responsible for neuron differentiation and projection guidance were down-regulated while oxidative stress and electron transport chain pathways were up-regulated based on the enrichment results of GSEA in the transcriptomic profiling. PCR analysis verified that CTN significantly down-regulated the expression of marker genes involved in neuron differentiation and synaptic signaling. CTN at the doses impairing cellular neurite outgrowth did not trigger mitochondrial oxidative stress and dysfunction. The neurotoxic mechanisms of CTN provide new information that is valuable in the assessment of CTN-related health risk for the general public.

How do the Local Physical, Biochemical, and Mechanical Properties of an Injectable Synthetic Anisotropic Hydrogel Affect Oriented Nerve Growth?

AbstractAs an injectable tissue regenerative platform, Anisogel aims to recapitulate the complex and anisotropic architecture of native extracellular matrix by the use of magneto‐responsive microgels, which are oriented under a low magnetic field of ≈100 mT, while a surrounding hydrogel matrix cross‐links around them. This system promotes the oriented growth of neurons when cultured in vitro. In this study, how the local microgel properties affect neurite outgrowth and orientation is aimed to understand using dorsal root ganglia from chicken embryos. When the surrounding matrix is a synthetic poly(ethylene glycol) hydrogel, the microgel concentration and length required to achieve oriented nerve growth is higher compared to fibrin‐based Anisogels. Microgels should be stiffer than the matrix for cells to sense the mechanical anisotropy but a wide range of microgel stiffness leads to similar cell alignment and growth. On the other hand, modification of the microgels with common extracellular matrix molecules enhances nerve growth but deteriorates nerve alignment compared to bioinert microgels in a cell adhesive surrounding gel. Finally, covalently coupling these microgels to the surrounding matrix reduces both cellular orientation and outgrowth suggesting a reduction in the ability of cells to sense the anisotropy.

Effect of Mechanical Mincing on Minimally Manipulated Articular Cartilage for Surgical Transplantation

Background: Point-of-care treatment options for medium to large symptomatic articular cartilage defects are limited. Minced cartilage implantation is an encouraging single-stage option, providing fresh viable autologous tissue with minimal morbidity and cost. Purpose: To determine the histological properties of mechanically minced versus minimally manipulated articular cartilage. Study Design: Controlled laboratory study. Methods: Remnant articular cartilage was collected from fresh femoral condylar allografts. Cartilage samples were divided into 4 groups: cartilage explants with or without fibrin glue and mechanically minced cartilage with or without fibrin glue. Samples were cultured for 42 days. Chondrocyte viability was assessed using live/dead assay. Cellular migration and outgrowth were monitored using bright-field microscopy. Extracellular matrix deposition was assessed via histological staining. Proteoglycan content and synthesis were assessed using dimethylmethylene blue assay and radiolabeled 35S-sulfate, respectively. Type II collagen (COL2A1) gene expression was analyzed via polymerase chain reaction. Results: The mean viability of minced cartilage particles (34% ± 14%) was not significantly reduced compared with baseline (46% ± 13%) on day 0 (P = .90). After culture, no significant difference in the percentage of live cells was appreciated between mechanically minced (58% ± 23%) and explant (73% ± 14%) cartilage in the presence of fibrin glue (P = .52). The addition of fibrin glue did not significantly affect the viability of cartilage samples. The qualitative assessment revealed comparable cellular migration and outgrowth between groups. Proteoglycan synthesis was not significantly different between groups. Histological analysis findings were positive for COL2A1 in all groups, and matrix formation was appreciated in all groups. COL2A1 expression in minced cartilage (1.72 ± 1.88) was significantly higher than in explant cartilage (0.15 ± 0.07) in the presence of fibrin glue (P = .01). Conclusion: Mechanically minced articular cartilage remained viable after 42 days of culture in vitro and was comparable with cartilage explants with regard to cellular migration, outgrowth, and extracellular matrix synthesis. Clinical Relevance: Mechanically minced articular cartilage is an encouraging intervention for the treatment of symptomatic cartilage defects. Further translational work is warranted to determine the viability of minced cartilage implantation as a single-stage therapeutic intervention in vivo.

Conical petal epidermal cells, regulated by the MYB transcription factor MIXTA, have an ancient origin within the angiosperms.

Conical epidermal cells occur on the tepals (perianth organs, typically petals and/or sepals) of the majority of animal-pollinated angiosperms, where they play both visual and tactile roles in pollinator attraction, providing grip to foraging insects, and enhancing colour, temperature, and hydrophobicity. To explore the evolutionary history of conical epidermal cells in angiosperms, we surveyed the tepal epidermis in representative species of the ANA-grade families, the early-diverging successive sister lineages to all other extant angiosperms, and analysed the function of a candidate regulator of cell outgrowth from Cabomba caroliniana (Nymphaeales). We identified conical cells in at least two genera from different families (Austrobaileya and Cabomba). A single SBG9 MYB gene was isolated from C. caroliniana and found to induce strong differentiation of cellular outgrowth, including conical cells, when ectopically expressed in Nicotiana tabacum. Ontogenetic analysis and quantitative reverse transcription-PCR established that CcSBG9A1 is spatially and temporally expressed in a profile which correlates with a role in conical cell development. We conclude that conical or subconical cells on perianth organs are ancient within the angiosperms and most probably develop using a common genetic programme initiated by a SBG9 MYB transcription factor.

CRISPR/Cas9-Mediated Insertion of HIV Long Terminal Repeat within BACH2 Promotes Expansion of T Regulatory-like Cells.

One key barrier to curative therapies for HIV is the limited understanding of HIV persistence. HIV provirus integration sites (ISs) within BACH2 are common, and almost all sites mapped to date are located upstream of the start codon in the same transcriptional orientation as the gene. These unique features suggest the possibility of insertional mutagenesis at this location. Using CRISPR/Cas9-based homology-directed repair in primary human CD4+ T cells, we directly modeled the effects of HIV integration within BACH2 Integration of the HIV long terminal repeat (LTR) and major splice donor increased BACH2 mRNA and protein levels, altered gene expression, and promoted selective outgrowth of an activated, proliferative, and T regulatory-like cell population. In contrast, introduction of the HIV-LTR alone or an HIV-LTR-major splice donor construct into STAT5B, a second common HIV IS, had no functional impact. Thus, HIV LTR-driven BACH2 expression modulates T cell programming and leads to cellular outgrowth and unique phenotypic changes, findings that support a direct role for IS-dependent HIV-1 persistence.

Effect of Electrical and Electromechanical Stimulation on PC12 Cell Proliferation and Axon Outgrowth.

Peripheral nerve injuries have become a common clinical disease with poor prognosis and complicated treatments. The development of tissue engineering pointed a promising direction to produce nerve conduits for nerve regeneration. Electrical and mechanical stimulations have been incorporated with tissue engineering, since such external stimulations could promote nerve cell proliferation, migration and differentiation. However, the combination of electrical and mechanical stimulations (electromechanical stimulation) and its effects on neuron proliferation and axon outgrowth have been rarely investigated. Herein, silver nanowires (AgNWs) embedded polydimethylsiloxane (PDMS) electrodes were developed to study the effects of electromechanical stimulation on rat pheochromocytoma cells (PC12 cells) behaviors. AgNWs/PDMS electrodes demonstrated good biocompatibility and established a stable electric field during mechanical stretching. PC12 cells showed enhanced proliferation rate and axon outgrowth under electrical stimulation alone, and the cell number significantly increased with higher electrical stimulation intensity. The involvement of mechanical stretching in electrical stimulation reduced the cell proliferation rate and axon outgrowth, compared with the case of electrical stimulation alone. Interestingly, the cellular axons outgrowth was found to depend on the stretching direction, where the axons prefer to align perpendicularly to the stretch direction. These results suggested that AgNWs/PDMS electrodes provide an in vitro platform to investigate the effects of electromechanical stimulation on nerve cell behaviors and can be potentially used for nerve regeneration in the future.

Dexmedetomidine does not compromise neuronal viability, synaptic connectivity, learning and memory in a rodent model

Recent animal studies have drawn concerns regarding most commonly used anesthetics and their long-term cytotoxic effects, specifically on the nervous tissue. It is therefore imperative that the search continues for agents that are non-toxic at both the cellular and behavioural level. One such agent appears to be dexmedetomidine (DEX) which has not only been found to be less neurotoxic but has also been shown to protect neurons from cytotoxicity induced by other anesthetic agents. However, DEX’s effects on the growth and synaptic connectivity at the individual neuronal level, and the underlying mechanisms have not yet been fully resolved. Here, we tested DEX for its impact on neuronal growth, synapse formation (in vitro) and learning and memory in a rodent model. Rat cortical neurons were exposed to a range of clinically relevant DEX concentrations (0.05–10 µM) and cellular viability, neurite outgrowth, synaptic assembly and mitochondrial morphology were assessed. We discovered that DEX did not affect neuronal viability when used below 10 µM, whereas significant cell death was noted at higher concentrations. Interestingly, in the presence of DEX, neurons exhibited more neurite branching, albeit with no differences in corresponding synaptic puncta formation. When rat pups were injected subcutaneously with DEX 25 µg/kg on postnatal day 7 and again on postnatal day 8, we discovered that this agent did not affect hippocampal-dependent memory in freely behaving animals. Our data demonstrates, for the first time, the non-neurotoxic nature of DEX both in vitro and in vivo in an animal model providing support for its utility as a safer anesthetic agent. Moreover, this study provides the first direct evidence that although DEX is growth permissive, causes mitochondrial fusion and reduces oxygen reactive species production, it does not affect the total number of synaptic connections between the cortical neurons in vitro.

Extended Large Area Si/ZnO Heterojunction Biosensor for Assessing Functional Behavior of Primary Cortical Neuronal Cells

In this study, we report the poly-L-lysine (PLL) coated extended large area p-Si/n-ZnO heterojunction based biosensing device to analyze the influence of functional behavior of primary cortical neuronal cells. Wherein, the proliferation of rat embryonic neurons and neural stem cells is highly influenced by the physicochemical and structural properties of the underlying substrate mimicking the extracellular microenvironment. We observed a significant increase in the impedance values while adhesion of the neuronal cells on the substrate; however, it further got significantly decreased with the progression of various cellular functions neurite outgrowth and network formation. Such neuronal processes might have increased the propagation of flow of current by reducing the resistivity of the PLL treated n-ZnO thin film sensing area. Additionally, through the Nyquist plot, we observed a noticeable decrease in the magnitude of impedance values of the fabricated device. Hence, we believe that the fabricated PLL coated extended large area p-Si/n-ZnO heterojunction biosensor can serve as a favourable device to monitor the influence of functional behavior of neuronal cells.

Chondroitinase ABC/galectin-3 fusion proteins with hyaluronan-based hydrogels stabilize enzyme and provide targeted enzyme activity for neural applications

Objective. Chondroitinase ABC (ChABC) has emerged as a promising therapeutic agent for central nervous system regeneration. Despite multiple beneficial outcomes for regeneration, translation of this enzyme is challenged by poor pharmacokinetics, localization, and stability. Approach. This study explored the function and in vitro application of engineered ChABC fused to galectin-3 (Gal3). Two previously developed ChABC-Gal3 oligomers (monomeric and trimeric) were evaluated for functionality and kinetics, then applied to an in vitro cellular outgrowth model using dorsal root ganglia (DRGs). The fusions were combined with two formulations of hyaluronan (HA)-based scaffolds to determine the extent of active enzyme release compared to wild type (WT) ChABC. Main Results. Monomeric and trimeric ChABC-Gal3 maintained digestive capabilities with kinetic properties that were substrate-dependent for chondroitin sulfates A, B, and C. The fusions had longer half-lives at 37 °C on the order of seven fold for monomer and twelve fold for trimer compared to WT. Both fusions were also effective at restoring DRG outgrowth in vitro. To create a combination approach, two triple-component hydrogels containing modified HA were formulated to match the mechanical properties of native spinal cord tissue and to support astrocyte viability (>80%) and adhesion. The hydrogels included collagen-I and laminin mixed with either 5 mg ml−1 of glycidyl methacrylate HA or 3 mg ml−1 Hystem. When combined with scaffolds, ChABC-Gal3 release time was lengthened compared to WT. Both fusions had measurable enzymatic activity for at least 10 d when incorporated in gels, compared to WT that lost activity after 1 d. These longer term release products from gels maintained adequate function to promote DRG outgrowth. Significance. Results of this study demonstrated cohesive benefits of two stabilized ChABC-Gal3 oligomers in combination with HA-based scaffolds for neural applications. Significant improvements to ChABC stability and release were achieved, meriting future studies of ChABC-Gal3/hydrogel combinations to target neural regeneration.

Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus.

PurposeWe aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus.MethodsThe study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase–mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment.ResultsWe demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment.ConclusionsThis chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment.Translational RelevanceThe cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.

Autonomous spheroid formation by culture plate compartmentation

Scaffold-free 3D cell cultures (e.g. pellet cultures) are widely used in medical science, including cartilage regeneration. Their drawbacks are high time/reagent consumption and lack of early readout parameters. While optimisation was achieved by automation or simplified spheroid generation, most culture systems remain expensive or require tedious procedures. The aim of this study was to establish a system for resource efficient spheroid generation with additional early readout parameters. This was achieved by a new approach for spheroid generation via self-assembly from monolayer via compartmentation of cell culture surfaces utilising laser engraving (grid plates). The compartmentation triggered contraction and rolling up of the cell monolayer, finishing in condensation into a spheroid in human adipose-derived stem cell (ASC/TERT1) and human articular chondrocytes (hACs)-ASC/TERT1 co-cultures, when cultivated on grid plates under chondrogenic conditions. Plates with 1 and 3 mm grid size yielded stable diameters (about 140 µm and 300 µm, respectively). ASC/TERT1 spheroids fully formed within 3 weeks while co-cultures took 1–2 weeks, forming significantly faster with increasing hAC ratio (p < 0.05 and 0.01 for 1:1 and 1:4 ASC/TERT1:hAC ratio, respectively). Co-cultures showed slightly lower spheroid diameters, due to earlier spheroid formation and incomplete monolayer formation. However, this was associated with a more homogeneous matrix distribution in the co-culture. Both showed differentiation capacity comparable to standard pellet culture in (immune-)histochemistry and RT-qPCR. To assess usability for cartilage repair, spheroids were embedded into a hydrogel (fibrin), yielding cellular outgrowth and matrix deposition, which was especially pronounced in co-cultures. The herein presented novel cell culture system is not only a promising tool for autonomous spheroid generation with the potential of experimental and clinical application in tissue engineering, but also for the generation of ‘building blocks’ for subsequential biofabrication strategies such as bioprinting.

Spherowell, a novel system inducing autonomous spheroid formation of cell monolayers

Purpose: Cell spheroids are the most frequently used system for in vitro chondrogenic differentiation and are gaining increasingly importance in clinical applications. However, drawbacks of currently used systems are the lack of early-readout parameters and time as well as reagent intensive procedures. While this can be partially alleviated by process automation, it is often not feasible due to high cost of necessary equipment. Herein we present a completely new concept of spheroid formation (SpheroWell) allowing for mass-production of chondrogenic spheroids from monolayer via growth surface compartmentation with promising applications in cartilage regeneration and in vitro screening applications. Methods: Standard 60 mm cell culture dishes were compartmentalized with a 1 mm or 3 mm raster by laser engraving. Telomerase immortalized adipose derived stromal/stem cells (ASC/TERT1) were seeded in mono-culture and in co-culture with primary human articular chondrocytes (hAC) at a total cell number of 1 x 106 cells/dish on compartmentalized dishes. Co-cultures were conducted at 1:4, 1:1 and 4:1 hAC:ASC ratios. Cells were cultured in differentiation media containing low concentrations of TGFβ-3 and BMP-6 (1 ng/ml). The spheroid formation process was analysed using time-laps imaging and bright field-/confocal microscopy and the resulting spheroid sizes were measured. Chondrogenic differentiation and internal structure were compared to standard pellet culture using qRT-PCR (collagen type 1/2 (Col1/2) and aggrecan (Agg)) and (immuno-) histochemistry (Alcian blue and collagen type II). As a model for tissue regeneration spheroids were embedded into fibrin hydrogel and the cellular outgrowth and matrix deposition were analysed. Finally the system was assessed for its usability as an in vitro testing system using the spheroid formation process as an early readout parameter by assessing the effect of interleukin-1β (IL-1β) on the formation kinetics. Results: After initial seeding, spheroids self-assembled in a multi-phase process. After a short proliferation phase, edge regions (especially in compartment corners) began to retract, followed by roll-up of the monolayer and final condensation into a spheroid (Figure 1). For ASC/TERT1 mono-cultures this process took about 3 weeks, while addition of hAC accelerated the formation time to 1-2 weeks in a cell-ratio dependent manner. However, while ASC and co-cultures up to 1:1 ratio formed pellets of constant size, hAC ratios over 50% lead to variable and smaller spheroids due to faster and incomplete monolayer formation. Apart from cell ratios, spheroid sizes could be controlled by varying the raster size, yielding highly reproducible mean diameters of 340.9 and 134.4 (SD < 10%) for 3 mm and 1 mm raster respectively (with ASC mono-cultures). When comparing spheroids from raster plates with standard pellet culture comparable differentiation capacity was visible despite the 2D-phase of the initial monolayer in SpheroWells. Internal structures revealed differences with spheroids showing strands of matrix homogenously distributed over the cross-section, while pellets generally showed a pronounced aligned cortex region. When embedding spheroids from raster plates into fibrin hydrogels cellular outgrowth into the hydrogel could be observed, with outgrowth speed being controllable via hydrogel density. Matrix deposition could be observed around the outgrowing cells, which was especially pronounced in co-cultures. Finally, first results using the system as an in vitro testing system were obtained. Treatment of raster plate cultures with IL-1β showed a strong reduction in spheroid formation time serving as early readout parameters, while as expected showing abolishment of Col2 expression in endpoint analysis. Conclusions: The new culture system, SpheroWell, is highly efficient in spheroid generation, saving time and culture medium and allowing for additional early read outs via tracking the spheroid formation process. The ability to produce and maintain hundreds of spheroids within one dish, which can be easily integrated into pre-existing production and research workflows makes it attractive for high throughput testing and clinical spheroid application.

An exosomal-carried short periostin isoform induces cardiomyocyte proliferation.

Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity.Methods: CPC were derived as the cellular outgrowth of ex vivo cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein.Results: Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both in vitro and in vivo, in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes.Conclusions: Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.

I. How can you choose the fate of iPSCs and stem cells, Regeneration or Carcinogenesis? A hypothetical insight.: II. Modelling human beta cell development with pluripotent stem cells.

It is nowadays taken granted that induced pluripotent stem cells (iPSCs) are available for the regeneration therapy since iPSCs differentiate into any kind of phenotypes. If iPSCs can choose their fate in every way of differentiation why they do not choose cancer phenotype. As a body develops for one fertilized egg, embryonic stem cell must choose every phenotype of tissues such as blood, neuron, lung, liver, pancreas and so on depending on the stages. And sometimes the cells get cancer. So do iPSCs because iPSCs are almost equivalent to embryonic cells. Then how can the safety of the regeneration therapy be maintained with iPSCs? When inducing the differentiation of iPSCs it is considered important to choose the proper conditions of culture such as 3D-platform for embryoid, supplement of cytokines and growth factors, inhibition of signaling and so on. On the other hand, several conditions have been reported to induce cancer stem cells. The cancer inducing conditions are possibly summarized as the factors chronically exposed to iPSCs. It is further worthwhile noticing that the conditions do not appear to induce mutations but affecting the epigenetics. Collectively, to secure the safety of regeneration therapy, it appears the best way to avoid the conditions to induce cancer stem cells. Further insights in details will be discussed in the lecture. Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of the pancreatic beta cells and loss of insulin. Using the Edmonton protocol, donor-derived islets seeded into the liver successfully restore glycemia in 58% of T1D patients. However, donor scarcity, risks associated with immunosuppressants and poor engraftment limit this therapeutic application to a small number of patients. To overcome these challenges, the developmental potential of human embryonic stem cells and human induced pluripotent stem cells is being harnessed to produce surrogate islets in vitro. We and others have been able to mimic human embryonic development and generate pancreatic progenitors (PP) that have the ability to mature into insulin-producing beta-like cells both in vitro and in vivo. Transplantation of pancreatic progenitors in the kidney capsule of immunodeficient mice leads to formation of islet-like structures that secrete human insulin. However, there are some limitations to the use of pancreatic progenitors for the treatment of T1D. First and foremost, their safety as the PP population can be heterogenous and highly proliferative, which might lead to formation of cellular outgrowth or teratoma after transplantation. Second, while insulin-producing cells develop in vivo 6 weeks after transplantation, restoration of normoglycemia occurs ~5 months later, suggesting that these "early" insulin-producing cells are immature, or poorly connected to the host vasculature. We have been addressing these two limitations and developed approaches to 1) improve safety by identifying markers to purify the PP populations and 2) accelerate functionality by improving vascularization at the time of transplantation.

Cryopreserved, Thin, Laser-Etched Osteochondral Allograft maintains the functional components of articular cartilage after 2\u2009years of storage

BackgroundDespite improvements in treatment options and techniques, articular cartilage repair continues to be a challenge for orthopedic surgeons. This study provides data to support that the 2-year Cryopreserved, Thin, Laser-Etched Osteochondral Allograft (T-LE Allograft) embodies the necessary viable cells, protein signaling, and extracellular matrix (ECM) scaffold found in fresh cartilage in order to facilitate a positive clinical outcome for cartilage defect replacement and repair.MethodsViability testing was performed by digestion of the graft, and cells were counted using a trypan blue assay. Growth factor and ECM protein content was quantified using biochemical assays. A fixation model was introduced to assess tissue outgrowth capability and cellular metabolic activity in vitro. Histological and immunofluorescence staining were employed to confirm tissue architecture, cellular outgrowth, and presence of ECM. The effects of the T-LE Allograft to signal bone marrow-derived mesenchymal stem cell (BM-MSC) migration and chondrogenic differentiation were evaluated using in vitro co-culture assays. Immunogenicity testing was completed using flow cytometry analysis of cells obtained from digested T-LE Allografts and fresh articular cartilage.ResultsAverage viability of the T-LE Allograft post-thaw was found to be 94.97 ± 3.38%, compared to 98.83 ± 0.43% for fresh articular cartilage. Explant studies from the in vitro fixation model confirmed the long-term viability and proliferative capacity of these chondrocytes. Growth factor and ECM proteins were quantified for the T-LE Allograft revealing similar profiles to fresh articular cartilage. Cellular signaling of the T-LE Allograft and fresh articular cartilage both exhibited similar outcomes in co-culture for migration and differentiation of BM-MSCs. Flow cytometry testing confirmed the T-LE Allograft is immune-privileged as it is negative for immunogenic markers and positive for chondrogenic markers.ConclusionsUsing our novel, proprietary cryopreservation method, the T-LE Allograft, retains excellent cellular viability, with native-like growth factor and ECM composition of healthy cartilage after 2 years of storage at − 80 °C. The successful cryopreservation of the T-LE Allograft alleviates the limited availably of conventionally used fresh osteochondral allograft (OCA), by providing a readily available and simple to use allograft solution. The results presented in this paper supports clinical data that the T-LE Allograft can be a successful option for repairing chondral defects.