Peripheral neuropathies associated with DNA repair disorders.

Repair of genomic DNA is a fundamental housekeeping process that quietly maintains the health of our genomes. The consequences of a genetic defect affecting a component of this delicate mechanism are quite harmful, characterized by a cascade of premature aging that injures a variety of organs, including the nervous system. One part of the nervous system that is impaired in certain DNA repair disorders is the peripheral nerve. Chronic motor, sensory, and sensorimotor polyneuropathies have all been observed in affected individuals, with specific physiologies associated with different categories of DNA repair disorders. Cockayne syndrome has classically been linked to demyelinating polyneuropathies, whereas xeroderma pigmentosum has long been associated with axonal polyneuropathies. Three additional recessive DNA repair disorders are associated with neuropathies, including trichothiodystrophy, Werner syndrome, and ataxia-telangiectasia. Although plausible biological explanations exist for why the peripheral nerves are specifically vulnerable to impairments of DNA repair, specific mechanisms such as oxidative stress remain largely unexplored in this context, and bear further study. It is also unclear why different DNA repair disorders manifest with different types of neuropathy, and why neuropathy is not universally present in those diseases. Longitudinal physiological monitoring of these neuropathies with serial electrodiagnostic studies may provide valuable noninvasive outcome data in the context of future natural history studies, and thus the responses of these neuropathies may become sentinel outcome measures for future clinical trials of treatments currently in development such as adeno-associated virus gene replacement therapies.

Agitation in a Microcarrier-based Spinner Flask Bioreactor Modulates Homeostasis of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are well known in cell therapy due to their secretion of trophic factors, multipotent differentiation potential, and ability for self-renewal. As a result, the number of clinical trials has been steadily increasing over the last decade highlighting the need for in vitro systems capable of producing large quantities of cells to meet growing demands. However, hMSCs are highly sensitive to microenvironment conditions, including shear stress caused by dynamic bioreactor systems, and can lead to alteration of cellular homeostasis. In this study, hMSCs were expanded on microcarriers within a 125 mL spinner flask bioreactor system. Our results demonstrate a three-fold expansion over seven days. Furthermore, our results show that culturing hMSCs in the microcarrier-based suspension bioreactor (compared to static planar culture) results in smaller cell size and higher levels of reactive oxidative species (ROS) and ROS regulator Sirtuin-3, which have implications on the nicotinamide adenine dinucleotide metabolic pathway and metabolic homeostasis. In addition, hMSCs in the bioreactor showed the increased Prostaglandin E2 secretion as well as reduced the Indoleamine-pyrrole 2,3-dioxygenase secretion upon stimulus with interferon gamma. The results of this study provide understanding of potential hMSC physiology alterations impacted by bioreactor microenvironment during scalable production of hMSCs for biomanufacturing and clinical trials.

Parental preferences in treatment of acute uncomplicated appendicitis comparing surgery to conservative management with antibiotics and their views on research participation.

Studies show that conservative management in acute uncomplicated appendicitis (AUA) is an alternative to surgery. This study aims to determine factors affecting parental preference in management of AUA and their decision for research participation. We conducted surveys on parents whose children were admitted with suspicion of appendicitis but later confirmed not to have appendicitis. Information on appendicectomy versus conservative treatment with antibiotics was provided using a fixed script and standard information leaflet. Questionnaires covered factors influencing decisions, opinions regarding research, treatment preference and demographic data. We excluded parents not fluent in English. Of 113 respondents, 71(62.8%) chose antibiotics, 39(34.5%) chose appendicectomy, and 3(2.7%) had no preference. Reasons given for choosing antibiotics were fear of surgical risks and preferring less invasive treatment. Those choosing appendicectomy expressed preference for definitive treatment and fear of recurrence. Majority were against randomisation (n = 89, 78.8%) and blinding (n = 90, 79.7%). Over half found difficulty involving their child in research (n = 65, 57.5%). Most thought that research is important (66.4%) and beneficial to others (59.3%). Parents who perceived their child as healthy found research riskier (p = 0.039). Educated parents were more likely to find research beneficial to others (p = 0.012) but less accepting of randomisation (p = 0.001).Conclusion: More parents appear to prefer conservative treatment for acute uncomplicated appendicitis. Researchers must consider parental concerns regarding randomisation and blinding.What is Known:• Conservative management of acute uncomplicated appendicitis in paediatric patients is safe and effective, sparing the child the need for an operation; however, neither conservative nor surgical management is proven to be superior.• Randomised controlled trials provide the highest level of evidence, but it is challenging to recruit paediatric patients as participants in such clinical trials.What is New:• More parents prefer conservative management of uncomplicated appendicitis over surgical management for their children due to fear of surgical risks and complications• Randomisation in trial design is significantly associated with a parent's decision to reject their child's participation in a clinical trial.

Public health practitioners' views of the 'Making Every Contact Count' initiative and standards for its evaluation.

BACKGROUND: National Health Service England encourages staff to use everyday interactions with patients to discuss healthy lifestyle changes as part of the 'Making Every Contact Count' (MECC) approach. Although healthcare, government and public health organisations are now expected to adopt this approach, evidence is lacking about how MECC is currently implemented in practice. This study explored the views and experiences of those involved in designing, delivering and evaluating MECC. METHODS: We conducted a qualitative study using semi-structured interviews with 13 public health practitioners with a range of roles in implementing MECC across England. Interviews were conducted via telephone, transcribed verbatim and analysed using an inductive thematic approach. RESULTS: Four key themes emerged identifying factors accounting for variations in MECC implementation: (i) 'design, quality and breadth of training', (ii) 'outcomes attended to and measured', (iii) 'engagement levels of trainees and trainers' and (iv) 'system-level influences'. CONCLUSIONS: MECC is considered a valuable public health approach but because organisations interpret MECC differently, staff training varies in nature. Practitioners believe that implementation can be improved, and an evidence-base underpinning MECC developed, by sharing experiences more widely, introducing standardization to staff training and finding better methods for assessing meaningful outcomes.

Effects of Drug Particle Size and Lipid Additives on Drug Release from Paraffin Wax Formulations Prepared by Spray Congealing Technique.

Paraffin wax is a hydrophobic meltable material that can be suitably used in spray congealing to develop drug-loaded microparticles for sustained release, taste-masking or stability enhancement of drugs. However, these functional properties may be impaired if the drug particles are not completely embedded. Moreover, highly viscous melts are unsuitable for spray dispersion. In this study, the effects of drug particle size and lipid additives, namely stearic acid (SA), cetyl alcohol (CA) and cetyl esters (CE), on melt viscosity and extent of drug particles embedment were investigated. Spray congealing was conducted on the formulations, and the resultant microparticles were analysed for their size, drug content, extent of drug particles embedment and drug release. The melt viscosity increased with smaller solid inclusions while lipid additives decreased the viscosity to varying extents. The spray-congealed microparticle size was largely dependent on the viscosity. The addition of lipid additives to paraffin wax enabled more complete embedment of the drug particles. CA produced microparticles with the lowest drug release, followed by SA and CE. The addition of CA and CE enhanced the drug release and showed potential for taste-masking. Judicious choice of drug particle size and matrix materials is important for successful spray congealing to produce microparticles with the desired characteristics.

Structural correlates of literacy difficulties in the second language: Evidence from Mandarin-speaking children learning English.

Several neuroimaging studies have explored the neural basis of literacy difficulties in the second language (L2). However, it remains unclear whether the associated neural alterations are related to literacy abilities in the first language (L1). Using magnetic resonance imaging, we explore this issue with two experiments in Mandarin-speaking children learning English as second language. In the first experiment, we investigated children with literacy difficulties in L2 and L1 (poor in both, PB) and children with literacy difficulties only in L2 (poor in English, PE). We compared the brain structure in these two groups to a control literacy (CL) group. The results showed that the CL group had significantly less gray matter volume in the left supramarginal gyrus compared to the PB group and moderately less gray matter volume compared to the PE group. In addition, the PB group had significant greater gray matter volume in the left medial fusiform gyrus compared to the PE group and had marginally greater gray matter volume compared to the CL group. In the second experiment, we explored the relationship between the two atypical regions and literacy abilities in the two languages in an independent sample consisting of children with typical literacy. Correlation analyses revealed that the left supramarginal gyrus was significantly associated with literacy performance only in the second language, English, whereas the left medial fusiform gyrus did not correlate with the performances in either L1 or L2. Taken together, these findings suggest that literacy difficulties in an alphabetic L2 are associated with a structural abnormality in the left supramarginal gyrus, a region implicated in phonological processing, which is independent of literacy abilities in the native language.

Metabolic Reconfiguration Supports Reacquisition of Primitive Phenotype in Human Mesenchymal Stem Cell Aggregates.

Spontaneous aggregation and the associated enhancement of stemness have been observed in many anchorage dependent cells. Recently, aggregation of human mesenchymal stem cells (hMSCs) in nonadherent culture has been shown to reverse expansion-induced heterogeneity and loss of stemness and reprogram the hMSC to reacquire their primitive phenotype, a phenomenon that can significantly enhance therapeutic applications of hMSC. The objective of this study was to investigate the mechanistic basis underlying the connection between multicellular aggregation and stemness enhancement in hMSC by testing the hypothesis that cellular events induced during three-dimensional aggregation on nonadherent substratum induces changes in mitochondrial metabolism that promote the expression of stem cell genes Oct4, Sox2, and Nanog. Our results show that aggregation changes mitochondrial morphology and reduces mitochondrial membrane potential, resulting in a metabolic reconfiguration characterized by increased glycolytic and anaplerotic flux, and activation of autophagy. We further demonstrate that interrupting mitochondrial respiration in two-dimensional planar culture with small molecule inhibitors partially recapitulates the aggregation-mediated enhancement in stem cell properties, whereas enhancement of mitochondrial oxidative phosphorylation in the aggregated state reduces the aggregation-induced upregulation of Oct4, Sox2, and Nanog. Our findings demonstrate that aggregation-induced metabolic reconfiguration plays a central role in reacquisition of primitive hMSC phenotypic properties. Stem Cells 2017;35:398-410.

Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor.

Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both spinner flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell®. The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.

Gastrointestinal transit time of radiopaque ingested foreign bodies in children: experience of two paediatric tertiary centres.

Introduction: Foreign body (FB) ingestion is a common paediatric emergency. While guidelines exist for urgent intervention, less is known of the natural progress of FBs passing through the gastrointestinal tract (GIT). We reviewed these FB transit times in an outpatient cohort. Methods: A retrospective review was performed on all children (≤18 years) treated for radiopaque FB ingestion at two major tertiary paediatric centres from 2015 to 2016. Demographic data, FB types, outcomes and hospital visits (emergency department [ED] and outpatient) were recorded. All cases discharged from the ED with outpatient follow-up were included. We excluded those who were not given follow-up appointments and those admitted to inpatient wards. We categorised the outcomes into confirmed passage (ascertained via abdominal X-ray or reported direct stool visualisation by patients/caregivers) and assumed passage (if patients did not attend follow-up appointments). Results: Of the 2,122 ED visits for FB ingestion, 350 patients who were given outpatient follow-up appointments were reviewed (median age 4.35 years [range: 0.5-14.7], 196 [56%] male). The largest proportion (16%) was aged 1-2 years. Coins were the most common ingested FB, followed by toys. High-risk FB (magnets or batteries) formed 9% of cases (n=33). The 50th centile for FB retention was 8, 4 and 7 days for coins, batteries and other radiopaque FBs, respectively; all confirmed passages occurred at 37, 7 and 23 days, respectively. Overall, 197 (68%) patients defaulted on their last given follow-up. Conclusion: This study provides insight into the transit times of FB ingested by children, which helps medical professionals to decide on the optimal time for follow-up visits and provide appropriate counsel to caregivers.

Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors.

Human mesenchymal stem cells (hMSCs) have demonstrated great potential to be used as therapies for many types of diseases. Due to their immunoprivileged status, allogeneic hMSCs therapies are particularly attractive options and methodologies to improve their scaling and manufacturing are needed. Microcarrier-based bioreactor systems provide higher volumetric hMSC production in automated closed systems than conventional planar cultures. However, more sophisticated bioprocesses are necessary to successfully convert from planar culture to microcarriers. This article summarizes key steps involved in the planar culture to microcarrier hMSC manufacturing scheme, from seed train, inoculation, expansion and harvest. Important bioreactor parameters, such as temperature, pH, dissolved oxygen (DO), mixing, feeding strategies and cell counting techniques, are also discussed.

Intrinsic Cerebro-Cerebellar Functional Connectivity Reveals the Function of Cerebellum VI in Reading-Related Skills.

The engagement of the cerebellum VI in reading was reported in both typically developing and dyslexic readers. However, it is still not clear how the cerebellum VI contributes to reading. Here we have examined the correlation of intrinsic cerebro-cerebellar functional connectivity with two critical reading-related skills-phonological awareness (PA) and rapid automatized naming (RAN)-with fMRI technology. Specifically, we tested the hypothesis that the cerebellum may contribute to reading either by phonological skills or by automatizing skills. We chose the left and right cerebellum VI as ROIs, and we calculated the intrinsic cerebro-cerebellar functional connectivity during a resting state. We further explored whether and how cerebro-cerebellar resting state functional connectivity (RSFC) is associated with individuals' reading-related skills including PA and RAN. The results showed that the functional connectivity between the left supramarginal gyrus and bilateral cerebellum VI was related to RAN, and the connectivity between the left insula and right cerebellum VI was related to PA. However, the effect of PA did not survive after the RAN was regressed out. Control analyses further confirmed that it was the intrinsic cerebro-cerebellar functional connectivity rather than the local cerebellar functionality that associated with phonological awareness ability and rapid automatized naming ability. For the first time, the relationship between cerebro-cerebellar resting state functional connectivity and specific reading-related skills has been explored, and this has deepened our understanding of the way the cerebellum VI is involved in reading.

Influence of Microenvironment on Mesenchymal Stem Cell Therapeutic Potency: From Planar Culture to Microcarriers.

Human mesenchymal stem cells (hMSCs) are a promising candidate in cell therapy as they exhibit multilineage differentiation, homing to the site of injury, and secretion of trophic factors that facilitate tissue healing and/or modulate immune response. As a result, hMSC-derived products have attracted growing interests in preclinical and clinical studies. The development of hMSC culture platforms for large-scale biomanufacturing is necessary to meet the requirements for late-phase clinical trials and future commercialization. Microcarriers in stirred-tank bioreactors have been widely utilized in large-scale expansion of hMSCs for translational applications because of a high surface-to-volume ratio compared to conventional 2D planar culture. However, recent studies have demonstrated that microcarrier-expanded hMSCs differ from dish- or flask-expanded cells in size, morphology, proliferation, viability, surface markers, gene expression, differentiation potential, and secretome profile which may lead to altered therapeutic potency. Therefore, understanding the bioprocessing parameters that influence hMSC therapeutic efficacy is essential for the optimization of microcarrier-based bioreactor system to maximize hMSC quantity without sacrificing quality. In this review, biomanufacturing parameters encountered in planar culture and microcarrier-based bioreactor culture of hMSCs are compared and discussed with specific focus on cell-adhesion surface (e.g., discontinuous surface, underlying curvature, microcarrier stiffness, porosity, surface roughness, coating, and charge) and the dynamic microenvironment in bioreactor culture (e.g., oxygen and nutrients, shear stress, particle collision, and aggregation). The influence of dynamic culture in bioreactors on hMSC properties is also reviewed in order to establish connection between bioprocessing and stem cell function. This review addresses fundamental principles and concepts for future design of biomanufacturing systems for hMSC-based therapy.

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

Development of a microdevice-based human mesenchymal stem cell-mediated drug delivery system.

Cell-mediated drug delivery systems utilize living cells as vehicles to achieve controlled delivery of drugs. One of the systems features integrating cells with disk-shaped microparticles termed microdevices into cell-microdevice complexes that possess some unique advantages over their counterparts. Human mesenchymal stem cells (hMSCs) have been extensively studied as therapeutic cells and used as carrier cells for drug-loaded nanoparticles or other functional nanoparticles. This article presents the development of a microdevice-based hMSC-mediated drug delivery system for the first time. This study revealed that the microdevices could be attached to the hMSCs in a controlled and versatile manner; the produced hMSC-microdevice complexes were stable over cultivation and trypsinization, and the microdevice attachment did not affect the viability and proliferation of the hMSCs. Moreover, cultured microdevice-bound hMSCs retained their abilities to migrate on a flat surface, form a spheroid, and actively dissociate from the spheroid. These results indicate that this microdevice-based hMSC-mediated system promises to be further developed into a clinically viable drug delivery system.

Neural Differentiation of Spheroids Derived from Human Induced Pluripotent Stem Cells-Mesenchymal Stem Cells Coculture.

Organoids, the condensed three-dimensional (3D) tissues emerged at the early stage of organogenesis, are a promising approach to regenerate functional and vascularized organ mimics. While incorporation of heterotypic cell types, such as human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs)-derived neural progenitors aid neural organ development, the interactions of secreted factors during neurogenesis have not been well understood. The objective of this study is to investigate the impact of the composition and structure of 3D hybrid spheroids of hiPSCs and hMSCs on dorsal cortical differentiation and the secretion of extracellular matrices and trophic factors in vitro. The hybrid spheroids were formed at different hiPSC:hMSC ratios (100:0, 75:25, 50:50, 25:75, 0:100) using direct mixing or pre-hiPSC aggregation method, which generated dynamic spheroid structure. The cellular organization, proliferation, neural marker expression, and the secretion of extracellular matrix proteins and the cytokines were characterized. The incorporation of MSCs upregulated Nestin and ß-tubulin III expression (the dorsal cortical identity was shown by Pax6 and TBR1 expression), matrix remodeling proteins, and the secretion of transforming growth factor-ß1 and prostaglandin E2. This study indicates that the appropriate composition and structure of hiPSC-MSC spheroids promote neural differentiation and trophic factor and matrix secretion due to the heterotypic cell-cell interactions.

Aggregation kinetics of human mesenchymal stem cells under wave motion.

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three-dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate.

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and tissue engineering and are being tested in clinical trials for a wide range of diseases. Originally isolated and expanded as plastic adherent cells, hMSCs have intriguing properties of in vitro self-assembly into three-dimensional (3D) aggregates that improve a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition. While cell-cell contacts and cell-extracellular matrix interactions mediate 3D cell aggregation, the adaptive changes of hMSC cytoskeleton during self-assembly and associated metabolic reconfiguration may also influence aggregate properties and functional activation. In this study, we investigated the role of actin in regulating 3D hMSC aggregate compaction, fusion, spreading and functional activation. Individual hMSC aggregates with controlled initial cell number were formed by seeding a known number of hMSCs (500, 2000, and 5000 cells/well) in multi-well plates of an ultra-low adherent surface to form multicellular aggregates in individual wells. To assess the influence of actin-mediated contractility on hMSC aggregation and properties, actin modulators, including cytochalasin D (cytoD), nocodazole, lysophosphatidic acid (LPA), and Y-27632, were added at different stages of aggregation and their impacts on hMSC aggregate compaction and apoptosis were monitored. The results suggest that actin-mediated contractility influences hMSC aggregation, compaction, fusion, and spreading on adherent surface. Formation of multi-cellular aggregates significantly upregulated caspase 3/7 expression, expression of C-X-C chemokine receptor type 4 (CXCR-4), cell migration, secretion of prostaglandin E2 (PGE-2) and interleukin 6 (IL-6), and resistance to in vitro ischemic stress. The functional enhancement, however, is dependent on caspase activation, because treatment with Q-VD-OPh, a pan-caspase inhibitor, attenuated CXCR-4 and cytokine secretion. Importantly, comparable ATP/cell levels and significantly reduced mitochondrial membrane potential in aggregates of different sizes suggest that altered mitochondria bioenergetics on 3D aggregation is the primary inducer for apoptosis. Together, the results suggest multicellular aggregation as an effective and nongenetic strategy for hMSC functional activation.