Tissue Engineering Part A

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

Producing bone and cartilage in a controlled and localized manner remains a significant challenge in regenerative medicine. This study investigated the ability of keratin- and polyethylene glycol (PEG)-based degradable hydrogels to deliver bone morphogenetic protein 2 (BMP-2) and leukocyte cell-derived chemotaxin 1 (LECT-1; also known as chondromodulin-1) intramuscularly to induce ectopic tissue formation. Adult male CD-1 mice received intramuscular implants of keratin-PEG gels containing a fixed dose of BMP-2 and increasing amounts of LECT-1. After two weeks, implants and surrounding muscle were analyzed using computed tomography (CT) and histology. The results showed that BMP-2 is necessary for forming new bone and cartilage, whereas LECT-1 alone appeared to trigger muscle dedifferentiation without ossification or chondrogenesis. Co-delivery of BMP-2 and LECT-1 enhanced bone and cartilage formation in a dose-dependent manner: higher LECT-1 doses led to proportionally more ectopic cartilage (linear correlation, r2 {approx} 90%), while bone formation peaked at the third LECT-1 dose at approximately twice the volume of the BMP-2-only group. These findings indicate that muscle-resident cells may be capable of reverting and switching to mesenchymal lineages, recapitulating endochondral ossification. The platform offers a promising strategy for growing bone and cartilage autografts within skeletal muscle bundles.

ABSTRACTThe repair of large bone defects remains a major clinical challenge, in part due to inadequate vascularization and poor integration of graft materials. Tissue engineering strategies that recapitulate the developmental process of endochondral ossification, whereby a cartilage template remodels into bone, have shown significant potential in pre-clinical models of large bone defect healing. However, successfully scaling these approaches to clinically relevant sizes will require the development of strategies to support the rapid vascularization of the graft following implantation in vivo. Here, mechanically reinforced templates were first fabricated by integrating hypertrophic cartilage microtissues derived from human mesenchymal stem/stromal cells (MSCs) within an osteoconductive 3D-printed polycaprolactone (PCL) framework coated with nano-hydroxyapatite (nanoHA). In vitro the cartilage microtissues fused and generated an extracellular matrix rich in sulphated glycosaminoglycans and collagen. To prevascularize these constructs, vascular microtissues derived from a co-culture of endothelial cells and MSCs were incorporated into a central channel within the construct, which generated a microvascular network within the graft in vitro. Following subcutaneous implantation, hypertrophic cartilage templates with ( vascular-channel group) and without ( empty-channel group) this central vascularized channel supported endochondral bone formation. Quantitative microCT and histological analyses revealed significantly greater remaining bone in the empty-channel group, whereas the vascular-channel group supported enhanced vascularization and remodeling of the graft in vivo. These findings support the continued development and testing of a modular biofabrication strategy that combine self-organizing hypertrophic cartilage and vascular microtissues with osteoconductive 3D-printed architectures to generate scalable, prevascularised hypertrophic cartilage templates for endochondral bone repair. Key-words: spheroids, microtissues, hypertrophic cartilage, vascularization, endochondral ossification, bone tissue engineering.

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

Biomaterial scaffolds for repairing traumatic muscle injuries require restoration of both the anisotropic architecture and basement membrane extracellular matrix cues critical to normal muscle function. To address this need, we establish a collagen-glycosaminoglycan (CG) scaffold platform pairing an aligned pore microstructure, produced via directional freeze-drying, with basement membrane protein functionalization via carbodiimide crosslinking. Laminin and/or collagen IV are successfully tethered and retained within CG scaffolds over 7 days without significantly altering pore size or alignment, confirming stable protein functionalization and preservation of scaffold architecture. Human muscle progenitor cells show excellent viability and metabolic activity in all scaffold groups, with collagen IV functionalization significantly enhancing myotube number and fusion index. Toward establishing scaffold compatibility with non-myogenic support cells, we show that neural stem cells remain viable and metabolically active across all scaffold conditions. Overall, these findings highlight the combination of aligned scaffold architecture and collagen IV functionalization as potentially impactful for skeletal muscle tissue engineering.

Granular extracellular matrix (gECM)-based biomaterials commonly contain polymer components to improve scaffold cohesion and handling during fabrication and use. However, these polymer hydrogel components may dilute ECM content and increase fabrication and regulatory complexity. This study evaluated whether particle-only gECM wafers could serve as a simplified alternative to hydrogel-based gECM scaffolds while maintaining structural, mechanical, and biological performance. Decellularized human cartilage and skin tissues were processed and fabricated into three scaffold formats: gECM hydrogels, freeze-dried gECM hydrogel wafers, and freeze-dried particle-only gECM wafers. Across fabrication methods, scaffold swelling, volume fraction, and stiffness were strongly influenced by both tissue type and fabrication approach. gECM hydrogels exhibited the greatest swelling and lowest stiffness, while gECM wafers displayed higher volume fractions and greater mechanical stiffness. Notably, gECM particle-only wafers achieved performance comparable to gECM hydrogel wafers despite the absence of a secondary polymer network. Particle-only wafers also maintained swelling behavior and structural properties over 3 months of dry storage at room temperature, with only modest decreases in stiffness. In vitro studies showed sustained cell viability over 14 days on particle-only wafers, with chondrocytes infiltrating cartilage wafers and fibroblasts remaining primarily surface-localized on skin wafers. In addition, particle-only wafers remained cohesive during implantation into a bovine cartilage defect model. These findings demonstrate that particle-only gECM wafers can achieve structural integrity, mechanical performance, and cytocompatibility without the need for an additional polymer network, highlighting a simplified and ECM-rich biomaterial platform. By eliminating polymer carriers and enabling dry storage with preserved function, this approach supports the development of off-the-shelf, translationally accessible gECM particle-only wafers for tissue engineering applications.

The human spine is a complex, coordinated biomechanical system. Physiologically, its tissues are also highly interdependent in terms of function and viability. The interaction between mechanical stress and biological/biochemical activity over time constitutes a key driver of spinal degeneration. Research to date providing mechanistic insights into this process has focused on individual components (vertebra and disc tissue analogues), in isolation or as basic functional units. However, many observations from individual units will not translate to whole spine behaviour. The intricate complexity of the spine requires novel experimental models (synthetic and biotic), which must consider the spine at an organ level and adopt an integrative approach that can capture the dynamics which govern its function. Here, we report the development of a biomimetic spinal model prototype, amenable to cellular integration, which is miniaturised to the in vitro scale to provide a controlled environment and testbed for axial biological mechanics. The research presented here encompasses more than a decade of systematic investigations during which the gradual emergence of key manufacturing innovations progressively enabled addressing an exceptionally complex bioengineering challenge - organotypic spine engineering. The model comprises the full anatomical range of spinal vertebrae/bones (C1 to Sacrum & Coccyx), reproduced using bioceramic materials, assembled in sequence into a relevant columnar architecture and mechanically connected end-to-end by biochemically active interfaces. A range of assessments examining anatomical design, material behaviour and manufacturing processes is presented. The work explores concepts such as longitudinal mechanobiology and multi-segment coupling as well as manufacturing strategies using autonomous materials and instrumentation. This prototype introduces for the first time columnar level behaviour and the ability to study time dependent adaptations. This model is important because it can support tissue maturation, evolving mechanical properties and adaptive behaviour and it represents an intermediate step between isolated skeletal tissue models and future organ-level spinal constructs.

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold tremendous promise for disease modeling, drug discovery, and cardiac regenerative therapies. However, the immature phenotype of hPSC-CMs remains a major barrier limiting their translational utility. Here, we performed integrated multi-omic profiling to identify molecular pathways and regulatory programs associated with hPSC-CM maturation during long-term culture. hPSC-CMs were cultured for 113 days and analyzed using metabolomics, proteomics, and transcriptomics across progressive stages of maturation. Long-term culture induced widespread multi-omic remodeling, including significant changes in 142/934 metabolites, 550/3,556 proteins, and 2,892/23,309 transcripts from Day 30 to Day 113. Metabolomic analyses revealed early increases in phospholipid biosynthesis and mitochondrial beta oxidation of fatty acids from Day 30 to Day 60, suggesting metabolic priming precedes later maturation events. In contrast, proteomic remodeling was more prominent during later stages of maturation and was characterized by enhanced calcium handling and cell cycle exit. Transcriptomic analyses demonstrated progressive increases in ion channel expression, t-tubule organization, fatty acid metabolism, creatine shuttle pathways, and cell cycle arrest programs. Transcriptomic and integrative multi-omic pathway analyses identified coordinated suppression of TGF{beta}, MAPK, Wnt, and Hedgehog signaling together with activation of integrin-related, respiratory electron transport, muscle contraction, and Slit-Robo signaling pathways during maturation. Moreover, multi-omic transcription factor activity analysis prioritized a GATA4-centered network of putative cardiomyocyte maturation regulators including SOX7, SOX18, TBX2, and ZFPM2 (FOG2). Together, these findings elucidate the degree and pace of hPSC-CM maturation during long-term culture and establish an integrated multi-omic framework for identifying strategies to accelerate hPSC-CM maturation.

Introduction: Critical-sized bone defects cannot heal spontaneously, requiring additional, often burdensome, treatment. Thus, various synthetic substitute materials have been investigated regarding their treatment capacity. Poly-L-lactic acid (PLLA) and polyglycolic acid (PGA) have emerged as promising biodegradable scaffold materials. The addition of inorganic materials such as calcium carbonate (CC) has also been shown to be advantageous. This study investigates the effect on bone regeneration of PLLA-PGA-CC scaffolds in critical-sized bone defects over a two-year observation period using sheep as an animal model. Methods: Critical-sized mandible angle defects were created in twelve female merino sheep. Mandibular defects were reconstructed with PLLA-PGA-CC scaffolds in four sheep, while the remaining eight served as negative control (defects left empty). The scaffolds were manufactured using computer-aided design and manufacturing, incorporating an interconnected porous structure and fixated with polyether ether ketone cages. Bone regeneration was evaluated using computed tomography (CT) imaging at 3, 12, and 24 months postoperatively. Bone volume was assessed quantitatively. Additionally, a histological analysis was performed. Results: Surgical procedures were successful and without major complications. CT assessment showed more bone regeneration in the scaffold group (mean volume: 7,472 mm3) than in the control group (4,168 mm3, p = 0.1) at 24 months postoperatively. Resorption of the scaffolds and formation of compact lamellar bone tissue were confirmed by histological analysis. However, the osteoconductive properties of the scaffolds were limited, with only minimal ingrowth of bone tissue into the porous structure. In both groups, fibrous tissue infiltration and the formation of cyst-like cavities in the defect region were observed. Conclusion: PLLA-PGA-CC scaffolds were found to be biocompatible and enhanced bone regeneration compared to the control group. Due to fibrous tissue infiltration and the lack of osteoconductivity, the suitability of the material for critical-sized bone defect reconstruction is limited.

Objective: To evaluate the effect of residual hair (RH) biomaterial particulates, biphasic electrical stimulation (ES), and their combination (RHES) on the kinetics and quality of skin wound healing. Method: Eighteen adult albino mice received bilateral, splinted 10-mm full-thickness dorsal excisional wounds and were randomly assigned to one of three animal groups producing four wound-level treatment conditions: untreated control (-) (n = 12), RH (n = 12), ES (n = 6), and combined RHES (n = 6 wounds). Daily wound images were segmented using an AI-assisted workflow: a U-Net (ResNet34 encoder, ImageNet-pretrained, trained on a parallel single-expert tracing study with held-out validation Dice = 0.906) generated initial boundary predictions, each reviewed and corrected as needed. Wound size measures (perimeter, area, equivalent diameter [D_eq], circularity, aspect ratio) were normalized to the day-0 value of each wound and analyzed by linear mixed-effects regression with mouse identity as a random intercept and mouse body weight as a covariate. On day 7, wounds were excised, fixed, processed for histology, and analyzed by Masson's trichrome (collagen content in granulation tissue) and GAP-43 immunohistochemistry (a marker of regenerative cellular activity). Results: All three treatments significantly accelerated wound closure compared to (-) (Day x Treatment interaction {chi}2(3) = 36.4, ***p < 0.0001). The closure-rate advantages on the log-D_eq scale were ES -0.047/day (***p < 0.0001), RHES -0.029/day (***p = 0.0005), and RH -0.022/day (**p = 0.0015). By day 7, mean D_eq had decreased to 0.58 of the day-0 value in ES, 0.69 in RHES, 0.73 in RH, and 0.79 in (-). Tissue analyses revealed treatment-specific differences in healing quality: RH and RHES wounds contained 6.1x and 8.5x more collagen in granulation tissue than (-) (both **p = 0.002 vs (-); both **p = 0.009 vs ES), and showed approximately 16x and 27x greater mean GAP-43 expression than (-), respectively; the RHES increase remained significant after Bonferroni correction (adjusted *p = 0.042), whereas the RH increase did not (adjusted p = 0.058). ES alone did not significantly increase either collagen content or GAP-43 expression. Wound shape was more circular and more stable across days in RH-containing groups. Mouse body weight did not predict closure, whereas image-derived dryness, eschar coverage, and wound contraction were significant negative predictors of measured wound size. Conclusion: ES, RH, and RHES each significantly improve wound closure kinetics. The improvement appears mechanistically distinct: ES principally accelerates closure rate, while RH principally enhances tissue-level regenerative markers (collagen deposition and GAP-43 expression). RHES combines both advantages.

Bioengineered, transplantable kidney tissue using decellularized scaffolds offers a promising strategy to overcome the shortage of donor kidneys that limits organ transplantation for patients with end stage renal disease. These kidney scaffolds retain essential extracellular matrix architecture, providing a biologically active niche for recellularization. Successful generation of bioengineered kidney tissues includes enhanced patent vasculature and mature, functional nephrons with collecting ducts. Here, we report the development of engineered kidney tissue consisting of reconstituted kidney scaffolds and human pluripotent stem cell-derived nephron and ureteric bud progenitors. Structural analysis of recellularized kidney scaffolds showed advanced nephron structures that became more mature and exhibited interconnected nephron and collecting ducts. In vivo engraftment of reconstituted kidney scaffolds in mice led to vascularization, maturation, and secretory function. Notably, mouse-graft vascular anastomosis was evident with erythrocytes present in vasculature and nephron-secreted proteins detected in mouse urine, indicating functional integration. This approach demonstrates the feasibility to generate advanced bioengineered kidney tissues that offer a versatile platform for disease modeling, drug screening, and regenerative medicine.

Peripheral nerve injuries often result in prolonged skeletal muscle denervation, leading to progressive atrophy, fibrosis, neuromuscular instability, and loss of regenerative capacity before axons can reinnervate distal targets. Here, we developed a non-viral strategy using tissue nanotransfection (TNT) to deliver the neurogenic transcription factor cocktail Ascl1, Brn2, and Myt1l (ABM) directly to denervated skeletal muscle. In vitro, ABM-transfected myoblasts sustained expression of the reprogramming factors, acquired neuron-like morphologies, upregulated neuronal markers including Tuj1, Map2, and Syp, and exhibited electrophysiological properties consistent with membrane excitability. RNA sequencing confirmed broad activation of neurogenic transcriptional programs, with enrichment of pathways associated with neuronal fate commitment, neuron differentiation, axon guidance, synaptogenesis, and developmental signaling. In a mouse model of sciatic nerve transection, TNT enabled localized ABM expression in denervated gastrocnemius muscle. ABM-TNT treatment accelerated resolution of denervation-associated fibrillation potentials and showed trends toward improved twitch and tetanic torque, compound muscle action potential amplitudes, and muscle mass preservation. Transcriptomic profiling of treated muscles 5 weeks after injury revealed distinct gene expression programs enriched for muscle regeneration, neuromuscular organization, trophic support, extracellular matrix remodeling, angiogenesis, myogenesis, and metabolic adaptation. Network analyses further identified activation of neurogenic regulators, neurotrophic signaling, and vascular-support pathways. These findings establish TNT-mediated ABM delivery as a non-viral platform for inducing neurogenic and myoprotective programs in denervated muscle, suggesting a potential strategy to preserve muscle viability during the prolonged interval required for peripheral nerve regeneration.

Neural stem cell (NSC) transplantation is a promising strategy for repairing the injured spinal cord, but transplanted cells typically require immunosuppressive therapy to prevent rejection, even for induced pluripotent stem cell (iPSC)-derived autologous grafts. However, the effects of immunosuppressive drugs on neurite outgrowth and axonal regeneration, processes critical for neural circuit reconstruction, have not been fully characterized. In this study, we tested nine clinically relevant immunosuppressants on human iPSC-derived neurons and primary human spinal cord NSCs in vitro at concentrations approximating clinical exposure levels. The drug panel included FK-506 (tacrolimus), cyclosporine A (CsA), rapamycin, belatacept (Nulojix), etanercept (Enbrel), mycophenolate mofetil (CellCept), cyclophosphamide (Cytoxan), prednisone, and azathioprine (Imuran). Neurite outgrowth was quantified via automated high-content imaging. Multiple agents, including CsA, Imuran, Nulojix, and CellCept, induced significant reductions in neurite outgrowth in a cell type- and dose-dependent manner, with CsA producing the most robust and consistent inhibition across both cell lines. In contrast, FK-506 showed no significant effect on neurite extension at clinically relevant concentrations. Consistent with the in vitro results, human neural progenitor cell grafts in a rodent spinal cord injury model exhibited significantly reduced graft-derived axon extension in the host spinal cord when hosts were treated with CsA rather than FK-506. These findings demonstrate that immunosuppressant choice can profoundly influence neural graft integration and axonal regeneration. Our study underscores the importance of preclinical evaluation of immunosuppressive regimens and suggests that selecting agents such as FK-506 over CsA may improve outcomes in future stem cell-based therapeutic trials for spinal cord injury and related disorders of the central nervous system.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

Glioblastoma (GBM) is a highly aggressive brain tumor with a five-year survival rate of less than 5%. The current standard of care established 20 years ago includes maximal surgical resection and administration of alkylating agent temozolomide (TMZ). GBM is highly invasive, and GBM cells that evade surgical resection can become resistant to TMZ and develop new aggressive secondary tumors. Post-relapse there are few treatment options available to patients. Tissue engineering approaches suggest the opportunity to develop in vitro models of the GBM tumor microenvironment that may accelerate the discovery of novel therapies for GBM. Here, we report the adaptation of hydrogel microdroplets (microgels) to encapsulate GBM cells in a tailorable 3D matrix to assess patterns of growth and to screen TMZ drug response using patient-derived xenograft (PDX) specimens. We exploit a unique aspect of the microgel system to account for the cellular heterogeneity within the tumor microenvironment (TME). We combine cell-laden microgels generated from TMZ-resistant and TMZ responsive variants of the same PDX specimens to create heterogeneous populations with varying levels of drug sensitivity. We demonstrate a range of drug resistance phenotypes as a function of the ratio of TMZ-responsive to resistance cells and identify the population required for TMZ-resistance to overtake take the response. We then investigate the influence of tumor mimetic shifts in hyaluronic acid bioavailability and hypoxia on patterns of TMZ resistance. We show exposure to matrix-bound hyaluronan increases TMZ resistance and the glioma stem cell population in both cell variants. Lastly, we report an increase in TMZ sensitivity but divergent changes in the GSC subfraction for TMZ resistant vs responsive GBM in the presence of hypoxia. Together, we demonstrate the versatility of cell-laden microgel approach to replicate heterogenous tumor populations, model shifts in the tumor microenvironment, and rapidly screen therapeutic response.

While defined synthetic substrates can replace Matrigel for human induced pluripotent stem cell (hiPSC) culture and hiPSC-derived cardiomyocyte (hiPSC-CM) production, existing approaches culture cells on two-dimensional surfaces and yield structurally immature cardiomyocytes, limiting their use in disease modeling and regenerative medicine. Here, we developed a xeno-free, fully-defined cyclic RGD (cRGD)-functionalized alginate platform in which we encapsulated hiPSCs to support their expansion and in situ cardiac differentiation. cRGD functionalization was essential for hiPSC survival and pluripotency, with maximal support achieved at a low ligand density (25 M). In the presence of cRGD, hiPSC encapsulation into softer gels made from lower molecular weight alginates led to enhanced hiPSC expansion and improved cardiogenesis. Strikingly, differentiation in situ with 3D gels led to hiPSC-CM with higher structural maturity, including a markedly increased proportion of Desmin positive cardiomyocytes. Finally, after enzymatic retrieval from hydrogels, cardiomyocytes derived from softer gels formed tissue-engineered myocardium with superior contractile force compared to tissue fashioned from hiPSC-CM derived from more rigid gels. Together, these results demonstrate the promise of this defined, tunable platform for biomanufacturing of structurally mature cardiomyocytes from hiPSC.

Osteoarthritis (OA) drug development remains constrained by preclinical models that fail to recapitulate the multicellular interactions that regulate human joint inflammation and extracellular matrix degeneration in response to investigational drugs. Tanezumab, a humanized anti-nerve growth factor monoclonal antibody developed for non-opioid pain relief, advanced to late-stage clinical trials but was discontinued due to unresolved joint-localized safety concerns, including rapidly progressive OA. This study evaluated whether a human microfluidic joint-on-a-chip co-culture system could detect early biomarker responses to tanezumab exposure that were not apparent in conventional chondrocyte monoculture. Tanezumab was first tested in human chondrocyte monoculture under untreated and disease-like (i.e., IL-1{beta}-treated) conditions. Across a 20-analyte panel of inflammatory and matrix-remodeling biomarkers, statistically significant monoculture responses to tanezumab were limited to decreased IL-1{beta} from 335 to 132 pg/mL ([~]0.39-fold) and increased IL-8 from 575 to 675 pg/mL ([~]1.17-fold). Major OA-associated matrix-remodeling markers, including MMP-1, MMP-3, and MMP-13, remained largely unchanged, indicating that monoculture conditions are insufficiently sensitive to detect clinically predictive drug-related molecular changes. Tanezumab was then evaluated in co-cultures containing chondrocytes, osteoblasts, fibroblast-like cells, and macrophages under low-inflammation (i.e., M0 macrophage-based) and high-inflammation (i.e., M1 macrophage-based) conditions. In the M0-based co-culture, tanezumab increased MMP-1 from [~]4.20 x 104 to [~]6.20 x 104 pg/mL ([~]1.48-fold), MMP-3 from [~]8.00 x 104 to [~]1.20 x 105 pg/mL ([~]1.50-fold), and MCP-1 from 2.85 x 103 to 4.31 x 103 pg/mL ([~]1.51-fold). In contrast, the M1-based co-culture showed decreases in MMP-13 from [~]1.66 x 104 to [~]1.17 x 104 pg/mL ([~]0.70-fold) and IFN-{gamma} from [~]1.95 x 104 to [~]1.56 x 104 pg/mL ([~]0.80-fold), changes that may appear beneficial despite the drugs known clinical risks. Collectively, these findings show that low-inflammation multicellular co-culture revealed coordinated matrix remodeling and inflammatory responses to NGF blockade that were missed in monoculture and were partly obscured in highly stimulated disease-like conditions. This platform may provide a useful, human-relevant approach for safety signal assessment and early evaluation of OA therapeutics within a defined context of use focused on joint-specific, tissue-level drug-response testing.

The African spiny mouse (Acomys cahirinus) exhibits a unique capacity among mammals for scarless tissue regeneration, making it a compelling model for investigating the cellular mechanisms underlying regenerative healing. To determine how cellular heterogeneity and specific phenotypes influence fibroblast behavior, we established an immortalized Acomys fibroblast line along with a CRISPR/Cas9-mediated Col3A1 knockout variant and a DNA damage-induced senescent population. Compared with Mus musculus, NIH 3T3 fibroblasts, Acomys cells displayed distinct morphology, similar migration speeds, reduced directional persistence, and greater biophysical heterogeneity. While previous studies have linked regenerative wound healing to the elevated expression of collagen type III (Col3A1), CRISPR-mediated knockout of Col3A1 in Acomys fibroblasts yielded comparable biophysical profiles to wild-type cells in 2D culture. To examine additional contributors to the enhanced wound-like matrix environment, we established a senescence model in which Acomys fibroblasts exhibited elevated resistance to DNA-damaging agents, complete loss of proliferation, and altered single-cell morphology. In 3D collagen gel contraction assays, Col3A1 knockout attenuated matrix remodeling capacity, whereas the introduction of a small fraction of senescent cells enhanced gel contraction and remodeling dynamics, suggesting that senescent fibroblasts can modulate collective matrix behaviors. Together, these findings demonstrate that both Col3A1 expression and senescence-associated cell states contribute to fibroblast-driven matrix remodeling, highlighting Acomys fibroblasts as a valuable model for investigating how cellular heterogeneity and senescence-associated cell phenotypes could influence regenerative wound healing.