Cellular therapies involving the co-delivery of cells with complementary pro-regenerative functionality hold promise as a strategy to promote soft tissue augmentation and regeneration. In particular, the co-delivery of adipose-derived stromal cells (ASCs) and endothelial colony-forming cells (ECFCs) has shown promise for regenerating stable blood vessels in vivo. The current study developed "cell-assembled" scaffolds for co-delivering human ASCs and ECFCs within a supportive decellularized adipose tissue (DAT) matrix, with the objective of enhancing their localized retention and augmenting their capacity to stimulate adipose tissue regeneration. Human ASCs and ECFCs were seeded separately onto human-derived DAT microcarriers under cell-type specific conditions. The cell-seeded microcarriers were then combined and cultured for 8 days under conditions that promoted matrix remodeling to fuse the microcarriers into 3D engineered tissues containing ASCs+ECFCs, ASCs alone, or ECFCs alone. Co-culture with ECFCs within the scaffolds was shown to modulate ASC pro-angiogenic gene expression, with some ECFCs forming tubule-like structures in vitro in both the ASC+ECFC and ECFC alone groups. In vivo bioluminescence imaging using a dual luciferase reporter system showed that co-delivery with ASCs enhanced ECFC retention following subcutaneous implantation in athymic nu/nu mice, but co-delivery did not alter the localized retention of viable ASCs. Interestingly, while immunofluorescence staining for CD31 and microcomputed tomography angiography indicated that vascular regeneration was similar in the cell-assembled scaffolds containing ASC+ECFCs, ASCs alone, and ECFCs alone, histological staining revealed that extensive regions of the ECFC alone scaffolds had remodelled into adipose tissue at 29 days post-implantation. STATEMENT OF SIGNIFICANCE: Cellular therapies involving the co-delivery of complementary pro-regenerative cell types hold promise as a strategy to promote soft tissue regeneration. In particular, the co-delivery of adipose-derived stromal cells (ASCs) and endothelial colony forming cells (ECFCs) may enhance blood vessel regeneration in vivo, as well as promote ASC engraftment and adipogenic differentiation. The current study developed a modular bottom-up fabrication approach for generating "cell-assembled" scaffolds incorporating both human ASCs and ECFCs dispersed throughout a supportive human decellularized adipose tissue (DAT) matrix, which were compared to scaffolds incorporating ASCs alone or ECFCs alone. Co-delivery modulated ASC pro-angiogenic gene expression in vitro and enhanced viable ECFC retention in vivo, but interestingly, in vivo adipogenesis was augmented in the cell-assembled scaffolds incorporating ECFCs alone.
Skeletal muscle possesses a remarkable capacity for regeneration, driven by the activation and proliferation of Pax7-positive muscle stem cells within a dynamic niche that includes immune cells, fibro-adipogenic progenitors, endothelial cells, pericytes, and neural elements. Cellular senescence, a stress-induced program featuring stable cell-cycle arrest and the senescence-associated secretory phenotype (SASP), has emerged as a critical yet paradoxical regulator of this process. Accumulating evidence indicates that transient senescence, particularly in FAPs, macrophages, and other niche cells during acute muscle injury, plays a beneficial role in supporting muscle regeneration. These senescent cells promote cellular plasticity, enhance myoblast differentiation, facilitate phagocytic clearance of debris, and modulate inflammation and repair via timely SASP factor secretion. However, conflicting findings suggest that senescent cells exert detrimental effects, impairing regeneration by establishing a sustained pro-inflammatory and pro-fibrotic niche, especially when senescence persists in aged or dystrophic muscle. This review synthesizes the complex and contradictory roles of cellular senescence in skeletal muscle regeneration, underscores the distinction between transient pro-regenerative and persistent deleterious senescence, highlights the importance of cell-type-specific contributions, and emphasizes the need for precise characterization of senescent cell dynamics and fate. Resolving these discrepancies will be critical for developing targeted senotherapeutic strategies to enhance muscle regeneration in aging and degenerative diseases.
Over the past two decades there have been remarkable advances in stem cell biology, bioengineering, and lung regenerative research, transforming our understanding of pulmonary biology from development to repair, and disease. Strategies using endogenous lung progenitor cells, pluripotent stem cell technologies, and engineered tissue platforms have become central tools for interrogating lung biology. Major breakthroughs have included the identification of diverse cell populations that coordinate lung homeostasis and repair, facilitated by the extensive adoption of single cell, multiomic and spatialomics approaches. Simultaneous progress in biomaterials, organoid systems, decellularized lung scaffolds, and lung-on-chip platforms has uncovered how extracellular matrix composition, mechanical forces, and tissue architecture contribute to the regulation of cell fate and function. These advances have enabled increasingly physiologically relevant in vitro, and ex vivo models while informing tissue engineering strategies aimed ultimately at functional lung replacement. Translation toward the clinic has advanced through both cell-based and cell-free therapeutic strategies. Early efforts focused largely on mesenchymal stromal cell-based approaches and extracellular vesicles, which have demonstrated safety and context-dependent efficacy in inflammatory lung diseases, alongside emerging preclinical evidence of functional engraftment of induced pluripotent stem cell-derived lung lineages. The past twenty years of progress, captured at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, highlights the power of interdisciplinary collaboration in advancing lung regeneration from foundational discovery toward therapeutic reality.
Diabetes affects >500 million people worldwide. Despite insulin therapy, most patients develop devastating complications, emphasizing the need for curative strategies. Human multipotent stromal/stem cells (MSC) secrete factors that promote islet regeneration. We previously showed that intrapancreatic (iPan) delivery of Wnt-activated MSC-conditioned media (Wnt+ CM) stimulates islet regeneration without cell transfer. This study aimed to identify and functionally validate specific MSC-secreted proteins with islet regenerative potential. Comprehensive mass spectrometry-based, quantitative proteomic analyses comparing Wnt-pathway activated versus untreated MSC secretomes were cross-referenced with a prior dataset distinguishing regenerative from nonregenerative MSC CM. Proteins enriched in both conditions were iPan-injected individually or in combination into streptozotocin-treated NOD/SCID mice. Nonfasting glucose, glucose tolerance, beta cell mass, islet morphology, and islet cell proliferation were assessed at 4- and 32-days post-treatment. Cross-referenced secretome analyses identified eight proteins implicated in islet regeneration: CALU, CTSB, FAM3C, GAL1, PPIA, PSAP, SOD1, and TGM2. A single iPan-injection of the 8-protein combination significantly lowered hyperglycemia, improved glucose tolerance, and increased beta cell mass, comparable to Wnt+ CM. Regenerative effects such as increased beta cell proliferation appeared as early as day 4. Single-protein testing identified CALU and SOD1 as leading candidates, improving glucose tolerance and reducing nonfasting glucose. This study defines a set of MSC-secreted proteins that promote islet regeneration in vivo, supporting the development of protein-based biologics to preserve or restore beta cell function during diabetes, with potential applications alongside islet replacement therapies to enhance graft survival and function. Diabetes causes the loss of insulin-producing cells in the pancreas, and current treatments cannot restore islet function. This study identifies specific proteins secreted by human MSC that can regenerate islet cells without the need for cell transplantation. Remarkably, a defined eight-protein combination improved glucose control and beta cell mass following intrapancreatic injection into diabetic mice. These findings highlight a potentially safe, scalable, and immune-compatible protein-based approach to preserve or regenerate insulin-producing beta cell function, offering a promising path toward regenerative therapies for diabetes.
The olfactory epithelium (OE) maintains lifelong neurogenesis and shows strong regenerative capacity through the coordinated functions of horizontal basal cells (HBCs) and globose basal cells (GBCs). These progenitors are regulated by key transcriptional factors such as Sox2, p63, Pax6, Ascl1, Neurog1 and NeuroD1, as well as signaling pathways including Wnt/β-catenin, Notch, YAP and inflammation-related regulators, which together control lineage specification and injury-induced plasticity. A set of genes such as Lgr5, Tmem59, Notch1, and Chil4 play critical roles in OE homeostasis and regeneration, depending on a broader and highly dynamic network. Recent progress in single-cell transcriptomics, spatial transcriptomics and organoid models has revealed previously unrecognized cell states, differentiation routes and intercellular communications. This review summarizes the molecular and cellular mechanisms that support OE regeneration and highlights emerging technologies that advance understanding the process of olfactory epithelium regeneration and guiding future approaches for restoring olfactory function.
Cold atmospheric plasma regulates cell fate by modulating cellular oxidative stress levels, making it a promising new therapy in the field of cell bioengineering. While the regulatory effects and potential molecular mechanisms of the novel piezoelectric cold atmospheric plasma (Piezo-CAP, PiezoBrush PZ3 handheld device, Relyon Plasma, Germany) on the fate of neurogenic cells remain unclear. We utilized C17.2 neural stem cells, rat astrocytes, and SH-SY5Y neuroblastoma cells to evaluate the impact of Piezo-CAP on cell proliferation, apoptosis, and differentiation through CCK-8 assay, Calcein AM/PI double staining, immunofluorescence, and western blot techniques. The signal network of Piezo-CAP governing neurogenic cell proliferation and differentiation was thoroughly examined by the merging of RNA sequencing and bioinformatics analysis. The results demonstrated that Piezo-CAP has a dual regulatory influence on neurogenic cell fate, dependent on specific parameters and cell types. Significantly, Piezo-CAP enables the bidirectional differentiation of C17.2 neural stem cells into neurons and glial cells. The described effects are mechanistically associated with the buildup of intracellular reactive oxygen and nitrogen species (RONS) and may be enhanced by the activation of the PI3K/AKT signaling pathway. Transcriptome research further confirms that Piezo-CAP can influence signaling pathways related to the maintenance of pluripotency and differentiation, including JAK-STAT, Wnt, and Hippo signaling. In conclusion, Piezo-CAP, as an effective physical stimulation method, may accurately influence the destiny of neurogenic cells through essential signaling pathways facilitated by RONS, including PI3K/AKT. This research provides actual evidence and innovative strategies for the application of Piezo-CAP in neural therapy and neural tissue engineering.
The adult pancreas possesses limited capacity for regeneration. Evidence suggests conversion between terminally differentiated pancreatic cells, either through transdifferentiation or dedifferentiation, but the extent, cellular origins and physiological relevance of these processes are not fully understood. Key insights have emerged from studies of pancreatic injury and stress, which induce cellular reprogramming and expansion of the beta cell compartment. Additionally, model organisms such as zebrafish and axolotls exhibit a greater capacity for pancreatic regeneration, providing evolutionary perspectives. Understanding these mechanisms may enable manipulation of endogenous regenerative responses for translational applications. In this review, we summarise recent studies on pancreatic plasticity and the mechanisms underlying beta cell regeneration and discuss how these insights may guide new regenerative strategies for the treatment of diabetes.
The intestinal mucosa is a complex functional layer which is formed from a diverse range of cell types that include epithelial cells (within crypts and villi) and an array of mesenchymal cells. Many intestinal diseases involve loss of the surface mucosa which can be difficult to restore, and which delays healing and return to normal function. We reason that development of a transplantable intestinal mucosal tissue graft may be a potential therapeutic strategy to aid healing. To be clinically useful, such a tissue graft would need to be capable of rapid production, avoid the risk of host rejection and be demonstrably safe. To create a potential intestinal graft, we developed a novel early-stage human induced pluripotent stem cell (hiPSC) co-differentiation platform capable of generating multiple intestinal cell lineages (epithelial, mesenchymal and endothelial) in 8 days. This protocol is simple to implement, serum-free and greatly reduces the use of animal products. We confirmed the identity of cells by demonstrating that these cells had RNA and protein expression profiles typical of intestinal cell lineages. In particular, we used bulk and single-cell RNA sequencing to characterise global cellular transcriptional profiles robustly and showed that the cells have intestinal identity with early polarisation towards colonic differentiation. The results were replicated across multiple hiPSC lines and in an independent centre. We further cultured the derived cells on collagen hydrogels to form colon-like intestinal patches (CL-IPs). When transplanted into mouse subcutis, CL-IPs formed into colon-like tissue structures, including crypts, stromal and muscle layers. They also developed human-origin vasculature which underwent anastomosis with the murine vasculature to transport murine blood into the graft. Teratoma assays and molecular analyses showed no evidence of residual pluripotency. While at an early stage, this platform shows great potential for further development as a potential source for novel intestinal mucosal regeneration therapy. In addition, the platform is physiologically relevant and thus shows promise as the basis for a new generation of in vitro models of intestinal pathobiology.
The thymus is emerging as a model for studying organ regeneration and stem cell biology. While research has long focused on how antigen-presenting cells shape the T cell repertoire, recent discoveries unveil a far richer cellular landscape that challenges long-held views of thymus structure and function. This review traces the history of early thymic reconstitution assays, the paradigm of clonal stem cells and serial transplantation, assessing evidence for "stemness" within the thymus. A key focus is the paradox that an involuting thymus retains cells able to expand in culture and reconstitute organ function. We differentiate embryonic/fetal thymus development from postnatal homeostasis, emphasizing how the potency of epithelial progenitor/stem cells shifts with age or upon injury. The role of mesenchymal/interstitial cells and the extracellular milieu is considered alongside advances in organ reconstruction. We outline major unsolved questions in the field: thymus regeneration after childhood; the minimal components required to generate functional naïve T cells outside the body; and the potential of next-generation humanized mouse models to interrogate immune tolerance and novel immunotherapies. We argue that thymus research is entering a new era, one in which understanding and harnessing thymus regenerative potential could yield transformative advances in both basic science and clinical applications.
The function of the thymus, essential for T cell development, declines sharply with age. Genetic defects that disrupt thymic organogenesis or function lead to profound immunodeficiency and autoimmunity. Allogeneic thymus transplantation for congenital athymia, the only existing thymus replacement therapy, provides proof of concept for T cell reconstitution but remains constrained by donor availability and Human Leukocyte Antigen (HLA) mismatch. Advances in single-cell biology and stem cell engineering now make scalable thymus regeneration an attainable goal. In particular, induced pluripotent stem cell-derived thymic epithelial cells could restore endogenous T cell development and immune tolerance in an HLA-compatible manner. We propose that the thymus is uniquely suited for stem cell-based regeneration, with the potential to transform the treatment of immune deficiency, transplantation, cancer, autoimmunity, and aging itself.
The thymic medulla is essential for establishing central tolerance, orchestrating the development of a diverse yet self-tolerant T cell repertoire, and preventing autoimmunity. This process is primarily mediated through interactions between developing thymocytes and antigen-presenting cells, including thymic epithelial cells (TECs) and dendritic cells (DCs), with additional regulatory contributions from endothelial cells, mesenchymal cells, and macrophages. Despite its critical role, the complexity of late-stage thymocyte development and the dynamics of their medullary residency remain incompletely understood. Recent advances in single-cell, epigenomic, and transcriptomic technologies have begun to reveal previously unappreciated layers of cellular and molecular heterogeneity within the thymic medulla throughout life. In this review, we explore how the medulla shapes the fate of both conventional and non-conventional T cells, examine the diversity of thymocyte populations it supports, and discuss how this specialized microenvironment adapts during aging and regeneration.
Over the past two decades, progress in stem cell biology, bioengineering, and systems biology has improved our understanding of lung regeneration and repair. Building on work presented at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, this review examines the evolving trajectory of the field and outlines remaining challenges and opportunities for future research. We focus on three main areas: improving ex vivo lung models to better capture cellular heterogeneity and biomechanics; using single-cell, spatial, and computational approaches to support translation into clinical practice; and innovative therapeutic strategies, including gene therapies, epithelial cell therapies, immune cell engineering, extracellular matrix reconditioning, senescence targeting, and whole-organ bioengineering and xenotransplantation. Together, these approaches are shifting lung regeneration from descriptive studies toward precision, mechanism-driven therapies. Future progress in lung regenerative medicine will require integration of omics-driven insights with functional validation and biomaterial innovation to achieve meaningful clinical impact.
Type 1 diabetes (T1D) is characterised by destruction of pancreatic beta-cells by islet-infiltrating cytotoxic lymphocytes and elevated intra-islet secretion of pro-inflammatory cytokines. However, the underlying pathophysiological mechanisms remain incompletely understood. We hypothesised that abnormal elevation of islet NAD, via activation of NAMPT, plays a key role in driving islet autoimmune processes, leading to beta-cell death in T1D. Here, we report that NAMPT inhibition protects against pro-inflammatory cytokine (IL-1β, TNFα and IFNγ) mediated beta-cell dysfunction and apoptosis in isolated mouse and human islets. RNAseq revealed that NAMPT inhibition blocked cytokine-mediated gene expression linked to pro-inflammatory responses and leukocyte migration. In vivo, diabetes was induced in CD1 mice via multiple low-dose streptozotocin (MLDS) injections. MLDS mice were administered the NAMPT inhibitor FK866 (10 mg/kg; IP) or saline equivalent for 16 days. These experiments demonstrated that NAMPT inhibition improved glycaemic control and beta-cell survival and function in MLDS mice. FK866 also reduced proportions of islet-residing TNFα-producing CD4+T-cells and F4/80+macrophages, proliferation of spleen-derived CD4+ and CD8+T-cells and proliferation of islet-derived CD4+T-cells and F4/80+macrophages. Finally, we report that NAMPT inhibition was able to block pro-inflammatory cytokine-mediated migration of cytotoxic CD8+T-cells into isolated islets, using an in vitro transwell platform. This data supports a key immunomodulatory role for NAMPT in islet autoimmunity. NAMPT inhibition may be able to prevent beta-cell death and thus represent a novel therapeutic approach for T1D. The effects of increased NAD levels on islet inflammation require in-depth characterisation and caution should be exercised with regard to the use of NAD boosting supplements, particularly in individuals at risk of developing T1D.
Despite recent advances, treatment outcomes for adults with acute lymphoblastic leukaemia (ALL) remain poor. Although patients often exhibit an initial favourable response to chemotherapy, with substantial clearance of tumour cells, most patients eventually relapse. This indicates the persistence of a chemoresistant ALL subpopulation capable of driving disease regeneration. Growing evidence implicates interactions between leukaemia cells and the bone marrow (BM) niche in this process. Our findings show that BM-derived mesenchymal stem cells (MSCs) and adipocytes (BMAds) promote chemotherapy resistance in ALL cells via activation of the wingless-related integration site (WNT) signalling pathway. Chemotherapy-treated co-cultures of MSCs/BMAds and ALL cells exhibited upregulation of several WNT ligands in the stromal compartment. Notably, pharmacological inhibition of WNT signalling abrogated the stromal-mediated chemoprotection and enhanced ALL cell apoptosis in vitro. In vivo, WNT inhibition in a p185BCR-ABLArf-/- B-ALL mouse model sensitised leukaemia cells to chemotherapy, delaying relapse and extending survival. Collectively, these results support the therapeutic potential of WNT inhibitors as a strategy to block the cross-talk between the BM stroma and leukaemic cells and reduce ALL chemoresistance.
Human skeletal muscle stem cells, also called satellite cells, have great potential for treating neuromuscular diseases and combating muscle aging. However, progress toward clinical use has been limited by the scarcity of donor tissue and the technical challenges of isolating, preserving, and culturing these rare cells. In this study, we introduce a new combination of cell surface markers and a gold nanoparticle-based live Pax7 mRNA detection system, both of which enable efficient isolation of human satellite cells. Additionally, we show that extracellular matrix (ECM) components support the maintenance of human satellite cells and improve their function in vivo. These findings provide integrated methods to enhance the production of human muscle-forming cells for translational and clinical applications.
Peripheral nerve injuries impose a substantial strain on the lives of millions of people worldwide. Engineered neural tissues provide a promising avenue to improve peripheral nerve repair strategies, potentially allowing the precise seeding of cells and materials that support regenerative processes. Current optimization of cell-seeding strategies relies on testing the impact of relevant parameters in vivo, requiring considerable time and resources. We propose an alternative approach to the design of cellular hydrogels based on a mathematical cell-solute model, informed via in vitro experiment, to identify promising cell-seeding strategies in silico at a limited cost. These designs are manufactured using 3D-printed moulds and validated in vivo. We evaluate the regenerative potential of these designs by focusing on the impact of different cell-seeding strategies on vascular endothelial growth factor secretion and gradient generation, both crucial elements of regenerative angiogenesis in early nerve repair. In this way, we provide a first proof-of-concept of a digital twin for nerve tissue engineering, which uses in silico, in vitro and in vivo repair models.
Neural stem cells (NSCs) span a continuum of cellular states that share fundamental properties with their differentiated glial progeny. Recent single-cell studies have refined our understanding of the diversity within both NSC and glial populations, revealing a highly dynamic and interconnected landscape of cell identities. In this review, we examine the glial nature of NSCs, emphasizing their heterogeneity and the stem- and progenitor-like properties shared with the differentiated glia. We focus particularly on astrocytes and integrate evidence from invertebrate models demonstrating that glial cells possess an intrinsic capacity for neurogenesis. Together, these findings highlight areas of convergence between astrocyte plasticity and NSC-associated properties, with important implications for nervous system regeneration and brain cancer.
Axon regeneration is limited in the mammalian central nervous system1. Neurons must balance stress responses with regenerative demands after axonal injury2, but the mechanisms remain unclear. Here we identify aryl hydrocarbon receptor (AhR), a ligand-activated basic helix-loop-helix/PER-ARNT-SIM (bHLH-PAS) transcription factor, as a key regulator of this stress-growth switch. We show that ligand-mediated AhR signalling restrains axon growth, whereas neuronal deletion or pharmacological inhibition of AhR promotes axonal regeneration and functional recovery in both peripheral nerve and spinal cord injury models. Mechanistic studies reveal that axotomy-induced AhR activation in dorsal root ganglion neurons enforces proteostasis and stress-response programs to preserve tissue integrity. By contrast, AhR ablation redirects the neuronal response towards elevated de novo translation and pro-growth signalling, enabling axon regeneration. This growth-promoting effect requires HIF1α, with shared transcriptional targets enriched for metabolic and regenerative pathways. Single-cell and epigenomic analyses further revealed that the AhR regulon engages the integrated stress response and DNA hydroxymethylation to rewire neuronal injury-response programs. Together, our findings establish AhR as a neuronal brake on axon regeneration, integrating environmental sensing, protein homeostasis and metabolic signalling to control the balance between stress adaptation and axonal repair.
Lgr5 marks both adult intestinal stem cells and embryonic intestinal stem/progenitor cells. However, the stemness properties and physiological roles of embryonic intestinal Lgr5⁺ cells prior to villification (PVLCs) remain largely unknown. In this study, we show that PVLCs in the embryonic small intestine exhibit region-specific stemness, with progressively enhanced stemness potential from the proximal to distal region. Through inducible cell ablation and gene knockout experiments, we demonstrate that PVLCs regulate small intestinal morphogenesis via Hedgehog signaling in a region-dependent manner, with distal morphogenesis being more dependent on this mechanism. This study reveals the stemness and functional roles of PVLCs in the embryonic small intestine prior to villification, highlighting regionalized cellular heterogeneity as a critical determinant of intestinal morphogenesis.
The notochord is a defining feature of chordates. It acts as mechanical support and a source of signals to surrounding tissues during development. In mammals, notochord-derived cells persist within intervertebral discs, where they form the nucleus pulposus, the cartilage in between vertebrae units that provides the spine with flexibility. Here, we synthesise developmental knowledge with recent advances in notochord biology and insights from single-cell molecular approaches. We discuss the developmental processes from notochord initiation during gastrulation through to disc formation, highlighting signalling pathways that govern axial mesoderm specification and notochordal lineage commitment. Knowledge gained from in vivo studies has guided the development of pluripotent stem cell-based models in mice and humans, including monolayer and micropatterned systems and 3D organoids. These models recapitulate key developmental aspects of notochord formation and pave the way for disease modelling and regenerative applications. We discuss their relevance to the study of developmental disorders arising from notochord dysfunction and notochordal cell roles in disc homeostasis. Finally, we outline remaining questions and examine how developmental insights and stem cell innovations can advance our understanding of tissue formation, function and homeostasis while fostering the integration of basic mechanistic insights with translational applications.