Combined analysis of single-cell sequencing and bulk transcriptome sequencing reveals new mechanisms for non-healing diabetic foot ulcers.

Diabetic foot ulcers (DFUs) pose a significant challenge in diabetes care. Yet, a comprehensive understanding of the underlying biological disparities between healing and non-healing DFUs remains elusive. We conducted bioinformatics analysis of publicly available transcriptome sequencing data in an attempt to elucidate these differences. Our analysis encompassed differential analysis to unveil shifts in cell composition and gene expression profiles between non-healing and healing DFUs. Cell communication alterations were explored employing the Cellchat R package. Pseudotime analysis and cytoTRACE allowed us to dissect the heterogeneity within fibroblast subpopulations. Our findings unveiled disruptions in various cell types, localized low-grade inflammation, compromised systemic antigen processing and presentation, and extensive extracellular matrix signaling disarray in non-healing DFU patients. Some of these anomalies partially reverted in healing DFUs, particularly within the abnormal ECM-receptor signaling pathway. Furthermore, we distinguished distinct fibroblast subpopulations in non-healing and healing DFUs, each with unique biological functions. Healing-associated fibroblasts exhibited heightened extracellular matrix (ECM) remodeling and a robust wound healing response, while non-healing-associated fibroblasts showed signs of cellular senescence and complement activation, among other characteristics. This analysis offers profound insights into the wound healing microenvironment, identifies pivotal cell types for DFU healing promotion, and reveals potential therapeutic targets for DFU management.

Extracellular matrix remodelling pathway in peripheral blood mononuclear cells from severe COVID-19 patients: an explorative study.

There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.

Vesicle transport of matrix metalloproteinases.

Multicellular organisms consist of cells and extracellular matrix (ECM). ECM creates a cellular microenvironment, and cells locally degrade the ECM according to their cellular activity. A major group of enzymes that modify ECM belongs to matrix metalloproteinases (MMPs) and play major roles in various pathophysiological events. ECM degradation by MMPs does not occur in all cellular surroundings but only where it is necessary, and cells achieve this by directionally secreting these proteolytic enzymes. Recent studies have indicated that such enzyme secretion is achieved by targeted vesicle transport along the microtubules, and several kinesin superfamily proteins (KIFs) have been identified as responsible motor proteins involved in the processes. This chapter discusses recent findings of the vesicle transport of MMPs and their roles.

Integrating hydrogels manipulate ECM deposition after spinal cord injury for specific neural reconnections via neuronal relays.

A bioinspired hydrogel composed of hyaluronic acid-graft-dopamine (HADA) and a designer peptide HGF-(RADA)4-DGDRGDS (HRR) was presented to enhance tissue integration following spinal cord injury (SCI). The HADA/HRR hydrogel manipulated the infiltration of PDGFRß+ cells in a parallel pattern, transforming dense scars into an aligned fibrous substrate that guided axonal regrowth. Further incorporation of NT3 and curcumin promoted axonal regrowth and survival of interneurons at lesion borders, which served as relays for establishing heterogeneous axon connections in a target-specific manner. Notable improvements in motor, sensory, and bladder functions resulted in rats with complete spinal cord transection. The HADA/HRR + NT3/Cur hydrogel promoted V2a neuron accumulation in ventral spinal cord, facilitating the recovery of locomotor function. Meanwhile, the establishment of heterogeneous neural connections across the hemisected lesion of canines was documented in a target-specific manner via neuronal relays, significantly improving motor functions. Therefore, biomaterials can inspire beneficial biological activities for SCI repair.

Protective Effect of Nicotinamide Riboside on Glucocorticoid-Induced Glaucoma: Mitigating Mitochondrial Damage and Extracellular Matrix Deposition.

Purpose: Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG. Methods: Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed. Results: NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo. Conclusions: Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.

More Velcro for the TGF-ß1 straitjacket: A new antibody straps latent TGF-ß1 to the matrix.

In this issue of Science Signaling, Jackson et al. present a new antibody strategy to-quite literally-strap transforming growth factor-ß1 (TGF-ß1) to latent complexes in the extracellular matrix. The antibody has no effect on latent TGF-ß1 presented on the surface of immune cells and thus allows targeting of the detrimental effects of TGF-ß1 in fibrosis without affecting its beneficial immune-suppressing activities.

An antibody that inhibits TGF-ß1 release from latent extracellular matrix complexes attenuates the progression of renal fibrosis.

Inhibitors of the transforming growth factor-ß (TGF-ß) pathway are potentially promising antifibrotic therapies, but nonselective simultaneous inhibition of all three TGF-ß homologs has safety liabilities. TGF-ß1 is noncovalently bound to a latency-associated peptide that is, in turn, covalently bound to different presenting molecules within large latent complexes. The latent TGF-ß-binding proteins (LTBPs) present TGF-ß1 in the extracellular matrix, and TGF-ß1 is presented on immune cells by two transmembrane proteins, glycoprotein A repetitions predominant (GARP) and leucine-rich repeat protein 33 (LRRC33). Here, we describe LTBP-49247, an antibody that selectively bound to and inhibited the activation of TGF-ß1 presented by LTBPs but did not bind to TGF-ß1 presented by GARP or LRRC33. Structural studies demonstrated that LTBP-49247 recognized an epitope on LTBP-presented TGF-ß1 that is not accessible on GARP- or LRRC33-presented TGF-ß1, explaining the antibody's selectivity for LTBP-complexed TGF-ß1. In two rodent models of kidney fibrosis of different etiologies, LTBP-49247 attenuated fibrotic progression, indicating the central role of LTBP-presented TGF-ß1 in renal fibrosis. In mice, LTBP-49247 did not have the toxic effects associated with less selective TGF-ß inhibitors. These results establish the feasibility of selectively targeting LTBP-bound TGF-ß1 as an approach for treating fibrosis.

Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

ECM Microenvironment in Vascular Homeostasis: New Targets for Atherosclerosis.

Alterations in vascular extracellular matrix (ECM) components, interactions, and mechanical properties influence both the formation and stability of atherosclerotic plaques. This review discusses the contribution of the ECM microenvironment in vascular homeostasis and remodeling in atherosclerosis, highlighting Cartilage oligomeric matrix protein (COMP) and its degrading enzyme ADAMTS7 as examples, and proposes potential avenues for future research aimed at identifying novel therapeutic targets for atherosclerosis based on the ECM microenvironment.

[Formation, organization and function of invadosomes in cell motility and tumor invasion]. / Invadosomes - Entre mobilité et invasion, naviguer dans la dualité des fonctions cellulaires.

Invadosome is an umbrella term used to describe a family of cellular structures including podosomes and invadopodia. They serve as contact zones between the cell plasma membrane and extracellular matrix, contributing to matrix remodeling by locally enriched proteolytic enzymes. Invadosomes, which are actin-dependent, are implicated in cellular processes promoting adhesion, migration, and invasion. Invadosomes, which exist in various cell types, play crucial roles in physiological phenomena such as vascularization and bone resorption. Invadosomes are also implicated in pathological processes such as matrix tissue remodeling during metastatic tumor cell invasion. This review summarizes basic information and recent advances about mechanisms underlying podosome and invadopodia formation, their organization and function.

Title: Invadosomes - Entre mobilité et invasion, naviguer dans la dualité des fonctions cellulaires. Abstract: Le terme « invadosome ¼ désigne une famille de structures cellulaires, comprenant les podosomes et les invadopodes, qui constituent des zones de contact entre la membrane plasmique des cellules et la matrice extracellulaire. Ces structures contribuent au remodelage de la matrice grâce à un enrichissement local en enzymes protéolytiques qui dégradent ses constituants fibrillaires. Les invadosomes, présents dans des types cellulaires variés, contribuent à des processus physiologiques, tels que la vascularisation, ou pathologiques, comme l'invasion des tissus par les cellules métastatiques.

Alterations in the Structure, Composition, and Organization of Galactosaminoglycan-Containing Proteoglycans and Collagen Correspond to the Progressive Stages of Dupuytren's Disease.

Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.

Breast Cancer: Extracellular Matrix and Microbiome Interactions.

Breast cancer represents the most prevalent form of cancer and the leading cause of cancer-related mortality among females worldwide. It has been reported that several risk factors contribute to the appearance and progression of this disease. Despite the advancements in breast cancer treatment, a significant portion of patients with distant metastases still experiences no cure. The extracellular matrix represents a potential target for enhanced serum biomarkers in breast cancer. Furthermore, extracellular matrix degradation and epithelial-mesenchymal transition constitute the primary stages of local invasion during tumorigenesis. Additionally, the microbiome has a potential influence on diverse physiological processes. It is emerging that microbial dysbiosis is a significant element in the development and progression of various cancers, including breast cancer. Thus, a better understanding of extracellular matrix and microbiome interactions could provide novel alternatives to breast cancer treatment and management. In this review, we summarize the current evidence regarding the intricate relationship between breast cancer with the extracellular matrix and the microbiome. We discuss the arising associations and future perspectives in this field.

[Preparation and biological characteristics of extracellular matrix vesicle mimetics].

Objective: To investigate the characteristics of extracellular matrix vesicle mimetics prepared by mechanical extrusion and their effects on the cell viability and osteogenic differentiation potential of human periodontal ligament stem cells (PDLSC). Methods: PDLSC derived extracellular matrix vesicles were prepared by collagenase digestion, while the cell derived vesicle mimetics were simulated by mechanical extrusion. The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. MVs, O-MVs, CVMs and O-CVMs do not affect the cell viability of PDLSC, and can promote the osteogenic differentiation potential of PDLSC to a certain extent.

Diffusion-tensor magnetic resonance imaging as a non-invasive assessment of extracellular matrix remodeling in lumbar paravertebral muscles of rats with sarcopenia.

BACKGROUND: Extracellular matrix (ECM) remodeling in skeletal muscle is a significant factor in the development of sarcopenia. This study aims to evaluate changes in ECM remodeling in the lumbar paravertebral muscles of sarcopenic rats using diffusion-tensor magnetic resonance imaging (DT-MRI) and compare them with histology. METHODS: Twenty 6-month-old female Sprague Dawley rats were randomly divided into the dexamethasone (DEX) group and the control (CON) group. Both groups underwent 3.0T MRI scanning, including Mensa, T2WI, and DT-MRI sequences. The changes in muscle fibers and extracellular matrix (ECM) of the erector spinal muscle were observed using hematoxylineosin and sirius red staining. The expressions of collagen I, III, and fibronectin in the erector spinae were detected by western blot. Pearson correlation analysis was employed to assess the correlation between MRI quantitative parameters and corresponding histopathology markers. RESULTS: The cross-sectional area and fractional anisotropy values of the erector spinae in the DEX group rats were significantly lower than those in the CON group (p < 0.05). Hematoxylin eosin staining revealed muscle fiber atrophy and disordered arrangement in the DEX group, while sirius red staining showed a significant increase in collagen volume fraction in the DEX group. The western blot results indicate a significant increase in the expression of collagen I, collagen III, and fibronectin in the DEX group (p < 0.001 for all). Correlation coefficients between fractional anisotropy values and collagen volume fraction, collagen I, collagen III, and fibronectin were - 0.71, -0.94, -0.85, and - 0.88, respectively (p < 0.05 for all). CONCLUSIONS: The fractional anisotropy value is strongly correlated with the pathological collagen volume fraction, collagen I, collagen III, and fibronectin. This indicates that DT-MRI can non-invasively evaluate the changes in extracellular matrix remodeling in the erector spinal muscle of sarcopenia. It provides a potential imaging biomarker for the diagnosis of sarcopenia.

Timut Pepper Extract Slows Age-Dependent Decline of Mobility and Collagen Loss and Promotes Longevity.

Investigations into human longevity are increasingly focusing on healthspan enhancement, not just lifespan extension. Lifestyle modifications and nutritional choices, including food supplements, can significantly affect aging and general health. Phytochemicals in centenarians' diets, such as those found in Timut pepper, a Nepalese spice with various medicinal properties, may contribute to their longevity. Similarly, Sichuan pepper, a related species, has demonstrated anti-inflammatory and neuroprotective activities. With the broader purpose of uncovering a novel treatment to address aging and its comorbidities, this study aims to investigate the potential lifespan- and healthspan-promoting effects of Timut pepper using the model organism Caenorhabditis elegans. We show that Timut pepper extract extends C. elegans' lifespan at different maintenance temperatures and increases the proportion of active nematodes in their early adulthood. In addition, we show that Timut pepper extract enhances speed and distance moved as the nematodes age. Finally, Timut pepper extract assures extracellular matrix homeostasis by slowing the age-dependent decline of collagen expression.

Compressive instabilities enable cell-induced extreme densification patterns in the fibrous extracellular matrix: Discrete model predictions.

We present a new model and extensive computations that explain the dramatic remodelling undergone by a fibrous collagen extracellular matrix (ECM), when subjected to contractile mechanical forces from embedded cells or cell clusters. This remodelling creates complex patterns, comprising multiple narrow localised bands of severe densification and fiber alignment, extending far into the ECM, often joining distant cells or cell clusters (such as tumours). Most previous models cannot capture this behaviour, as they assume stable mechanical fiber response with stress an increasing function of fiber stretch, and a restriction to small displacements. Our fully nonlinear network model distinguishes between two types of single-fiber nonlinearity: fibers that undergo stable (supercritical) buckling (as in previous work) versus fibers that suffer unstable (subcritical) buckling collapse. The model allows unrestricted, arbitrarily large displacements (geometric nonlinearity). Our assumptions on single-fiber instability are supported by recent simulations and experiments on buckling of individual beams with a hierarchical microstructure, such as collagen fibers. We use simple scenarios to illustrate, for the first time, two distinct compressive-instability mechanisms at work in our model: unstable buckling collapse of single fibers, and snap-through of multiple-fiber groups. The latter is possible even when single fibers are stable. Through simulations of large fiber networks, we show how these instabilities lead to spatially extended patterns of densification, fiber alignment and ECM remodelling induced by cell contraction. Our model is simple, but describes a very complex, multi-stable energy landscape, using sophisticated numerical optimisation methods that overcome the difficulties caused by instabilities in large systems. Our work opens up new ways of understanding the unique biomechanics of fibrous-network ECM, by fully accounting for nonlinearity and associated loss of stability in fiber networks. Our results provide new insights on tumour invasion and metastasis.

The cysteine-rich virulence factor NipA of Arthrobotrys flagrans interferes with cuticle integrity of Caenorhabditis elegans.

Animals protect themself from microbial attacks by robust skins or a cuticle as in Caenorhabditis elegans. Nematode-trapping fungi, like Arthrobotrys flagrans, overcome the cuticle barrier and colonize the nematode body. While lytic enzymes are important for infection, small-secreted proteins (SSPs) without enzymatic activity, emerge as crucial virulence factors. Here, we characterized NipA (nematode induced protein) which A. flagrans secretes at the penetration site. In the absence of NipA, A. flagrans required more time to penetrate C. elegans. Heterologous expression of the fungal protein in the epidermis of C. elegans led to blister formation. NipA contains 13 cysteines, 12 of which are likely to form disulfide bridges, and the remaining cysteine was crucial for blister formation. We hypothesize that NipA interferes with cuticle integrity to facilitate fungal entry. Genome-wide expression analyses of C. elegans expressing NipA revealed mis-regulation of genes associated with extracellular matrix (ECM) maintenance and innate immunity.

Human amniotic membrane modulates collagen production and deposition in vitro.

Pathological fibrosis is a significant complication of surgical procedures resulting from the accumulation of excess collagen at the site of repair which can compromise the tissue architecture and severely impede the function of the affected tissue. Few prophylactic treatments exist to counteract this process; however, the use of amniotic membrane allografts has demonstrated promising clinical outcomes. This study aimed to identify the underlying mechanism of action by utilizing relevant models that accurately represent the pathophysiology of the disease state. This study employed a pro-fibrotic in vitro system using TGFß1 stimulation and macromolecular crowding techniques to evaluate the mechanism by which amniotic membrane allografts regulate collagen biosynthesis and deposition. Following treatment with dehydrated human amnion chorion membrane (DHACM), subsequent RNA sequencing and functional enrichment with Reactome pathway analysis indicated that amniotic membranes are indeed capable of regulating genes associated with the composition and function of the extracellular matrix. Furthermore, macromolecular crowding was used in vitro to expand the evaluation to include both the effects of DHACM and a lyophilized human amnion/chorion membrane (LHACM). DHACM and LHACM regulate the TGFß pathway and myofibroblast differentiation. Additionally, both DHACM and LHACM modulate the production, secretion, and deposition of collagen type I, a primary target for pathological fibrosis. These observations support the hypothesis that amniotic membranes may interrupt pathological fibrosis by regulating collagen biosynthesis and associated pathways.

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.