Skeletal Stem Cells Research Articles

Abstract 14625: Clinical Outcomes of Autologous Stem Cell Patch Implantation for Heart Failure Patients With Non-ischemic Dilated Cardiomyopathy

Background: Although pre-clinical researches proved that cardiac regenerative therapy with autologous skeletal stem cell (SSC) patch implantation healed non-ischemic dilated cardiomyopathy (NIDCM) animal model by anti-fibrotic effect and preservation of cytoskeletal proteins in distressed myocytes, its clinical effectiveness for NIDCM has not been clearly elucidated. We hypotheses SSC patch implantation for NIDCM is feasible, safe and have therapeutic efficacy and we aim to identify the predictor of responder of this treatment in NIDCM patients Methods: Twenty-four NIDCM patients with ejection fraction less than 35% underwent SSC patch transplantation (average: 3.9±0.4х10 8 cells) via the left thoracotomy without concomitant procedures. We identified thirteen responders (Group RP) and eleven non-responders (Group NR) using the combined indicator of major cardiac adverse event and incidence of heart failure event. Results: In Group RP, symptoms, exercise capacity and cardiac performance were improved postoperatively (NYHA functional class; 2.5±0.5 to 1.9±0.3, p<0.05, six-minute walk test; 471(370-541) to 525(425-555)m, p<0.05, LVSWI; 31.1(22.7-35.5) to 32.8(28-38.5)g/m 2 /beat, p=0.21). However, such improvement was not observed in Group NR. Overall survival rate was 90.9±8.7% at 5 years, which was superior to estimated survival rate using Seattle heart failure model (SHFM) of 70.9±5.4% in Group RP. However overall and estimated survival rate using SHFM was similar in Group NR (47.7±21.6% and 56.3±8.1%). Preoperative histological analysis demonstrated significant low level of hypertrophy of myocyte and interstitial fibrosis and high expression of histone methylation-related molecules in Group RP compared with Group NR. Multivariate regression model with BNP, PCWP and expression of histone H3 lysine 4 trimethylation (H3K4me3) strongly predicted the responder (BNP: OR 0.96, PCWP: OR, 0.58, H3K4me3; OR, 1.35, AUC in ROC, 0.96, p<0.001). Conclusions: This clinical trial demonstrated that SSC patch implantation might promise functional recovery and good clinical outcome in selected NIDCM patients with preoperatively preserved diastolic function and ability of protein synthesis in myocytes.

Advantages and Limitations of Cre Mouse Lines Used in Skeletal Research.

The Cre-LoxP technology permits gene ablation in specific cell lineages, at chosen differentiation stages of this lineage and in an inducible manner. It has allowed tremendous advances in our understanding of skeleton biology and related pathophysiological mechanisms, through the generation of loss/gain of function or cell tracing experiments based on the creation of an expanding toolbox of transgenic mice expressing the Cre recombinase in skeletal stem cells, chondrocytes, osteoblasts, or osteoclasts. In this chapter, we provide an overview of the different Cre-LoxP systems and Cre mouse lines used in the bone field, we discuss their advantages, limitations, and we outline best practices to interpret results obtained from the use of Cre mice.

Bone Marrow Stromal Cell Assays: In Vitro and In Vivo.

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). It is now apparent that cells with similar but not identical properties can be isolated from other skeletal compartments (growth plate, periosteum). The biological properties of BMSCs, and these related stem/progenitor cells, are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provide a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease.

Abstract IA-17: Mechanisms of pancreatic cancer-induced cachexia

Abstract Cachexia is a debilitating syndrome that results in severe, involuntary weight loss due to the depletion of skeletal muscle mass. Currently, no effective therapy exists to combat this malignant disorder. For pancreatic cancer, 85% of patients lose on average 14% of their pre-illness weight, and cachexia dramatically limits their ability to tolerate surgery, chemo- or radiotherapy. New therapies will likely evolve from an enhanced understanding of the mechanisms leading to muscle wasting and tumor development. Our laboratory has been examining the role of the NF-κB signaling pathway in tumorigenesis for several decades and that interest led us to discover the connection between NF-κB and muscle wasting in cancer cachexia and more recently in pancreatic cancer. We view the pathway as playing two separate functions in pancreatic cancer-induced cachexia. The first occurs at the level of skeletal muscle, or more preciously in skeletal muscle stem cells. We have found that during cancer progression, skeletal muscle undergoes a type of injury response leading to the activation of resident stem cells to engage in a regeneration program. NF-κB is activated in these stem cells and functions to inhibit regeneration, which contributes to the overall wasting process in cachexia. New data reveal that NF-κB activity in progenitor cells also regulates a local muscle inflammatory environment that might also contribute to skeletal muscle catabolism. The mechanism of this regulation will be discussed in more details. The second function of NF-κB signaling that we are pursuing focuses in the tumor microenvironment of pancreatic cancer. Using orthotopic mouse models of pancreatic cancer, we showed that NF-κB plays a critical role in protecting tumor cells from the surveillance property of anti-tumor macrophages. This occurs through the direct transcriptional regulation of the immunosuppressive cytokine, GDF15. Interestingly, circulating levels of GDF15 are elevated in cachectic patients and recent studies indicate that this cytokine might be an attractive therapeutic target in cancer cachexia. Together, we speculate that NF-κB functions in cancer cachexia by acting in muscle stem cells to block muscle repair, as well as promoting pancreatic cancer through the production of immunosuppressive genes such as GDF15. Citation Format: Benjamin Pryce, Nivedita Ratnam, Erin E. Talbert, Mary Dilhoff, Carl R. Schmidt, David J. Wang, Denis C. Guttridge. Mechanisms of pancreatic cancer-induced cachexia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-17.

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis

ObjectivesOsteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage,...

Membrane-type 1 Matrix Metalloproteinase Modulates Tissue Homeostasis by a Non-proteolytic Mechanism

SummaryMembrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14−/−) and man causes dwarfism, osteopenia, arthritis, and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP-2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here, we report that a mouse with the MT1-MMP Y573D mutation (Mmp14Y573D/Y573D) shows abnormalities similar to but also different from those of Mmp14−/− mice. Skeletal stem cells (SSC) of Mmp14Y573D/Y573D mice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the first in vivo demonstration that MT1-MMP modulates bone, cartilage, and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis.

Bone regeneration via skeletal cell lineage plasticity: All hands mobilized for emergencies

An emerging concept is that quiescent mature skeletal cells provide an important cellular source for bone regeneration. It has long been considered that a small number of resident skeletal stem cells are solely responsible for the remarkable regenerative capacity of adult bones. However, recent in vivo lineage-tracing studies suggest that all stages of skeletal lineage cells, including dormant pre-adipocyte-like stromal cells in the marrow, osteoblast precursor cells on the bone surface and other stem and progenitor cells, are concomitantly recruited to the injury site and collectively participate in regeneration of the damaged skeletal structure. Lineage plasticity appears to play an important role in this process, by which mature skeletal cells can transform their identities into skeletal stem cell-like cells in response to injury. These highly malleable, long-living mature skeletal cells, readily available throughout postnatal life, might represent an ideal cellular resource that can be exploited for regenerative medicine.

Skeletal Stem Cells-A Paradigm Shift in the Field of Craniofacial Bone Tissue Engineering.

Defects of the craniofacial skeleton arise as a direct result of trauma, diseases, oncological resection, or congenital anomalies. Current treatment options are limited, highlighting the importance for developing new strategies to restore form, function, and aesthetics of missing or damaged bone in the face and the cranium. For optimal reconstruction, the goal is to replace “like with like.” With the inherent challenges of existing options, there is a clear need to develop alternative strategies to reconstruct the craniofacial skeleton. The success of mesenchymal stem cell-based approaches has been hampered by high heterogeneity of transplanted cell populations with inconsistent preclinical and clinical trial outcomes. Here, we discuss the novel characterization and isolation of mouse skeletal stem cell (SSC) populations and their response to injury, systemic disease, and how their re-activation in vivo can contribute to tissue regeneration. These studies led to the characterization of human SSCs which are able to self-renew, give rise to increasingly fate restricted progenitors, and differentiate into bone, cartilage, and bone marrow stroma, all on the clonal level in vivo without prior in vitro culture. SSCs hold great potential for implementation in craniofacial bone tissue engineering and regenerative medicine. As we begin to better understand the diversity and the nature of skeletal stem and progenitor cells, there is a tangible future whereby a subset of human adult SSCs can be readily purified from bone or activated in situ with broad potential applications in craniofacial tissue engineering.

Radiation-Induced Damage to Prepubertal Pax7+ Skeletal Muscle Stem Cells Drives Lifelong Deficits in Myofiber Size and Nuclear Number.

SummaryDuring prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.

Extracellular serine and glycine are required for mouse and human skeletal muscle stem and progenitor cell function

ObjectiveSkeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. MethodsPrimary human MPCs (hMPCs) were used for in vitro experiments, and young (4–6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. ResultsWe identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. ConclusionsThese studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration.

New insights on the reparative cells in bone regeneration and repair.

Bone possesses a remarkable repair capacity to regenerate completely without scar tissue formation. This unique characteristic, expressed during bone development, maintenance and injury (fracture) healing, is performed by the reparative cells including skeletal stem cells (SSCs) and their descendants. However, the identity and functional roles of SSCs remain controversial due to technological difficulties and the heterogeneity and plasticity of SSCs. Moreover, for many years, there has been a biased view that bone marrow is the main cell source for bone repair. Together, these limitations have greatly hampered our understanding of these important cell populations and their potential applications in the treatment of fractures and skeletal diseases. Here, we reanalyse and summarize current understanding of the reparative cells in bone regeneration and repair and outline recent progress in this area, with a particular emphasis on the temporal and spatial process of fracture healing, the sources of reparative cells, an updated definition of SSCs, and markers of skeletal stem/progenitor cells contributing to the repair of craniofacial and long bones, as well as the debate between SSCs and pericytes. Finally, we also discuss the existing problems, emerging novel technologies and future research directions in this field.

Markers for Identification of Postnatal Skeletal Stem Cells In Vivo.

The adult skeleton contains stem cells involved in growth, homeostasis, and healing. Mesenchymal or skeletal stem cells are proposed to provide precursors to osteoblasts, chondrocytes, marrow adipocytes, and stromal cells. We review the evidence for existence and functionality of different skeletal stem cell pools, and the tools available for identifying or targeting these populations in mouse and human tissues. Lineage tracing and single cell-based techniques in mouse models indicate that multiple pools of stem cells exist in postnatal bone. These include growth plate stem cells, stem and progenitor cells in the diaphysis, reticular cells that only form bone in response to injury, and injury-responsive periosteal stem cells. New staining protocols have also been described for prospective isolation of human skeletal stem cells. Several populations of postnatal skeletal stem and progenitor cells have been identified in mice, and we have an increasing array of tools to target these cells. Most Cre models lack a high degree of specificity to define single populations. Human studies are less advanced and require further efforts to refine methods for identifying stem and progenitor cells in adult bone.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Sirtuin 1 deficiency decreases bone mass and increases bone marrow adiposity in a mouse model of chronic energy deficiency

Sirtuin of type 1 (Sirt1), a class III HDAC, is known to be involved in the regulation of differentiation of skeletal stem cells (SSCs) into osteoblasts and adipocytes. In caloric restriction, it has been shown that the expression and activity of Sirt1 is a tissue-dependent regulation. However, at present, no study has focused on the link between Sirt1, bone marrow adiposity (BMA) and osteoporosis related to anorexia nervosa (AN). Thus, the aims of this work were to (i) determine BMA and bone changes in a mouse model replicating the phenotypes of AN (separation-based anorexia model (SBA)); (ii) determine the expression of Sirt1 in bone marrow stromal cells (BMSCs) extracted from these mice and identify their differentiation capacities; (iii) study the effects of pharmacological activation and inhibition of Sirt1 on the osteoblastogenesis and adipogenesis of these cells and (iiii) delineate the molecular mechanism by which Sirt1 could regulate osteogenesis in an SBA model. Our results demonstrated that SBA protocol induces an increase in BMA and alteration of bone architecture. In addition, BMSCs from restricted mice present a down-regulation of Sirt1 which is accompanied by an increase in adipogenesis at expense of osteogenesis. After a 10-day organotypic culture, tibias from SBA mice displayed low levels of Sirt1 mRNA which are restored by resveratrol treatment. Interestingly, this recovery of Sirt1 levels also returned the BMA, BV/TV and Tb.Th in cultured tibias from SBA mice to normal levels. In contrast of down-regulation of Sirt1 expression induced by sirtinol treatment, stimulation of Sirt1 expression by resveratrol lead to a decrease in adipogenesis and increase in osteogenesis. Finally, to investigate the molecular mechanisms by which Sirt1 could regulate osteogenesis in the SBA model, the acetylation levels of Runx2 and Foxo1 transcription factors were determined. Our data show that this chronic energy deficiency in female mice causes a decrease in BMSC activity, resulting in critical changes to Runx2 and Foxo1 acetylation levels and thus to their activity. Altogether, these data suggest that Sirt1 could be considered as a potential therapeutic target in osteoporosis related to AN.

αE-catenin deletion in skeletal stem and progenitor cells (SSPCs) increases their adipogenic potential and protects against diet- or age-induced obesity and hyperglycemia

αE-catenin deletion in skeletal stem and progenitor cells (SSPCs) increases their adipogenic potential and protects against diet- or age-induced obesity and hyperglycemia

RhoA regulates the quiescence and cell fate of skeletal stem and progenitor cells (SSPCs)

RhoA regulates the quiescence and cell fate of skeletal stem and progenitor cells (SSPCs)

Chondrobags: A high throughput alginate-fibronectin micromass platform for in vitro human cartilage formation

The maintenance and expansion of the cells required for formation of tissue-engineered cartilage has, to date, proven difficult. This is, in part, due to the initial solid phase extracellular matrix demanded by the cells inhabiting this avascular tissue. Herein, we engineer an innovative alginate-fibronectin microfluidic-based carrier construct (termed a chondrobag) equipped with solid phase presentation of growth factors that support skeletal stem cell chondrogenic differentiation while preserving human articular chondrocyte phenotype. Results demonstrate biocompatibility, cell viability, proliferation and tissue-specific differentiation for chondrogenic markers SOX9, COL2A1 and ACAN. Modulation of chondrogenic cell hypertrophy, following culture within chondrobags loaded with TGF-β1, was confirmed by down-regulation of hypertrophic genes COL10A1 and MMP13. MicroRNAs involved in the chondrogenesis process, including miR-140, miR-146b and miR-138 were observed. Results demonstrate the generation of a novel high-throughput, microfluidic-based, scalable carrier that supports human chondrogenesis with significant implications therein for cartilage repair-based therapies.

A single bout of high-intensity exercise modulates the expression of vitamin D receptor and vitamin D-metabolising enzymes in horse skeletal muscle.

The expressions of vitamin D receptor (VDR) and vitamin D-metabolising enzymes (CYP27B1 and CYP24A1) in skeletal muscle have been reported. However, the regulation of this vitamin D system in horse skeletal muscle after high-intensity exercise has not yet been elucidated. To investigate the effect of high-intensity exercise on the expression of vitamin D system-related proteins in horse skeletal muscle and its associations with skeletal muscle stem cell (SMSC) activity and serum 25(OH)D level. Longitudinal study. Six healthy ponies (5 geldings, 1 mare; age 6.3±2.2years) were studied. Serum and muscle samples were taken from the jugular vein and gluteus medius respectively. Samples were collected at pre-exercise, post-exercise, 1 and 3weeks after a single bout of high-intensity exercise. Protein expression levels of VDR, CYP27B1, CYP24A1, OxPhos and Pax7 (SMSC marker) were determined using immunohistochemical analysis. Oxidative capacity and intramuscular glycogen content were evaluated using histochemical analysis. Blood biochemistry was analysed for lactate concentration and creatine kinase (CK), and 25(OH)D activity. High-intensity exercise significantly upregulated Pax7 and VDR protein expression, which correlated with significantly increased blood lactate and serum CK levels immediately post-exercise. Serum 25(OH)D2 level correlated with CYP27B1 protein expression in skeletal muscle, and it reduced significantly immediately post-exercise and at 1 and 3weeks post-exercise. However, CYP24A1 protein expression was unchanged throughout study periods. The healthy ponies could not represent a fit population of racehorses and eventers. The rapid increase in Pax7 and VDR protein expression along with serum CK level after high-intensity exercise demonstrated an association between SMSC activity and activation of the vitamin D system in response to muscle injury in horses. Moreover, a decrease in CYP27B1 protein expression, correlated with a reduction in serum 25(OH)D2 , may indicate a compromised vitamin D metabolism after high-intensity exercise.

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells.

Background: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Aims: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. Methods: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3–R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. Results: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. Conclusions: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Wnt-modified materials mediate asymmetric stem cell division to direct human osteogenic tissue formation for bone repair.

The maintenance of human skeletal stem cells (hSSCs) and their progeny in bone defects is a major challenge. Here, we report on a transplantable bandage containing a three-dimensional Wnt-induced osteogenic tissue model (WIOTM). This bandage facilitates the long-term viability of hSSCs (8 weeks) and their progeny, and enables bone repair in an in vivo mouse model of critical-sized calvarial defects. The newly forming bone is structurally comparable to mature cortical bone and consists of human and murine cells. Furthermore, we show that the mechanism of WIOTM formation is governed by Wnt-mediated asymmetric cell division of hSSCs. Covalently immobilizing Wnts onto synthetic materials can polarize single dividing hSSCs, orient the spindle and simultaneously generate a Wnt-proximal hSSC and a differentiation-prone Wnt-distal cell. Our results provide insight into the regulation of human osteogenesis and represent a promising approach to deliver human osteogenic constructs that can survive in vivo and contribute to bone repair.