Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

An immunologically active, adipose-derived extracellular matrix biomaterial for soft tissue reconstruction: concept to clinical trial

Soft tissue reconstruction remains an intractable clinical challenge as current surgical options and synthetic implants may produce inadequate outcomes. Soft tissue deficits may be surgically reconstructed using autologous adipose tissue, but these procedures can lead to donor site morbidity, require multiple procedures, and have highly variable outcomes. To address this clinical need, we developed an “off-the-shelf” adipose extracellular matrix (ECM) biomaterial from allograft human tissue (Acellular Adipose Tissue, AAT). We applied physical and chemical processing methods to remove lipids and create an injectable matrix that mimicked the properties of lipoaspirate. Biological activity was assessed using cell migration and adipogenesis assays. Characterization of regenerative immune properties in a murine muscle injury model revealed that allograft and xenograft AAT induced pro-regenerative CD4+ T cells and macrophages with xenograft AAT additionally attracting eosinophils secreting interleukin 4 (Il4). In immunocompromised mice, AAT injections retained similar volumes as human fat grafts but lacked cysts and calcifications seen in the fat grafts. The combination of AAT with human adipose-derived stem cells (ASCs) resulted in lower implant volumes. However, tissue remodeling and adipogenesis increased significantly in combination with ASCs. Larger injected volumes of porcine-derived AAT demonstrated biocompatibility and greater retention when applied allogeneicly in Yorkshire cross pigs. AAT was implanted in healthy volunteers in abdominal tissue that was later removed by elective procedures. AAT implants were well tolerated in all human subjects. Implants removed between 1 and 18 weeks demonstrated increasing cellular infiltration and immune populations, suggesting continued tissue remodeling and the potential for long-term tissue replacement.

Sonic Hedgehog acts as a macrophage chemoattractant during regeneration of the gastric epithelium

Sonic Hedgehog (Shh), secreted from gastric parietal cells, contributes to the regeneration of the epithelium. The recruitment of macrophages plays a central role in the regenerative process. The mechanism that regulates macrophage recruitment in response to gastric injury is largely unknown. Here we tested the hypothesis that Shh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMcre/+;Smof/f) was generated using transgenic mice bearing loxP sites flanking the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and LysMcre/+;Smof/f (SmoKO) mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post-injury. Bone marrow-derived macrophages (BM-Mø) were collected from control and SmoKO mice. Human-derived gastric organoid/macrophage co-cultures were established, and macrophage chemotaxis measured. Compared to control mice, SmoKO animals exhibited inhibition of ulcer repair and normal epithelial regeneration, which correlated with decreased macrophage infiltration at the site of injury. Bone marrow chimera experiments using SmoKO donor cells showed that control chimera mice transplanted with SmoKO bone marrow donor cells exhibited a loss of ulcer repair, and transplantation of control bone marrow donor cells to SmoKO mice rescued epithelial cell regeneration. Histamine-stimulated Shh secretion in human organoid/macrophage co-cultures resulted in macrophage migration toward the gastric epithelium, a response that was blocked with Smo inhibitor Vismodegib. Shh-induced macrophage migration was mediated by AKT signaling. In conclusion, Shh signaling acts as a macrophage chemoattractant via a Smo-dependent mechanism during gastric epithelial regeneration in response to injury.

The clinical potential of articular cartilage-derived progenitor cells: a systematic review

Over the past two decades, evidence has emerged for the existence of a distinct population of endogenous progenitor cells in adult articular cartilage, predominantly referred to as articular cartilage-derived progenitor cells (ACPCs). This progenitor population can be isolated from articular cartilage of a broad range of species, including human, equine, and bovine cartilage. In vitro, ACPCs possess mesenchymal stromal cell (MSC)-like characteristics, such as colony forming potential, extensive proliferation, and multilineage potential. Contrary to bone marrow-derived MSCs, ACPCs exhibit no signs of hypertrophic differentiation and therefore hold potential for cartilage repair. As no unique cell marker or marker set has been established to specifically identify ACPCs, isolation and characterization protocols vary greatly. This systematic review summarizes the state-of-the-art research on this promising cell type for use in cartilage repair therapies. It provides an overview of the available literature on endogenous progenitor cells in adult articular cartilage and specifically compares identification of these cell populations in healthy and osteoarthritic (OA) cartilage, isolation procedures, in vitro characterization, and advantages over other cell types used for cartilage repair. The methods for the systematic review were prospectively registered in PROSPERO (CRD42020184775).

Esophageal regeneration following surgical implantation of a tissue engineered esophageal implant in a pediatric model

Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an “in-life” monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.

A bioinspired gelatin-hyaluronic acid-based hybrid interpenetrating network for the enhancement of retinal ganglion cells replacement therapy

Biomaterial-based cell replacement approaches to regenerative medicine are emerging as promising treatments for a wide array of profound clinical problems. Here we report an interpenetrating polymer network (IPN) composed of gelatin-hydroxyphenyl propionic acid and hyaluronic acid tyramine that is able to enhance intravitreal retinal cell therapy. By tuning our bioinspired hydrogel to mimic the vitreous chemical composition and mechanical characteristics we were able to improve in vitro and in vivo viability of human retinal ganglion cells (hRGC) incorporated into the IPN. In vivo vitreal injections of cell-bearing IPN in rats showed extensive attachment to the inner limiting membrane of the retina, improving with hydrogels stiffness. Engrafted hRGC displayed signs of regenerating processes along the optic nerve. Of note was the decrease in the immune cell response to hRGC delivered in the gel. The findings compel further translation of the gelatin-hyaluronic acid IPN for intravitreal cell therapy.

Mesenchymal stromal cells mitigate liver damage after extended resection in the pig by modulating thrombospondin-1/TGF-β

Post-surgery liver failure is a serious complication for patients after extended partial hepatectomies (ePHx). Previously, we demonstrated in the pig model that transplantation of mesenchymal stromal cells (MSC) improved circulatory maintenance and supported multi-organ functions after 70% liver resection. Mechanisms behind the beneficial MSC effects remained unknown. Here we performed 70% liver resection in pigs with and without MSC treatment, and animals were monitored for 24 h post surgery. Gene expression profiles were determined in the lung and liver. Bioinformatics analysis predicted organ-independent MSC targets, importantly a role for thrombospondin-1 linked to transforming growth factor-β (TGF-β) and downstream signaling towards providing epithelial plasticity and epithelial-mesenchymal transition (EMT). This prediction was supported histologically and mechanistically, the latter with primary hepatocyte cell cultures. MSC attenuated the surgery-induced increase of tissue damage, of thrombospondin-1 and TGF-β, as well as of epithelial plasticity in both the liver and lung. This suggests that MSC ameliorated surgery-induced hepatocellular stress and EMT, thus supporting epithelial integrity and facilitating regeneration. MSC-derived soluble factor(s) did not directly interfere with intracellular TGF-β signaling, but inhibited thrombospondin-1 secretion from thrombocytes and non-parenchymal liver cells, therewith obviously reducing the availability of active TGF-β.

3D in vitro M2 macrophage model to mimic modulation of tissue repair

Distinct anti-inflammatory macrophage (M2) subtypes, namely M2a and M2c, are reported to modulate the tissue repair process tightly and chronologically by modulating fibroblast differentiation state and functions. To establish a well-defined three-dimensional (3D) cell culture model to mimic the tissue repair process, we utilized THP-1 human monocytic cells and a 3D collagen matrix as a biomimetic tissue model. THP-1 cells were differentiated into macrophages, and activated using IL-4/IL-13 (MIL-4/IL-13) and IL-10 (MIL-10). Both activated macrophages were characterized by both their cell surface marker expression and cytokine secretion profile. Our cell characterization suggested that MIL-4/IL-13 and MIL-10 demonstrate M2a- and M2c-like subtypes, respectively. To mimic the initial and resolution phases during the tissue repair, both activated macrophages were co-cultured with fibroblasts and myofibroblasts. We showed that MIL-4/IL-13 were able to promote matrix synthesis and remodeling by induction of myofibroblast differentiation via transforming growth factor beta-1 (TGF-β1). On the contrary, MIL-10 demonstrated the ability to resolve the tissue repair process by dedifferentiation of myofibroblast via IL-10 secretion. Overall, our study demonstrated the importance and the exact roles of M2a and M2c-like macrophage subtypes in coordinating tissue repair in a biomimetic model. The established model can be applied for high-throughput platforms for improving tissue healing and anti-fibrotic drugs testing, as well as other biomedical studies.

Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys

Localized cartilage lesions in early osteoarthritis and acute joint injuries are usually treated surgically to restore function and relieve pain. However, a persistent clinical challenge remains in how to repair the cartilage lesions. We expressed doublecortin (DCX) in human adipose-derived stromal/stem cells (hASCs) and engineered hASCs into cartilage tissues using an in vitro 96-well pellet culture system. The cartilage tissue constructs with and without DCX expression were implanted in the knee cartilage defects of rabbits (n = 42) and monkeys (n = 12). Cohorts of animals were euthanized at 6, 12, and 24 months after surgery to evaluate the cartilage repair outcomes. We found that DCX expression in hASCs increased expression of growth differentiation factor 5 (GDF5) and matrilin 2 in the engineered cartilage tissues. The cartilage tissues with DCX expression significantly enhanced cartilage repair as assessed macroscopically and histologically at 6, 12, and 24 months after implantation in the rabbits and 24 months after implantation in the monkeys, compared to the cartilage tissues without DCX expression. These findings suggest that hASCs expressing DCX may be engineered into cartilage tissues that can be used to treat localized cartilage lesions.

Current progress of rehabilitative strategies in stem cell therapy for spinal cord injury: a review

Stem cell-based regenerative therapy has opened an avenue for functional recovery of patients with spinal cord injury (SCI). Regenerative rehabilitation is attracting wide attention owing to its synergistic effects, feasibility, non-invasiveness, and diverse and systemic properties. In this review article, we summarize the features of rehabilitation, describe the mechanism of combinatorial treatment, and discuss regenerative rehabilitation in the context of SCI. Although conventional rehabilitative methods have commonly been implemented alone, especially in studies of acute-to-subacute SCI, the combinatorial effects of intensive and advanced methods, including various neurorehabilitative approaches, have also been reported. Separating the concept of combined rehabilitation from regenerative rehabilitation, we suggest that the main roles of regenerative rehabilitation can be categorized as conditioning/reconditioning, functional training, and physical exercise, all of which are indispensable for enhancing functional recovery achieved using stem cell therapies.