iMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.

Application of mesenchymal stem/stromal cells in periodontal regeneration: Opportunities and challenges.

Guided tissue regeneration (GTR) has been widely used in the periodontal treatment of intrabony and furcation defects for nearly four decades. The treatment outcomes have shown effectiveness in reducing pocket depth, improving attachment gain and bone filling in periodontal tissue. Although applying GTR could reconstruct the periodontal tissue, the surgical indications are relatively narrow, and some complications and race ethic problems bring new challenges. Therefore, it is challenging to achieve a consensus concerning the clinical benefits of GTR. With the appearance of stem cell-based regenerative medicine, mesenchymal stem/stromal cells (MSCs) have been considered a promising cell resource for periodontal regeneration. In this review, we highlight preclinical and clinical periodontal regeneration using MSCs derived from distinct origins, including non-odontogenic and odontogenic tissues and induced pluripotent stem cells, and discuss the transplantation procedures, therapeutic mechanisms, and concerns to evaluate the effectiveness of MSCs.

＜Editors' Choice＞ Mesenchymal stem/stromal cells generated from induced pluripotent stem cells are highly resistant to senescence.

The use of mesenchymal stem/stromal cells (MSCs) has attracted attention in the field of regenerative medicine based on their anti-inflammatory and tissue repair-promoting effects. Bone marrow is widely used as a source of MSCs; however, the performance of bone marrow (BM)-MSCs deteriorates as the cells age along with cell passaging. Recently, it has been reported that MSCs can be generated from induced pluripotent stem cells (iPSCs), which is expected to represent a new source of MSCs. However, few studies have investigated aging in iPSC-derived MSCs (iMSCs) and their functions. In this study, we investigated whether iMSCs overcome cellular senescence compared to that in BM-MSCs. Cellular senescence was quantitatively evaluated by staining iMSCs and BM-MSCs with fluorescein di-ß-D-galactopyranoside (FDG) and following flow cytometer analysis. The hepatocyte growth factor (HGF) concentration in the culture supernatant was also measured as a factor in the therapeutic efficacy of nephritis. The iMSCs did not reach their proliferation limit and their morphology did not change even after 10 passages. The FDG positivity of BM-MSCs increased with passaging, whereas that in iMSCs did not increase. The HGF concentration increased with passaging in iMSCs. In conclusion, our results suggest that iMSCs may be less susceptible to senescence than BM-MSCs and may be used in clinical applications.

Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells.

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.

Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair.

Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.

Selective vulnerability of human-induced pluripotent stem cells to dihydroorotate dehydrogenase inhibition during mesenchymal stem/stromal cell purification.

The use of induced mesenchymal stem/stromal cells (iMSCs) derived from human induced pluripotent stem cells (hiPSCs) in regenerative medicine involves the risk of teratoma formation due to hiPSCs contamination in iMSCs. Therefore, eradicating the remaining undifferentiated hiPSCs is crucial for the effectiveness of the strategy. The present study demonstrates the Brequinar (BRQ)-induced inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, selectively induces apoptosis, cell cycle arrest, and differentiation; furthermore, it promotes transcriptional changes and prevents the growth of 3-dimensional hiPSC aggregates. Contrastingly, BRQ-treated iMSCs showed no changes in survival, differentiation potential, or gene expression. The results suggest that BRQ is a potential agent for the effective purification of iMSCs from a mixed population of iMSCs and hiPSCs, which is a crucial step in successful iMSC-based therapy.

Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.

Nerve regeneration using the Bio 3D nerve conduit fabricated with spheroids.

Autologous nerve grafting is the gold standard method for peripheral nerve injury with defects. Artificial nerve conduits have been developed to prevent morbidity at the harvest site. However, the artificial conduit regeneration capacity is not sufficient. A Bio 3D printer is technology that creates three-dimensional tissue using only cells. Using this technology, a three-dimensional nerve conduit (Bio 3D nerve conduit) was created from several cell spheroids. We reported the first application of the Bio 3D nerve conduit for peripheral nerve injury. A Bio 3D nerve conduit that was created from several cells promotes peripheral nerve regeneration. The Bio 3D nerve conduit may be useful clinically to treat peripheral nerve defects.

Dental applications of induced pluripotent stem cells and their derivatives.

Periodontal tissue regeneration is the ideal tactic for treating periodontitis. Tooth regeneration is the potential strategy to restore the lost teeth. With infinite self-renewal, broad differentiation potential, and less ethical issues than embryonic stem cells, induced pluripotent stem cells (iPSCs) are promising cell resource for periodontal and tooth regeneration. This review summarized the optimized technologies of generating iPSC lines and application of iPSC derivatives, which reduce the risk of tumorigenicity. Given that iPSCs may have epigenetic memory from the donor tissue and tend to differentiate into lineages along with the donor cells, iPSCs derived from dental tissues may benefit for personalized dental application. Neural crest cells (NCCs) and mesenchymal stem or stomal cells (MSCs) are lineage-specific progenitor cells derived from iPSCs and can differentiate into multilineage cell types. This review introduced the updated technologies of inducing iPSC-derived NCCs and iPSC-derived MSCs and their application in periodontal and tooth regeneration. Given the complexity of periodontal tissues and teeth, it is crucial to elucidate the integrated mechanisms of all constitutive cells and the spatio-temporal interactions among them to generate structural periodontal tissues and functional teeth. Thus, more sophisticated studies in vitro and in vivo and even preclinical investigations need to be conducted.

Generation of human GAPDH knock-in reporter iPSC lines for stable expression of tdTomato in pluripotent and differentiated culture conditions.

Human induced pluripotent stem cells (iPSCs) can differentiate into multiple cell types and are utilized for research on human development and regenerative medicine. Here, we report the establishment of human GAPDH knock-in reporter iPSC lines (GAPDH-tdT1 and 2), via CRISPR/Cas9-mediated homologous recombination, that stably express tdTomato as a constitutive cell label in both iPSCs and their differentiated derivatives. These cell lines will provide useful tools to trace cell locations and fates in 2D cultures and 3D organoids and will facilitate in vivo experiments.

Generation of a human SOX10 knock-in reporter iPSC line for visualization of neural crest cell differentiation.

SOX10 (SRY-box transcription factor 10) is not only a definitive molecular marker of neural crest cells (NCCs) but also an essential transcription factor for the differentiation of NCCs in vertebrate embryogenesis. Here, we report the establishment of a human SOX10 knock-in reporter iPSC line (SOX10-tdT) by CRISPR/Cas9-mediated homologous recombination, in which the expression of SOX10 can be monitored as tdTomato fluorescence. This iPSC line can provide a useful tool to model the differentiation process of human NCCs in vitro.

Systemic Supplementation of Collagen VI by Neonatal Transplantation of iPSC-Derived MSCs Improves Histological Phenotype and Function of Col6-Deficient Model Mice.

Collagen VI is distributed in the interstitium and is secreted mainly by mesenchymal stromal cells (MSCs) in skeletal muscle. Mutations in COL6A1-3 genes cause a spectrum of COL6-related myopathies. In this study, we performed a systemic transplantation study of human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) into neonatal immunodeficient COL6-related myopathy model (Col6a1 KO /NSG) mice to validate the therapeutic potential. Engraftment of the donor cells and the resulting rescued collagen VI were observed at the quadriceps and diaphragm after intraperitoneal iMSC transplantation. Transplanted mice showed improvement in pathophysiological characteristics compared with untreated Col6a1 KO /NSG mice. In detail, higher muscle regeneration in the transplanted mice resulted in increased muscle weight and enlarged myofibers. Eight-week-old mice showed increased muscle force and performed better in the grip and rotarod tests. Overall, these findings support the concept that systemic iMSC transplantation can be a therapeutic option for COL6-related myopathies.

Bio-3D printing iPSC-derived human chondrocytes for articular cartilage regeneration.

Osteoarthritis is a leading cause of pain and joint immobility, the incidence of which is increasing worldwide. Currently, total joint replacement is the only treatment for end-stage disease. Scaffold-based tissue engineering is a promising alternative approach for joint repair but is subject to limitations such as poor cytocompatibility and degradation-associated toxicity. To overcome these limitations, a completely scaffold-free Kenzan method for bio-3D printing was used to fabricate cartilage constructs feasible for repairing large chondral defects. Human induced pluripotent stem cell (iPSC)-derived neural crest cells with high potential to undergo chondrogenesis through mesenchymal stem cell differentiation were used to fabricate the cartilage. Unified, self-sufficient, and functional cartilaginous constructs up to 6 cm2in size were assembled by optimizing fabrication time during chondrogenic induction. Maturation for 3 weeks facilitated the self-organisation of the cells, which improved the construct's mechanical strength (compressive and tensile properties) and induced changes in glycosaminoglycan and type II collagen expression, resulting in improved tissue function. The compressive modulus of the construct reached the native cartilage range of 0.88 MPa in the 5th week of maturation. This paper reports the fabrication of anatomically sized and shaped cartilage constructs, achieved by combining novel iPSCs and bio-3D printers using a Kenzan needle array technology, which may facilitate chondral resurfacing of articular cartilage defects.

Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice.

BACKGROUND: Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. METHODS: To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). RESULTS: All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. CONCLUSIONS: These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice.

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture.

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.

Challenges and Opportunities for Drug Repositioning in Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is an ultrarare congenital disease that progresses through intermittent episodes of bone formation at ectopic sites. FOP patients carry heterozygous gene point mutations in activin A receptor type I ACVR1, encoding the bone morphogenetic protein (BMP) type I serine/threonine kinase receptor ALK2, termed activin receptor-like kinase (ALK)2. The mutant ALK2 displays neofunctional responses to activin, a closely related BMP cytokine that normally inhibits regular bone formation. Moreover, the mutant ALK2 becomes hypersensitive to BMPs. Both these activities contribute to enhanced ALK2 signalling and endochondral bone formation in connective tissue. Being a receptor with an extracellular ligand-binding domain and intrinsic intracellular kinase activity, the mutant ALK2 is a druggable target. Although there is no approved cure for FOP yet, a number of clinical trials have been recently initiated, aiming to identify a safe and effective treatment for FOP. Among other targeted approaches, several repurposed drugs have shown promising results. In this review, we describe the molecular mechanisms underlying ALK2 mutation-induced aberrant signalling and ectopic bone formation. In addition, we recapitulate existing in vitro models to screen for novel compounds with a potential application in FOP. We summarize existing therapeutic alternatives and focus on repositioned drugs in FOP, at preclinical and clinical stages.

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction.