Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

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.

Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells.

Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.

Development of pluripotent stem cell-based human tenocytes.

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.

Olfactomedin-like 3 promotes PDGF-dependent pericyte proliferation and migration during embryonic blood vessel formation.

Pericytes promote vessel stability and their dysfunction causes pathologies due to blood vessel leakage. Previously, we reported that Olfactomedin-like 3 (Olfml3) is a matricellular protein with proangiogenic properties. Here, we explored the role of Olfml3 in a knockout mouse model engineered to suppress this protein. The mutant mice exhibited vascular defects in pericyte coverage, suggesting that pericytes influence blood vessel formation in an Olfml3-dependent manner. Olfml3-deficient mice exhibited abnormalities in the vasculature causing partial lethality of embryos and neonates. Reduced pericyte coverage was observed at embryonic day 12.5 and persisted throughout development, resulting in perinatal death of 35% of Olfml3-deficient mice. Cultured Olfml3-deficient pericytes exhibited aberrant motility and altered pericyte association to endothelial cells. Furthermore, the proliferative response of Olfml3-/- pericytes upon PDGF-B stimulation was significantly diminished. Subsequent experiments revealed that intact PDGF-B signaling, mediated via Olfml3 binding, is required for pericyte proliferation and activation of downstream kinase pathways. Our findings suggest a model wherein pericyte recruitment to endothelial cells requires Olfml3 to provide early instructive cue and retain PDGF-B along newly formed vessels to achieve optimal angiogenesis.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

Induced pluripotent stem cell-derived mesenchymal stem cells prolong hind limb survival in a rat vascularized composite allotransplantation model.

BACKGROUND: The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model. MATERIALS AND METHODS: One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 106 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed. RESULTS: The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 µm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively). CONCLUSIONS: iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings.

Clumps of Mesenchymal Stem Cell/Extracellular Matrix Complexes Generated with Xeno-Free Conditions Facilitate Bone Regeneration via Direct and Indirect Osteogenesis.

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis.

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-ß signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.

Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.

The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

New Protocol to Optimize iPS Cells for Genome Analysis of Fibrodysplasia Ossificans Progressiva.

Successful in vitro disease-recapitulation using patient-specific induced pluripotent stem cells (iPSCs) requires two fundamental technical issues: appropriate control cells and robust differentiation protocols. To investigate fibrodysplasia ossificans progressiva (FOP), a rare genetic disease leading to extraskeletal bone formation through endochondral ossification, gene-corrected (rescued) iPSC clones (resFOP-iPSC) were generated from patient-derived iPSC (FOP-iPSC) as genetically matched controls, and the stepwise induction method of mesenchymal stromal cells (iMSCs) through neural crest cell (NCC) lineage was used to recapitulate the disease phenotype. FOP-iMSCs possessing enhanced chondrogenic ability were transcriptionally distinguishable from resFOP-iMSCs and activated the SMAD1/5/8 and SMAD2/3 pathways at steady state. Using this method, we identified MMP1 and PAI1 as genes responsible for accelerating the chondrogenesis of FOP-iMSCs. These data indicate that iMSCs through NCC lineage are useful for investigating the molecular mechanism of FOP and corresponding drug discovery.

[Application of disease-specific iPS cells for intractable diseases-from pathomechanisms to drug discovery].

Genetic diseases affecting bone and cartilage, which are main components of the locomotive system, are extremely diverse. Even if the causative genes are known, detail pathomechanisms are not yet disclosed in most of them and no effective treatments are established. One of such condition is fibrodysplasia ossificance progressive, which is characterized by systemic ectopoic bone formation and caused by mutations of ACVR1/ALK2 gene encoding one of typeⅠBMP receptors. Using patient-derived iPS cells, we have succeeded to recapitulate the disease in vitro and found a unexpected molecular mechanism that Activin-A induced the BMP signal through mutant receptors. This novel finding provides us with a key to discover drugs for this condition.

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.

Rat tracheal cartilage regeneration using mesenchymal stem cells derived from human iPS cells.

Tracheal cartilage provides structural support to the airways to enable breathing. However, it can become damaged or impaired, sometimes requiring surgical resection and reconstruction. Previously, we clinically applied an artificial trachea composed of a polypropylene mesh and collagen sponge, with a favorable postoperative course. However, the artificial trachea presents a limitation, as the mesh is not biodegradable and cannot be used in pediatric patients. Compared to a polypropylene mesh, regenerated cartilage represents an ideal material for reconstruction of the damaged trachea. The use of mesenchymal stem cells (MSCs) as a source for cartilage regeneration has gained widespread acceptance, but challenges such as the invasiveness of harvesting and limited cell supply, persist. Therefore, we focused on the potential of human induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) for tracheal cartilage regeneration. In this study, we aimed to regenerate tracheal cartilage on an artificial trachea as a preliminary step to replace the polypropylene mesh. iMSCs were induced from hiPSCs through neural crest cells and transplanted with a polypropylene mesh covered with a collagen sponge into the damaged tracheal cartilage in immunodeficient rats. Human nuclear antigen (HNA)-positive cells were observed in all six rats at 4 weeks and in six out of seven rats at 12 weeks after transplantation, indicating that transplanted iMSCs survived within the tracheal cartilage defects of rats. The HNA-positive cells co-expressed SOX9, and type II collagen was detected around HNA-positive cells in four of six rats at 4 weeks and in three of seven rats at 12 weeks after transplantation, reflecting cartilage-like tissue regeneration. These results indicate that the transplanted iMSCs could differentiate into chondrogenic cells and promote tracheal cartilage regeneration. iMSC transplantation thus represents a promising approach for human tracheal reconstruction.

Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells.

Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT-SSX. Although precise function of SYT-SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT-SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT-SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT-SSX2 gene. SYT-SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT-SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT-SSX, respectively. Association of these genes with SYT-SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT-SSX, and more than half of SYT-SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT-SSX function, and demonstrated how our new system will help to overcome this issue.

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.

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.