As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Mesenchymal stem cells (MSCs) are used as a major source for cell therapy, and its application is expanding in various diseases. On the other hand, reliable method to evaluate quality and therapeutic properties of MSC is limited. In this study, we focused on TWIST1 that is a transcription factor regulating stemness of MSCs and found that the transmembrane protein LRRC15 tightly correlated with the expression of TWIST1 and useful to expect TWIST1-regulated stemness of MSCs. The LRRC15-positive MSC populations in human and mouse bone marrow tissues highly expressed stemness-associated transcription factors and therapeutic cytokines, and showed better therapeutic effect in bleomycin-induced pulmonary fibrosis model mice. This study provides evidence for the important role of TWIST1 in the MSC stemness, and for the utility of the LRRC15 protein as a marker to estimate stem cell quality in MSCs before cell transplantation.

Nowadays, ordinary people can travel in space, and the possibility of extended durations in an environment such as moon of the Earth and Mars with higher space radiation exposures compared to past missions, is increasing. Until now, the physical doses of space radiation have been measured, but measurement of direct biological effects has been hampered by its low dose and low dose-rate effect. To assess the biological effects of space radiation, we launched and kept frozen mouse embryonic stem (ES) cells in minus eighty degree Celsius freezer in ISS (MELFI) on the International Space Station (ISS) for a maximum of 1,584 days. The passive dosimeter for life science experiments in space (PADLES) was attached on the surface of the sample case of the ES cells. The physical dosimeter measured the absorbed dose in water. After return, the frozen cells were thawed and cultured and their chromosome aberrations were analyzed. Comparative experiments with proton and iron ion irradiation were performed at particle accelerators on Earth. The wild-type ES cells showed no differences in chromosomal aberrations between the ground control and ISS exposures. However, we detected an increase of chromosome aberrations in radio-sensitized histone H2AX heterozygous-deficient mouse ES cells and found that the rate of increase against the absorbed dose was 1.54-fold of proton irradiation at an accelerator. On the other hand, we estimated the quality factor of space radiation as 1.48 ± 0.2. using formulas of International Commission of Radiation Protection (ICRP) 60. The relative biological effectiveness (RBE) observed from our experiments (1.54-fold of proton) was almost equal (1.04-fold) to the physical estimation (1.48 ± 0.2). It should be important to clarify the relation between biological effect and physical estimates of space radiation. This comparative study paves a way to reveal the complex radiation environments to reduce the uncertainty for risk assessment of human stay in space.

Sarcopenia is an aging-associated attenuation of muscular volume and strength and is the major cause of frailty and falls in elderly individuals. The number of individuals with sarcopenia is rapidly increasing worldwide; however, little is known about the underlying mechanisms of the disease. Sarcopenia often copresents with obesity, and some patients with sarcopenia exhibit accumulation of peri-organ or intra-organ adipose tissue as ectopic fat deposition, including atrophied skeletal muscle. In this study, we showed that transplantation of the perimuscular adipose tissue (PMAT) to the hindlimb thigh muscles of young mice decreased the number of integrin α7/CD29-double positive muscular stem/progenitor cells and that the reaction was mediated by PMAT-derived exosomes. We also found that the inhibition of cell proliferation was induced by Let-7d-3p miRNA that targets HMGA2, which is an important transcription factor for stem cell self-renewal, in muscular stem/progenitor cells and the composite molecular reaction in aged adipocytes. Reduction of Let-7 miRNA repressor Lin28 A/B and activation of nuclear factor-kappa B signaling can lead to the accumulation of Let-7d-3p in the exosomes of aged PMAT. These findings suggest a novel crosstalk between adipose tissue and skeletal muscle in the development of aging-associated muscular atrophy and indicate that adipose tissue-derived miRNAs may play a key role in sarcopenia.

OBJECTIVES: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis. METHODS: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq). RESULTS: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of Klf2 in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2. CONCLUSIONS: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.

Osteoarthritis (OA), the most prevalent joint disease, is characterized by the progressive loss of articular cartilage. Autophagy, a lysosomal degradation pathway, maintains cellular homeostasis, and autophagic dysfunction in chondrocytes is a hallmark of OA pathogenesis. However, the cause of autophagic dysfunction in OA chondrocytes remains incompletely understood. Recent studies have reported that decidual protein induced by progesterone (C10orf10/DEPP) positively regulates autophagic functions. In this study, we found that DEPP was involved in mitochondrial autophagic functions of chondrocytes, as well as in OA pathogenesis. DEPP expression decreased in human OA chondrocytes in the absence or presence of pro-inflammatory cytokines, and was induced by starvation, hydrogen peroxide (H2 O2 ), and hypoxia (cobalt chloride). For functional studies, DEPP knockdown decreased autophagic flux induced by H2 O2 , whereas DEPP overexpression increased autophagic flux and maintained cell viability following H2 O2 treatment. DEPP was downregulated by knockdown of forkhead box class O (FOXO) transcription factors and modulated the autophagic function regulated by FOXO3. In an OA mouse model by destabilization of the medial meniscus, DEPP-knockout mice exacerbated the progression of cartilage degradation with TUNEL-positive cells, and chondrocytes isolated from knockout mice were decreased autophagic flux and increased cell death following H2 O2 treatment. Subcellular fractionation analysis revealed that mitochondria-located DEPP activated mitochondrial autophagy via BCL2 interacting protein 3. Taken together, our data demonstrate that DEPP is a major stress-inducible gene involved in the activation of mitochondrial autophagy in chondrocytes, and maintains chondrocyte viability during OA pathogenesis. DEPP represents a potential therapeutic target for enhancing autophagy in patients with OA.

BACKGROUND: In order to enhance cartilage regeneration, surface modification of the cubic micro-cartilage with the collagenase treatment was tested and its efficacy to tissue engineer ear cartilage was investigated. MATERIALS AND METHODS: Harvested cubic micro-cartilages were treated with collagenase with different digestion time (0, 15, 60, and 120 min). Histological, ultrastructural (SEM and TEM), and Western blot analyses were carried out. Subsequently, A total of 45 dogs were used to tissue engineer ear cartilage. Using collagenase-treated micro-cartilage, the ear cartilage regeneration with the prepared dilution (8, 12.5, 25, 50, 100%) of micro-cartilage block seeding was performed to determine the minimum amount of cartilage tissue required for ear tissue-engineering (n = 6 at each point in each group). At 10 weeks after surgery, samples were resected and subjected to histochemical and immune-histological evaluation for cartilage regeneration. RESULTS: In vitro study on micro-cartilage morphology and western blot analysis showed that collagenase digestion was optimal at 60 min for cartilage regeneration. In vivo evaluation on the reduced proportions of micro-cartilage block seeding onto implant scaffolds under 60-min collagenase digestion determined the minimum amount of cartilage tissue necessary to initiate a one-step ear cartilage regeneration in a canine autologous model, which was 12.5-25% of the original ear size. CONCLUSION: Tissue-engineering ear cartilage from limited volume of donor cartilage can possibly be achieved by the collagenase treatment on micro-cartilage to expand cartilage regeneration capacity, application of cytokine sustained-release system, and seeding on a suitable ear scaffold material.

Frailty of the locomotory organs has become a widespread problem in the geriatric population. The major factor leading to frailty is an age-associated decrease in muscular mass and a reduced number of muscular cells and myofibers. In aged muscular tissues, muscular satellite cells (MuSCs) are reduced due to abnormalities in their self-renewal and the induction of apoptosis. However, the molecular mechanisms connecting aging-associated physiological changes and the reduction of MuSCs are largely unknown. NIMA-related kinase 2 (Nek2), a member of the Nek family of serine/threonine kinases, was found to be downregulated in aged MuSCs/progenitors. Further, Nek2 downregulation was found to inhibit self-renewal and apoptotic cell death by activating the p53-dependent checkpoint. Attenuated NEK2 expression was also observed in the muscular tissues of elderly donors, and its function was confirmed to be conserved in humans. Overall, this study proposes a novel mechanism for inducing muscular atrophy to understand aging-associated muscular diseases.

PURPOSE: Treatment with KRAS G12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non-small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRAS G12C NSCLC cells. EXPERIMENTAL DESIGN: Clones of sotorasib-sensitive KRAS G12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy number evaluation. The underlying mechanisms of resistance were investigated using immunological examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo Results: Unbiased screening detected subclonal evolution of MET amplification in KRAS G12C NSCLC cells that had developed resistance to sotorasib in vitro MET knockdown using siRNA restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK-ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS-MEK-ERK as well as AKT signaling. MET/KRAS G12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice. CONCLUSIONS: MET amplification leads to the development of resistance to KRAS G12C inhibitors in NSCLC. Dual blockade of MET and KRAS G12C could be a treatment option for MET amplified, KRAS G12C-mutated NSCLC.

BACKGROUND: Using the rat sciatic nerve model, sliced nerves of different thickness was combined to a biodegradable nerve conduit and the amount of nerve fragment necessary to promote nerve regeneration was investigated. MATERIALS AND METHODS: Harvested sciatic nerve (n = 6) was processed in sliced nerve of the different width; 2, 1, 0.5 mm, respectively. Western blot analysis was carried out to determine protein expression of Erk1/2. Subsequently, a total of 246 rats were used to create a 10 mm gap in the sciatic nerve. A polyglycolic acid-based nerve conduit was used to bridge the gap, with one sliced (width; 2, 1, 0.5 mm) or two (width; 1 mm × 2) incorporated within the conduit (n = 6 at each point in each group). At 2, 4, 8, and 20 weeks after surgery, samples were resected and subjected to immune-histological, transmission electron microscopic, and motor functional evaluation for nerve regeneration. RESULTS: Western blot analysis demonstrated Erk1/2 expressions were significantly increased in the groups of 2-mm and 1-mm width and attenuated in the 0.5-mm width group (p < .05). The immune-histological study showed the migration of Schwann cells and axon elongation were significantly extended in the groups of 2-mm, 1-mm, and 1 mm × 2 width at 4 weeks (p < .01), in which nerve conduction velocity was marked at 20 weeks (p < .01) after implantation. CONCLUSION: When nerve tissue was inserted in the biodegradable nerve conduit as a sliced nerve, the method of inserting two sheets with a slice width of 1 mm most strongly accelerated motor function.

BACKGROUND: The main function of folate receptor α (FOLRα) has been considered to mediate intracellular folate uptake and induce tumor cell proliferation. Given the broad spectrum of expression among malignant tumors, including gastric cancer (GC) but not in normal tissue, FOLRα represents an attractive target for tumor-selective drug delivery. However, the efficacy of anti-FOLRα monoclonal antibodies (mAbs) has not been proved so far, with the reason for this failure remaining unclear, raising the need for a better understanding of FOLRα function. METHODS: The distribution of FOLRα in GC cells was evaluated by immunohistochemistry. The impacts of FOLRα expression on the survival of GC patients and GC cell lines were examined with the Gene Expression Omnibus database and by siRNA of FOLRα. RNA-sequencing and Microarray analysis was conducted to identify the function of FOLRα. Proteins that interact with FOLRα were identified with shotgun LC-MS/MS. The antitumor efficacy of the anti-FOLRα mAb farletuzumab as well as the antibody-drug conjugate (ADC) consists of the farletuzumab and the tublin-depolymerizing agent eribulin (MORAb-202) was evaluated both in vitro and in vivo. RESULTS: FOLRα was detected both at the cell membrane and in the cytoplasm. Shorter overall survival was associated with FOLRα expression in GC patients, whereas reduction of FOLRα attenuated cell proliferation without inducing cell death in GC cell lines. Transcriptomic and proteomic examinations revealed that the FOLRα-expressing cancer cells possess a mechanism of chemotherapy resistance supported by MDM2, and FOLRα indirectly regulates it through a chaperone protein prohibitin2 (PHB2). Although reduction of FOLRα brought about vulnerability for oxaliplatin by diminishing MDM2 expression, farletuzumab did not suppress the MDM2-mediated chemoresistance and cell proliferation in GC cells. On the other hand, MORAb-202 showed significant antitumor efficacy. CONCLUSIONS: The ADC could be a more reasonable choice than mAb as a targeting agent for the FOLRα-expressing tumor.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

BACKGROUND: Using the rat sciatic nerve model, the difference in outcome using a nerve segment either sliced open or minced with a blade incorporated into a nerve conduit were compared and the relative effects upon the rate and completeness of the nerve regeneration was determined. MATERIALS AND METHODS: A 10-mm gap was created in the rat sciatic nerve and bridged with a biodegradable nerve conduit. Segments of the resected nerve (2-mm lengths) were prepared by either slicing the nerve with one longitudinal cut or by scalpel mincing of the nerve tissue, with insertion of the prepared nerve segment into the center of the conduit. Flow cytometry and Western blotting of these preparations were performed to measure viable cells and to examine the expression of Erk1/2 for neural regeneration potential with both treatments. in vivo nerve regeneration was evaluated at 2, 4, 8, and 20 weeks, using immunohistochemistry, transmission electron microscopy, muscle wet weight, and nerve conduction velocity determination. RESULTS: The sliced nerve group showed significantly greater Schwann cell migration with the subsequent axonal elongation at 4 weeks after implantation, in comparison to the minced nerve group and controls (unaltered conduit grafts). By 20 weeks anterior tibial muscle weight and nerve conduction velocity were also greater in the sliced nerve group in comparison to the other groups (p < .05). CONCLUSION: These findings suggest that insertion of a sliced section of nerve into a biodegradable nerve conduit can shorten the time for and improve the quality of nerve regeneration.

Removal of dysfunctional mitochondria is essential step to maintain normal cell physiology, and selective autophagy in mitochondria, called mitophagy, plays a critical role in quality control of mitochondria. While in several diseases and aging, disturbed mitophagy has been observed. In stem cells, accumulation of damaged mitochondria can lead to deterioration of stem cell properties. Here, we focused on miR-155-5p (miR-155), one of the most prominent miRNAs in inflammatory and aged tissues, and found that miR-155 disturbed mitophagy in mesenchymal stem cells (MSCs). As a molecular mechanism of miR-155-mediated mitophagy suppression, we found that BCL2 associated athanogene 5 (BAG5) is a direct target of miR-155. Reduction of BAG5 resulted in destabilization of PTEN-induced kinase (PINK1) and consequently disrupted mitophagy. Our study suggests a novel mechanism connecting aging and aging-associated inflammation with mitochondrial dysfunction in stem cells through a miRNA-mediated mechanism.

Inflammation-induced reactive oxygen species (ROS) are implicated in cellular dysfunction and an important trigger for aging- or disease-related tissue degeneration. Inflammation-induced ROS in stem cells lead to deterioration of their properties, altering tissue renewal or regeneration. Pathological ROS generation can be induced by multiple steps, and dysfunction of antioxidant systems is a major cause. The identification of the central molecule mediating the above-mentioned processes would pave the way for the development of novel therapeutics for aging, aging-related diseases, or stem cell therapies. In recent years, microRNAs (miRNAs) have been shown to play important roles in many biological reactions, including inflammation and stem cell functions. In inflammatory conditions, certain miRNAs are highly expressed and mediate some cytotoxic actions. Here, we focused on miR-155, which is one of the most prominent miRNAs in inflammation and hypothesized that miR-155 participates to inflammation-induced ROS generation in stem cells. We observed mesenchymal stem cells (MSCs) from 1.5-year-old aged mice and determined that antioxidants, Nfe2l2, Sod1, and Hmox1, were suppressed, while miR-155-5p was highly expressed. Subsequent in vitro studies demonstrated that miR-155-5p induces ROS generation by suppression of the antioxidant genes by targeting the common transcription factor C/ebp beta Moreover, this mechanism occurred during the cell transplantation process, in which ROS generation is triggering loss of transplanted stem cells. Finally, attenuation of antioxidants and ROS accumulation were partially prevented in miR-155 knockout MSCs. In conclusion, our study suggests that miR-155 is an important mediator connecting aging, inflammation, and ROS generation in stem cells.

Background: There is growing evidence that the subacromial injection of hyaluronic acid (HA) is effective for pain relief in rotator cuff tears; however, its effect on tendon-to-bone healing remains unknown. Purpose: To examine the effect of HA on the chondrogenesis of mesenchymal stem cells (MSCs) in vitro and on tendon-to-bone healing in a rotator cuff repair model. Study Design: Controlled laboratory study. Methods: Bilateral complete tears of the infraspinatus tendon were made in rabbits and subsequently repaired. Before closure, 1 mL HA was applied to the repaired site, and phosphate-buffered saline was used in the opposite side as a control. Biomechanical, histological, and immunohistochemical analyses were performed at 4, 8, and 12 weeks after surgery. After euthanizing each animal, the bone marrow was isolated from the femoral bone in the same rabbits. Then, MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. Results: At 4 and 8 weeks after surgery, ultimate load-to-failure was significantly greater in the HA group than in the control group (45.61 +/- 9.0 N vs 32.42 +/- 9.4 N at 4 weeks, 90.7 +/- 16.0 N vs 66.97 +/- 10.0 N at 8 weeks; both P < .05) but not at 12 weeks after surgery (109.6 +/- 40.2 N vs 108.1 +/- 42.6 N, P > .05). Linear stiffness was not significant throughout the time point evaluation. The chondroid formation area at the tendon-bone interface stained by safranin O (control vs HA group) was 0.33% +/- 0.7% versus 13.5% +/- 12.3% at 4 weeks after surgery (P < .05) and 3.0% +/- 5.9% versus 12.9% +/- 12.9% at 8 weeks after surgery (P < .05), but there was no significant difference at 12 weeks after surgery. Maturity of collagen at the repaired site stained by PicroSirius Red (control vs HA group) was 16.2 +/- 10.6 versus 43.5 +/- 21.3 at 4 weeks after surgery (P < .05), but there were no significant differences at 8 and 12 weeks after surgery. MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. The number of CD44-positive cells (control vs HA group) was 8.3% +/- 1.4% versus 26.2% +/- 5.2% at 3 days after surgery (P < .05), 1.8% +/- 1.1% versus 26.6% +/- 11.6% at 4 weeks after surgery (P < .05), 0.6% +/- 0.9% versus 0.5% +/- 0.6% at 8 weeks after surgery (P > .05), and 1.8% +/- 4.0% versus 5.4% +/- 4.2% at 12 weeks after surgery (P > .05). Compared with the control group, HA significantly increased the volume of cartilaginous pellet produced by MSCs (0.0016 +/- 0.0015 mm(3) at 0 mg/mL of HA, 0.0041 +/- 0.0023 mm(3) at 1.0 mg/mL, and 0.0041 +/- 0.0018 mm(3) at 4.0 mg/mL), with increased mRNA expression (relative ratio to control) of type 2 collagen (1.34 +/- 0.38), SOX9 (1.58 +/- 0.31), and aggrecan (1.30 +/- 0.22) genes in the pellet (P < .01). Conclusion: HA accelerated tendon-to-bone healing in the rotator cuff repair model, enhancing the biomechanical strength and increasing chondroid formation and tendon maturity at the tendon-bone interface. Based on the data of in vitro experiments, HA-activated MSCs may play a crucial role in the acceleration of tendon-to-bone healing.

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Intracerebral inflammation resulting from injury or disease is implicated in disruption of neural regeneration and may lead to irreversible neuronal dysfunction. Analysis of inflammation-related microRNA profiles in various tissues, including the brain, has identified miR-155 among the most prominent miRNAs linked to inflammation. Here, we hypothesize that miR-155 mediates inflammation-induced suppression of neural stem cell (NSC) self-renewal. Using primary mouse NSCs and human NSCs derived from induced pluripotent stem (iPS) cells, we demonstrate that three important genes involved in NSC self-renewal (Msi1, Hes1 and Bmi1) are suppressed by miR-155. We also demonstrate that suppression of self-renewal genes is mediated by the common transcription factor C/EBP beta, which is a direct target of miR-155. Our study describes an axis linking inflammation and miR-155 to expression of genes related to NSC self-renewal, suggesting that regulation of miR-155 may hold potential as a novel therapeutic strategy for treating neuroinflammatory diseases.

A high-throughput test of cell growth inhibition was performed using mouse embryonic stem (ES) cells to assess chemical toxicities. We herein demonstrated using a 96-well culture plate approach and the MTT assay that this method was suitable for prioritization of chemicals for their cytotoxic properties. In order to categorize chemicals, we used p53 gene-modified mouse ES cells as well as wild-type ES cells. The p53 gene is a well-known tumor suppressor and controls programmed cell death (apoptosis) and cellular senescence that is triggered by DNA-damaging agents such as alkylating agents and radiation. In the present study, p53-deficient ES cells were found to be more resistant to a tumor initiator, diethylnitrosamine (DEN), than wild-type ES cells, suggesting the inhibition of apoptosis or senescence by a dysfunction in p53. Chromosome aberrations were more frequently detected in p53-deficient ES cells than in wild-type cells, indicating genomic instability due to the deletion of p53. Other tumor initiators, methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU), did not reveal apparent differences in cytotoxicity between wild-type and p53-deficient ES cells. Thus, ES test system using gene-modified ES cells may be used to categorize chemicals by detecting their characteristic effects on apoptosis, genotoxic potentials as well as general cytotoxicity.

The cell adhesion molecule Cadherin 2 (Cdh2) plays important roles in somatic cell adhesion, proliferation and migration. Cdh2 is also highly expressed in mouse epiblast stem cells (mEpiSCs), but its function in these cells is unknown. To understand the function of Cdh2 in mEpiSCs, we compared the expression of pluripotency-related genes in mEpiSCs and mouse embryonic stem cells (mESCs) after either Cdh2 knockdown or Cdh2 over-expression. Introduction of specific siRNA against Cdh2 led to attenuation of pluripotency-related genes. Pluripotent gene expression was not recovered by overexpression of Cdh1 following Cdh2 knockdown. Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs. Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation. Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

One important pharmacological function of hyaluronic acid (HA) in chondrocytes is reduction of cellular superoxide generation and accumulation. Here we demonstrated a relationship between HA supplementation and accumulation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), which is a master transcription factor in cellular redox reactions, in cultured chondrocytes derived from bovine joint cartilage. In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. In HA-treated chondrocytes, Akt was phosphorylated, and inhibition of Akt activity or suppression of HA receptors CD44 and/or RHAMM with siRNAs prevented HA-mediated Nrf2 accumulation. Furthermore, Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. These results show that HA might contribute to ROS reduction through Nrf2 regulation by activating Akt. Our study suggests a new mechanism for extracellular matrix (ECM)-mediated redox systems in chondrocytes. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY license.

Oxidative stress within the arthritis joint has been indicated to be involved in generating mediators for tissue degeneration and inflammation. COX-2 is a mediator in inflammatory action, pain and some catabolic reactions in inflamed tissues. Here, we demonstrated a direct relationship between oxidative stress and Cox-2 expression in the bovine synovial fibroblasts. Furthermore, we elucidated a novel mechanism, in which oxidative stress induced phosphorylation of MAPKs and NF-jB through TAK1 activation and resulted in increased Cox-2 and prostaglandin E2 expression. Finally, we demonstrated that ROS-induced Cox-2 expression was inhibited by supplementation of an antioxidant such as N-acetyl cysteamine and hyaluronic acid in vitro and in vivo. From these results, we conclude that oxidative stress is an important factor for generation of Cox-2 in synovial fibroblasts and thus its neutralization may be an effective strategy in palliative therapy for chronic joint diseases. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY license.

Purpose: In 2013, we reported that the local renin-angiotensin system (RAS) can modulate the hypertrophic differentiation of chondrocytes activating angiotensin II type 1 receptor (AT1R) or angiotensin II type 2 receptor (AT2R). However, the details of the modulation have not been revealed. On the other hand, many researchers have already reported that the local RAS can modulate hypertrophic changes modulating the generations of reactive oxygen species (ROS) in vascular endothelial cells. The purpose of our study is to reveal whether or not the local RAS modulates the hypertrophic differentiation of chondrocytes modulating the generations of ROS.

Methods: We cultured ATDC5 cell line under the inducing of 10 μg/ml bovine insulin for 14 days after they had reached confluence. Then, we added 1.0 μg/ml angiotensin II, 1.0 μg/ml Olmesartan and 1.0 μg/ml PD123319 to activate AT1R or AT2R separately. 6 times after the activation of the receptors, we measured the concentrations of ROS. We also administered vitamin C, vitamin E and hyaluronic acid (HA) and measured the concentrations of ROS and the expressions of type X collagen (Col.X), matrix metalloproteinase 13 (MMP13) and runt-related transcription factor (Runx2).

Results: The generations of ROS were downregulated activating AT1R and were conversely upregulated activating AT2R. In the upregulated hypertrophic differentiation caused by activating AT2R, the expressions of Col.x, MMP13 and Runx2 and the generations of ROS were downregulated administering vitamin C, vitamin E and HA. However, in the downregulated hypertrophic differentiation caused by activating AT1R, we did not find significant changes in the the expressions of Col.x, MMP13 and Runx2 and the generations of ROS administering vitamin C, vitamin E and HA .

Conclusions: The local RAS could modulate the hypertrophic differentiation of chondrocytes modulating the generations of ROS. Upregulation of hypertrophic differentiation of chondrocytes cased by activating AT2R could be suppressed by administering vitamin C, vitamin E and HA.

Purpose: Recently, the local renin-angiotensin system (RAS) has attracted many researchers in many pathophysiological issues. Also in orthopedics, expression of the local RAS was found in fracture callus, bone tissues and arthritic synovium. In the last year, we reported that the local RAS expressed in chondrocytes of the epiphyseal plates of mice. The purpose of this study is to reveal immunohistological localization of the RAS components in the limb buds of mice where another physiological hypertrophic differetiation occurs and to analyze function of the local RAS in the processes of hypertrophic differentiation using ATDC5 cell line.

Methods: The limb buds of 15-day-viviparous mice were immunostained with antibodies to angiotensinogen, angiotensinogen converting enzyme 1 (ACE1), angiotensinII type 1 receptor (AT1R) and angiotensinII type 2 receptor (AT2R). We cultured ATDC5 cell line and evaluated expression of angiotensinogen, ACE1, AT1R and AT2R during the hypertrophic term using quantitative real-time PCR and Western blot analysis. Then, we separately stimulated AT1R and AT2R in hypertrophic term using angiotensin II, olmesartan and PD123319 and evaluated expression of type X collagen using quantitative real-time PCR and Western blot analysis.

Results: In the limb buds of mice, angiotensinogen and AT1R expressed in the resting chondrocytes, the proliferative chondrocytes and hypertrophic chondrocytes; however, ACE1 and AT2R expressed only in the hypertrophic chondrocytes. In ATDC5 cell line, angiotensinogen and AT1R expressed mainly in the proliferative term and slightly in the hypertrophic term; conversely, ACE1 and AT2R expressed mainly in the hypertrophic term and slightly in the proliferating term. In the hypertrophic term, expression of type X collagen were downregulated activating AT1R and were conversely upregulated activating AT2R.

Conclusions: The local RAS also expresses in the limb buds of mice. In the hypertrophic term of the chondrocyte differentiation, activating AT1R decelerates the hypertrophic differentiation; conversely, activating AT2R accelerates the hypertrophic differentiation. Therefore, the local RAS might regulate the chondrocyte hypertrophic differentiation.

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology and can strongly influence stem cell fate. Here we demonstrate that severe low O2 concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs) and that MSCs induced under severe hypoxic conditions function as normal MSCs that is, they repopulate after cloning, express specific markers (vimentin, CD29, CD90, CD105, and CD140a) and differentiate into adipocytes, osteoblasts, and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs. © 2013 Cognizant Comm. Corp.

Excessive mechanical stress on the cartilage causes the degradation of the matrix, leading to the osteoarthritis (OA). Matrix metalloproteinases 13 (MMP13) is a major catalytic enzyme in OA and p38 plays an important role in its induction. However, precise pathway inducing p38 activation has not been elucidated. We hypothesized here that the small GTPase Rho and its effector ROCK might function in upper part of the mechanical stress-induced matrix degeneration pathway. Bovine metacarpal phalangeal articular cartilage explants were loaded with 1 MPa dynamic compression for 6 h with or without a ROCK specific inhibitor Y27632 or/and a p38 specific inhibitor SB202190. Then p38 phosphorylation and MMP13 expression were assessed by western blot or/and quantitative RT-PCR. Rho-activity was measured by pull-down assay using glutathione S-transferase fusion protein of Rho binding domain. Cyclic compression caused Rho activation, p38 phosphorylation and MMP13 expression. Both Y27632 and SB202190 were found to block the mechanical stress-enhanced p38 phosphorylation and subsequent MMP13 expression. The present results show that p38 phosphorylation and MMP13 expression are regulated by Rho/ROCK activation, and support the potential novel pathway that Rho/ROCK is in the upper part of the mechanical stress-induced matrix degeneration cascade in cartilage comprised of p38 and MMP13.

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state. © Copyright 2012, Mary Ann Liebert, Inc.

Purpose: Recently, the local renin-angiotensin system (RAS) has attracted many researchers in many pathophysiological issues. In Orthopedics, expression of local RAS was found in bone tissues, fracture callus and arthritic synovium. The purpose of this study is to reveal immunohistological localization of the RAS components in the epiphyseal plates of mice and to analyze function of the local RAS in the processes of hypertrophic differentiation using ATDC5 chondroprogenitor cells.

Methods: The epiphyseal plates of 8-week-old mice was immunostained with antibodies to angiotensinogen, angiotensinogen converting enzyme 1 (ACE1), angiotensinII type 1 receptor (AT1R) and angiotensinII type 2 receptor (AT2R). We cultured ATDC5 in long term and evaluated the expression of angiotensinogen, ACE1, AT1R, AT2R and type 2 collagen (COL2) by real- time PCR and Western blot analysis.

Results: In the epiphyseal plates of mice, angiotensinogen and AT1R expressed in the resting chondrocytes, the proliferative chondrocytes and hypertrophic chondrocytes; however, ACE1 and AT2R expressed only in the hypertrophic chondrocytes. In ATDC5 chondroprogenitor cells, the local RAS components expressed both in proliferative and hypertrophic differentiating stages.

Conclusions: The local RAS expresses in the epiphyseal plates of mice and might play an important role in the process of hypertrophic differentiation.

Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the sternness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, ART phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the ART phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator. (C) 2011 Elsevier Inc. All rights reserved.

Recently, several research groups have shown that germ cells can be produced in vitro from pluripotent embryonic stem cells (ESCs). In the mouse, live births of offspring using germ cells induced from ESCs in vitro have been reported. Furthermore, some efficient methods for inducing the useful number of germ cells from ESCs have also been developed. On the other hand, in primates, despite the appearances of germ cell-like cells including meiotic cells were observed by spontaneous differentiation or introducing transgenes, it has not been determined whether fully functional germ cells can be derived from ESCs. To elucidate the property for the germ cells induced from primate ESCs, specification of the promoting factors for the germ cell development and improving the efficiency of germ cell derivation are essential. Leukemia inhibitory factor (LIF) has been reported as one of the important factors for mouse primordial germ cell (PGC) survival in vitro. However, the effects of LIF on germ cell formation from pluripotent cells of primates have not been examined. The aim of this study is to determine whether LIF addition can improve in vitro germ cell production from cynomolgus monkey ESCs (cyESCs). After 8 days of differentiation, LIF added culture induced dome-shaped germ cell colonies as indicated by the intense expression of alkaline phosphatase activity (ALP). These cells also demonstrate high-level expression of the germ cell-marker VASA, OCT-4, and BLIMP-1, and show SSEA-1 expression that supports their early stage germ cell identity. Finally, we observed that adding LIF to differentiating cultures inhibited meiotic gene expressions and increased the percentage of ALP-positive cells, and demonstrate that the addition of LIF to differentiation media increases differentiation of early germ cells from the cyESCs.

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals. (C) 2010 Elsevier Inc. All rights reserved.

Abnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear. Mechanical compression was loaded on the bovine cartilage using the Biopress system. Proteoglycan (PG) and reactive oxygen species (ROS) synthesis were measured with [ (35)S] incorporation and fluorescent dye, respectively. Accumulation of peroxynitrite was determined with western blotting using nitrotyrosine antibody. Mechanical compression inhibited PG synthesis and enhanced ROS. Externally added HA reversed stress-inhibited PG synthesis and attenuated ROS synthesis. HA also significantly decreased the generation of nitrotyrosine. HA neutralized stress-enhanced ROS synthesis and resulted in the reversing of PG synthesis. These data suggest that HA plays an anabolic effect as an antioxidant.

As stem cells can regenerate damaged tissue, their therapeutic potential on brain damage has been investigated. In this study, the effects of embryonic stem cell transplantation on brain damage were investigated by using a photochemically induced thrombotic brain damage model. Mice with systemic transplantation of embryonic stem cells expressing enhanced green fluorescence protein on day 1 showed a smaller brain lesion size on day 8 than the control mice. The smaller lesion was accompanied by the increase in the number of microvessels at the border of the damaged area. Inside and around the damaged lesion, no EGFP-positive cells were observed. These findings suggested that embryonic stem cell transplantation reduced the brain lesion through the acceleration of angiogenesis by endogenous endothelial cells. NeuroReport 21:575-579 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Despite recent cell-engineering advances, treatment and repair of cartilage remains challenging. Although stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a prominent strategy, the major problem of limited proliferative capacity of autologous cells has been unsolved. Recently, an induced pluripotent stem (iPS) cell line was suggested as an alternative way to cure various human diseases due to their potential proliferating infinitely while possessing the capacity to form all types of cells. However, the method to induce lineage-restricted differentiation has not been well examined or established. Here, we suggest a simple method to induce mesenchymal progenitors possessing chondrogenic property from mouse iPS cells. The MSC-like cells produced in our study expressed some MSC markers, and could also differentiate to osteoblast and adipocyte. The present study demonstrates the property of iPS cells as an alternative candidate for treatment of articular disorders, and suggests an effective approach for preparing chondrocyte from iPS cells. Copyright 2010, Mary Ann Liebert, Inc.

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.

Objective. Findings of recent in vivo and in vitro studies suggest that oxidized low-density lipoprotein (ox-LDL) plays a role in the degeneration of cartilage. The purpose of this study was to determine whether ox-LDL induces chondrocyte senescence through binding to lectin-like ox-LDL receptor 1 (LOX-1). Methods. The effects of ox-LDL on senescence of cultured bovine articular chondrocytes (BACs) were investigated by observing senescence-associated (SA) beta-galactosidase (beta-gal) activity, cell proliferation activity, and telomerase activity. Telomerase activity was measured after adding LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]) or after adding insulin-like growth factor 1 (IGF-1; an activator of PI3K) plus ox-LDL to the culture medium to elucidate the involvement of the PI3K/Akt pathway. Immunoblot analysis was used to investigate whether ox-LDL affects the phosphorylation of Akt. To ascertain whether these effects were attributable to ox-LDL binding to LOX-1, BACs were preincubated with TS-20, an anti-bovine LOX-1 blocking antibody. Results. The activity of SA beta-gal was increased and the incorporation of bromodeoxyuridine into BACs was decreased by ox-LDL in a dose-dependent manner. The telomerase activity of BACs was suppressed by the addition of ox-LDL in a time- and dose-dependent manner. LY294002 suppressed the telomerase activity of BACs, and IGF-1 reversed the ox-LDL-induced suppression of telomerase activity. In addition, ox-LDL rapidly decreased the amount of phosphorylated Akt in BACs. Pretreatment of cultured BACs with TS-20 recovered these effects. Conclusion. These data show that ox-LDL binding to LOX-1 induces stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway. Oxidized LDL may play an important role in the pathogenesis of osteoarthritis by inducing chondrocyte senescence.

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs Could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible, tissues for transplantation.

Non-human primates are suitable models for preclinical research aimed at cell-replacement therapies. Recently, it has been reported that Rho-associated kinase inhibitor Y-27632 markedly reduced dissociation-induced apoptosis of human embryonic stem (hES) cells, and is expected as a novel supplement for hES cell maintenance or differentiation inductions; however, the effects of the chemical are still to be determined in model animals. Here, we demonstrated the effect of Y-27632 on cynomolgus monkey ES (cyES) cells. Also, in cyES cells, Y-27632 treatment dramatically improved the efficiency of colony formation from single cells without affecting the pluripotent state and karyotype. Y-27632 supplementation was also effective for feeder-free culture and differentiation induction. Neural stem cells directly induced from cyES cells could give rise to neurons, astrocytes and dopamine producing cells. The present result not only suggests that the chemical was effective for improving the culture system of primate ES cells, but also the similarity between cyES and hES cells regarding the reactions to the chemical, which might be further evidence that cyES cells are superior models for hES cells.

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become Cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they call also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin oil inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.

Background: Autologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 ( Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined. Methods: Synovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg- 2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes. Results: SP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1% FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1-all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte. Conclusion: In the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.

To clarify the mechanism that impairs development of in vitro grown (IVG) oocytes, we assessed whether the developmental disability of IVG oocytes is caused by cytoplasmic dysfunction. First, we assessed the cleavage of nuclear-substituted oocytes cultured in vitro. The nuclei, but not the cytoplasm, of the IVG oocytes were able to support subsequent cleavage after artificial activation. The mitochondrial activity of the oocytes increased as the follicles grew. However, the mitochondrial activity of the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 mu m. Furthermore, the expression levels of mitochondrial transcriptional factor A (TFAM) in the oocytes increased in a similar manner. However, the expression levels of TFAM in the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 mu m. Taken together, these results indicate that the low developmental competence of IVG oocytes is caused by a cytoplasm deficiency due to low mitochondrial activity.

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development. © Mary Ann Liebert, Inc.

Embryonic stem cells (ESCs) are a good material for the study of mammalian development, production of genetically modified animals, and drug discovery because they proliferate infinitely while maintaining a multilinege differentiation potency and a normal karyotype. However, ethical considerations limit the use of human embryos for the establishment of ESCs. Recently, ESCs have been produced from blastomeres divided by biopsy in mice and humans. The method is expected to be less controversial because it does not destroy the embryo. However, no one has yet produced both a pup and an ESC from a single embryo. Here, we describe the production of individual/ESC pairs from each of three embryos out of 20 attempts, and is thus considered efficient. Blastomere-derived ESC could differentiate some types of tissues and contribute to chimera mouse. These results show that each blastomere at two-cell stage possesses pluripotency and separated blastomeres maintain viability to develop to a pup or pluripotent ESC. © 2007 Mary Ann Liebert, Inc.

KRAS阻害剤ソトラシブ（AMG510）の耐性機序の解明、さらに耐性克服治療の開発につなげることが目的。 研究方法としてKRAS変異陽性でソトラシブ感受性肺癌細胞株H23及び同耐性株H23ARC10を用いて耐性の原因遺伝子の探索を行う。すでに網羅的な遺伝子解析によって耐性株でMET遺伝子増幅の出現を確認。さらに同耐性株に対しMET阻害剤の有効性をマウスモデルで確認。これらの研究成果はすでに論文発表をおこなった（Suzuki S, Yonesaka K, et al. Clin Cancer Research2021)。MET遺伝子増幅はSOS1、SHP2を介し、変異型及び野生型RASシグナルの増強をもたらした。このためSOS1、SHP2阻害剤とソトラシブの併用にも有効性を認めた。 現在あらたに樹立したソトラシブ耐性株H2122ARについて耐性機序の解明と耐性克服治療の検討を行っている。すなわち同耐性株について次世代シークエンサーによる遺伝子解析、マイクロアレイによる発現解析を実施。同定された遺伝子の異常に関連した阻害剤の投与試験を行っている。またその遺伝子について機能解析も行い、耐性のメカニズムを確認中である。同時にマウスモデル試験も開始しており、今後の耐性克服治療の臨床応用を目指した実験を行っている。またKRAS変異陽性患者由来の血液、腫瘍組織を収集しておりこれらについての遺伝子解析も予定している。

間葉系幹細胞(Mesenchymal stem cell : MSC、stromal stem cell)は骨髄や脂肪などから分離される組織幹細胞であり、優れたサイトカイン産生能力と分化能を有する事から様々な疾患に対する再生医療材料としての期待が高まっている。一方で、多能性幹細胞とは異なり、ドナー年齢や細胞の継代数などにより劣化しやすく、維持・供給が安定しないという課題がある。また、品質を評価するマーカーが存在しないため、供給された細胞の品質がばらつくという問題も抱えている。申請者らは、転写因子Twist1がMSCの幹細胞性に極めて重要な分子であり、MSCの性能を評価する上で有用であることを発見している。体内において、MSCは骨髄、脂肪組織、筋組織等の血管周囲や間質に見られる。体外培養時と同様、体内においてもMSCは加齢や炎症などストレスの影響を受けるため、若齢患者から得られるMSCと高齢患者から得られるMSCでは性質、品質が全く異なることがわかってきている。一方で、体内に存在するMSCは、存在場所、ストレス蓄積量、それまでの分裂回数など複数の要素によってその品質が非常にヘテロな状態になっており、たとえ高齢ドナーであっても適した細胞マーカーに基づき評価分別できれば、より高い治療効果を安定的に得られると考えられる。本研究では、同知見を有用な医療技術として発展させる為、Twist1を操作しヒトMSCの作製・評価を目的として行う。

間葉系幹細胞(Mesenchymal Stem Cell：MSC)を用いた再生医療が次々と進められているが、 その細胞供給には根本的な問題がある。新たなMSCの供給源としてヒト多能性幹細胞からの分化誘導が進められているが、普及には至っていない。その理由として、ヒトMSCの発生機序、詳細な性質と正しい分類、由来組織や時期、培養方法による性質の変化など細胞そのものに対する基本的理解が進んでいないことにある。一方で、ヒトの発生解析は、倫理的にも技術的にも実施困難である。本研究では、ヒトNaive型多能性幹細胞を出発点とした試験管内における初期胚発生モデルGastruloidの構築と、これを用いた間葉系幹細胞の発生機序の解明を行う。Gastruloidは三次元的にヒト初期胚発生を模した培養技術であり、現存する発生モデルとしては最もin vivoに近い。本研究では、発生初期がブラックボックスに覆われるヒト間葉系原基について、ヒトNaive型多能性幹細胞より誘導したGastruloidを用い、発生機序の解明と新しい分化誘導法の確立を試みる。 令和3年度では、本研究の重要なツールであるGastruloid誘導法の確立を試みた。すでにいくつかのGastruloid法は報告されているが、確立されたとはいえない。そこで、Wnt/beta catenin経路の活性化あるいは不活性化による誘導法のどちらが適しているか検討した。すると、マウス多能性幹細胞ではどちらの方法も可能であったが、ヒト多能性幹細胞では、Wnt/beta catenin経路の阻害ではGastruloidの誘導ができなかった。これは、多能性幹細胞の状態が大きく影響していることが示唆される。正常な発生過程を体外で作り出すためには多能性幹細胞の状態を考慮する必要があると考えられる。

高齢化が急速に進行する中で、加齢性組織変性・疾患とその主因の一つである幹細胞老化を理解し方策を講じることは重要な課題である。筋骨格組織における幹細胞としては筋衛星細胞（Muscular Satellite Cell: MuSC）や間葉系幹細胞（Mesenchymal stem cell: MSC）が知られており、加齢に伴う細胞数の減少、自己複製能や分化能の低下が知られている。一方、老化研究においては体外培養下での培養細胞の表現系が個体の老化を反映しないため、転写解析、エピジェネティック解析を始め、モデル研究から得られた知見については明確な結論が得られている機序は限られていた。本研究では、ATACseq、NET-CAGEseq、IP質量分析といったオミックス解析を駆使しながら、幹細胞老化の本質となるメカニズムを明らかにすることを目的とする。さらに、新しい遺伝子治療技術SRVなどを取り入れ、加齢性に減少したクロマチン修飾因子、転写活性化因子の補完をはじめとする老化形質の制御法の開発を試みる。 本年度は、老化モデルマウスよりFACSで分離した筋MuSC、骨髄MSCを用いてトランスクリプトーム解析を行った。その結果、加齢MuSCで著しく発現が低下する分子としてNek2を抽出した。モデル細胞を用いた検討において、Nek2がG2期制御と非対称分裂の制御によって幹細胞の自己複製/分化の制御に関わっており、老化筋組織ではその調節が破綻している可能性を見出した。 また、昨年度までに申請者らが報告した炎症メディエターMAP3KであるTAK1と相互作用する分子をIP質量分析にて検索し、核内タンパク質Lyarを同定した。Lyarは老化骨髄MSCで発現が低下しており、トランスクリプトーム解析の結果、Lyarの機能は脂肪分化制御にあることを発見している。モデル細胞を用いた解析により、Lyarの減少は脂肪分化の促進をもたらすことが明らかとなり、老化骨髄でみられる脂肪組織蓄積を引き起こしうる新たな分子機序が示された。

組織幹細胞の老化は、組織恒常性の低下を引き起こす要因の一つであると考えられている。酸化ストレス(Reactive oxygen species: ROS)の蓄積は細胞老化の代表的な形質であり機能低下の主因である。劣化したミトコンドリア、ペルオキシダーゼの残留は強大なROSの発生源であるが、それを引き起こす機構はよくわかっていない。今回、老化細胞で特異的に発現が上昇するmiRNA（Aging-associated miRNA: Ag-miRNA）が劣化ミトコンドリアやペルオキシダーゼ分解の調節因子であるという仮説を立てた。本研究では骨髄内の間葉系幹細胞(Mesenchymal stem cell、MSC)をモデルにAg-miRNAを同定し、その働きを明らかにすることにより幹細胞老化の新しい機構を明らかにすることが本研究の目的である。 昨年度までに、骨髄MSCで加齢性に蓄積するAg-miRNAの同定と解析を進め、miR-155、MIR-142がそれぞれミトコンドリアとペルオキシソームの更新を阻害し、活性酸素の発生を誘導しうることを発見、報告した。一方で、老化組織環境とmiR-155やmiR-142の発現を結びつける機序には不明な点が多い。そこで、昨年度以降はmiR-155、142以外のAg-miRNAの解析に加え、組織環境とAg-miRNA発現の関係性について検討を進めている。

BMMSCs are multipotent stem cells capable of differentiation into a variety of cell types, proliferation, and production of clinically useful secretory factors. These advantages make BMMSCs highly useful for cell transplantation therapy. However, the molecular network underlying BMMSC proliferation remains poorly understood. Here, we showed that Tak1 is a critical molecule that regulates the activation of cell cycling and that Tak1 inhibition leads to quiescence in BMMSCs both in vivo and in vitro.Tak1 was phosphorylated by growth factor stimulations, allowing it to bind and stabilize Yap1/Taz, which could then be localized to the nucleus. We also demonstrated that the quiescence induction by inhibiting Tak1 increased oxidized stress tolerance and improved BMMSC engraftment in intrabone marrow cell transplantation models. This study reveals a novel pathway controlling BMMSC proliferation and suggests a useful method to improve the therapeutic effect of BMMSC transplantation.

再生医療の中心的な細胞材料である間葉系幹細胞の治療効果を向上させるとともに、新規の細胞原料を開発するため、転写因子Twist1の機能解明を行う。

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent stem cells capable of differentiation into a variety of cell types, proliferation, and production of clinically useful secretory factors. However, the molecular network underlying BMMSC proliferation remains poorly understood. Here, we showed that Tgfβ-activated kinase (Tak1) is a critical molecule that regulates the activation of cell cycling and that Tak1 inhibition leads to quiescence in BMMSCs. Mechanistically, Tak1 was phosphorylated by growth factor stimulations, binding with Yap1/Taz, and supported their nuclear localization through stabilization of Yap1/Taz in proliferating BMMSCs. Furthermore, we also demonstrated that the cell-cycle synchronization in quiescence by Tak1 inhibition significantly improved engraftment after intra-bone marrow cell transplantation of BMMSCs. This study may suggest a novel central pathway controlling the BMMSC proliferation and useful pre-treatment for cell transplantation.

本研究では運動器の再生や維持に重要な間葉系幹細胞におけるNrf2の機能、老化との関連性、発現制御機構と発現をコントロールする介入法について検討する。 研究初年度は、Nrf2の機能と老化との関わりについて検討をおこなった。老化では慢性的な老化関連性炎症が生じていることがわかってきている。我々は本研究において、老化組織ならびに炎症刺激においてmiR-155が上昇していることを発見した。また、miR-155がNrf2を標的としその発現を低下させていることを発見した。現在、研究成果を論文投稿中である。現在、マウス筋前駆細胞株C2C12を用いて詳細な検討を行っている。なお、今後の解析に必要なNrf2発現ベクター、ノックダウンシステム等は今年度に準備を完了した。

H27年度に確立した方法によりfeeder lessのiPS細胞からMSC様の細胞を誘導し、それらの性状解析を行った。iPS細胞から2週間でCD44, CD73, CD90, CD105が陽性の純度が高いMSC様細胞が得られることがわかり、多能性マーカー（TRA-1-60, Oct3/4, Nanog, Sox2, Lin28a）の発現消失および減少が確認された。次にこれらの細胞の骨、軟骨、脂肪細胞への分化能を調べたところ、それぞれの系列への分化能は有するものの生体から採取したMSCと比較すると、その分化能は低く、再生医療に用いるに十分な細胞が得られたとは言えなかった。ここで用いたiPS細胞からのMSC様細胞誘導法は血清培地を用いるだけの簡便な手法であるが、iPS細胞の自発的な分化に依存した方法で、生体内の発生を模倣した誘導系とは言い難い。そこで、骨軟骨系列への分化能を高く有するMSC様細胞を誘導するには、骨軟骨の発生を模倣した誘導系が必要であると考え、初期胚の胚様の中でも神経堤発生経路に着目した。神経堤細胞はヒトiPS細胞から高い効率で誘導可能であるだけでなく、その後の増幅培養が可能である事が知られており、応用利用するに有望な細胞である。神経堤を介して誘導したMSC様細胞の軟骨疾患への応用を検討するため、京都大学iPS細胞研究所よりヒトiPS細胞由来神経堤細胞の提供を受け、その細胞を血清培地で培養する事でMSC様細胞へ誘導した。神経堤細胞を血清培地で培養後1-2週以内にMSCマーカー(CD44, 73, 105)陽性かつ神経堤マーカー(CD271)陰性の細胞集団が再現性高く得られることがわかり、さらに多能性マーカー（TRA-1-60, Oct3/4, Nanog, Sox2, Lin28a）の発現は著しく消失していることから安全性についても鑑みられた。さらに神経堤由来MSC様細胞がin vitroの分化誘導において高い骨分化能、軟骨分化能を示すことを確認した。

間葉系幹細胞（MSCs）は再生医療の中心的材料として注目されているが、培養による形質の劣化が顕著であり、最適な培養系、安定的な分化誘導法が必要とされている。一方で、MSCの維持・発生に関わる分子経路は明らかになっていない。申請者は、上皮間葉転換の制御因子であるTwist1に着目し、①幹細胞維持に関わる遺伝子の発現と相関すること、②エピジェネティック修飾因子と相互作用すること、③ヒト単球において、リプログラミング因子とともにTwist1を発現させると、MSC様細胞に直接転換するという結果を得た。以上から、Twist1はMSCにおいて重要な因子であることが明らかになった。

非侵襲的な変形性膝関節症（OA）の治療においては、破壊責任分子経路の同定とこれに介入する方法を確立する必要がある．本研究では細胞内にストレスを伝達し軟骨に組織破壊的変化をもたらす分子としてRho/ROCK経路とそれを取り巻く分子経路に着目した。活性酸素によるストレスはRho/ROCK、TAK1の活性化を誘導し、軟骨基質の発現抑制と滑膜炎症変化を誘導した。一方でRac、Nrf2の活性化は軟骨基質の発現、抗ストレス分子の発現を誘導した。これらの分子はいずれもストレス応答経路として重要であり、薬理的介入は新規のOA治療戦略として有望である。

新規間葉系幹細胞（MSC）ソースの探索を目的に、多能性幹細胞からのMSCの誘導と評価を実施した。SCIDマウス内でのテラトーマ形成を介したin vivoでの分化誘導、低酸素培養によるin vitroでの誘導両方法により、典型的なMSCの形質を示す多能性幹細胞由来MSC様細胞（pcMSC）が得られた。In vitro誘導pcMSCは移植後の腫瘍形成性が認められず、軟骨再生に寄与した。しかし、in vivo誘導pcMSCにおいては継代培養により高い腫瘍原性が出現した。以上より、多能性幹細胞は有望なMSCソースとなりうるが、腫瘍原性などが異なる場合があるため慎重な検討を要するという結論が得られた。

今までに確立したヒト滑膜間葉系幹細胞（MSC）由来三次元人工組織（Tissue Engineered Construct, TEC）を作製する技術を用いて、ウサギ胚性幹細胞由来MSCでTEC（ES-TEC）を作成した。ES-TECは軟骨分化誘導後に滑膜MSC-TEC(Sy-TEC)と比べ軟骨関連遺伝子の発現が有意に高値であった。ウサギ膝関節骨軟骨欠損へのTEC/β-TCP複合体移植を行いに修復組織を検討したところES-TECでSy-TECに比しては硝子軟骨様組織による修復が促進されており、臨床応用が期待できる。またiPS細胞を用いて短期間でTECを作成する技術も開発した。

本研究の目的は、酸化低密度リポ蛋白(酸化LDL)が軟骨組織の老化変性に関与することを、レクチン様酸化LDL受容体―1(LOX-1)knockout (KO) mouseを用いてin vivoで明らかにすることである.トレッドミルを用いてCB57/BL6マウスを強制走行(週3回、2週間に1Km)させると、非侵襲的に変形性膝関節症を生じることが明らかになった.また、これらの変化は走行週数、一回当たり走行距離により調節可能であった.そこでLOX-1 KO mouseを強制走行させ、膝軟骨変性における酸化LDL/LOX-1系の関与を明らかにすることを試みたが、両マウスの変形の程度において2週間の走行では有意な差は無かった.次に、6ヶ月、1年、1.5年と長期間自然飼育したwild typeとKO mouseの膝軟骨変性を観察した.その結果、飼育開始後1、1.5年において軟骨変性がKO mouseにおいて有意に抑制されていた.さらに、Zymosan関節内投与による関節炎モデルマウスにおいても、LOX-1 KOにより滑膜炎症、軟骨変性の抑制が認められ、LOX-1と関節症変化の関連が示された.以上の結果より、酸化LDL、および酸化LDLの受容体LOX-1は血管内皮細胞の老化促進因子であるのみならず軟骨細胞の老化変性にも関与を示し、変形性関節症の薬物的治療に新たな可能性を示すことができたと考える.

本研究では、ヒトiPS細胞からの効率的な軟骨細胞誘導法の開発と誘導軟骨細胞の特性解析を実施した。1) iPS細胞から間葉系幹細胞(MSCs)を誘導し、段階的に軟骨細胞を誘導することで、効率的な軟骨細胞の獲得を試みた。誘導環境を低酸素状態とすることで、高率にMSC-like性質を有する細胞が得られることを見出した。2)誘導したMSCsについて発現解析を実施し、誘導MSCsが骨髄由来MSCsに類似した発現パターンを示すこと、骨芽細胞、軟骨細胞、脂肪細胞への誘導が可能であることを示した。また、3)未分化iPS細胞およびiPS由来軟骨細胞に機械的ストレスを加え、未分化維持分子機構に及ぼす影響を検討し、機械的ストレスの負荷は未分化性の制御に影響しうることを明らかにした。

現在、関節軟骨損傷に対する再生医療では自家軟骨細胞が用いられているが、細胞材料としての限界から幹細胞への期待が高まっている。本研究では幹細胞の分離培養方法の開発、細胞特性の解明および多能性幹細胞の可能性に関する検討を行った。その結果、血清低減下など組織幹細胞の未分化維持および増殖に重要な因子が抽出され、これらの知見を応用する事で、多能性幹細胞から有用な細胞を選択的に誘導する方法が開発された。

関節軟骨移植(ACI)は運動器疾患に対する代表的な再生医療である.本研究では,ACIの力学的環境に関する検討を行なった.培養軟骨片への圧迫負荷により,プロテオグリカン(PG)合成の抑制と活性酸素量の増加を認めた.そこで,酸化ストレスの緩和を目的にヒアルロン酸(HA)の添加を検討した.HAは活性酸素を抑制し,PG産生量を回復させた.このことから,ACIの環境改善におけるHA投与の有用性が示唆された.