Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

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.

LRRC15 expression indicates high level of stemness regulated by TWIST1 in mesenchymal stem cells.

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.

Fibrinogen supports self-renewal of mesenchymal stem cells under serum-reduced condition through autophagy activation.

Mesenchymal stem cells (MSCs) are somatic stem cells used in cell transplantation therapy for tissue injuries and inflammatory diseases because of their ability to support tissue regeneration and to suppress inflammation. While their applications are expanding, needs for automation of culture procedures with reduction of animal-derived materials to meet stable quality and suppliability are also increasing. On the other hand, the development of molecules that safely support cell adherence and expansion on a variety of interfaces under the serum-reduced culture condition remains a challenge. We report here that fibrinogen enables MSC culture on various materials with low cell adhesion property even under serum-reduced culture conditions. Fibrinogen promoted MSC adhesion and proliferation by stabilizing basic fibroblast growth factor (bFGF), which was secreted in the culture medium by autocrine, and also activated autophagy to suppress cellar senescence. Fibrinogen coating allowed MSCs expansion even on the polyether sulfone membrane that represents very low cell adhesion, and the MSCs showed therapeutic effects in a pulmonary fibrosis model. This study demonstrates that fibrinogen is currently the safest and most widely available extracellular matrix and can be used as a versatile scaffold for cell culture in regenerative medicine.

Comparison of biological measurement and physical estimates of space radiation in the International Space Station.

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.

Adipose-derived exosomes block muscular stem cell proliferation in aged mouse by delivering miRNA Let-7d-3p that targets transcription factor HMGA2.

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.

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

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.

C10orf10/DEPP activates mitochondrial autophagy and maintains chondrocyte viability in the pathogenesis of osteoarthritis.

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.

Surface modification of the cubic micro-cartilage by collagenase treatment and its efficacy in cartilage regeneration for ear tissue engineering.

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.

Depletion of NIMA-related kinase Nek2 induces aberrant self-renewal and apoptosis in stem/progenitor cells of aged muscular tissues.

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.

KRAS Inhibitor Resistance in MET-Amplified KRAS G12C Non-Small Cell Lung Cancer Induced By RAS- and Non-RAS-Mediated Cell Signaling Mechanisms.

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 immunologic 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 small interfering RNA (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.

Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

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.

Folate receptor α increases chemotherapy resistance through stabilizing MDM2 in cooperation with PHB2 that is overcome by MORAb-202 in gastric cancer.

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.

Efficacy of sliced nerves of different thickness in a biodegradable nerve conduit to promote Schwann cell migration and axonal growth: An experimental study in the rat model.

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.

Use of sliced or minced peripheral nerve segments for nerve regeneration through a biodegradable nerve conduit: A preliminary study in the rat.

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.

miR-142 induces accumulation of reactive oxygen species (ROS) by inhibiting pexophagy in aged bone marrow mesenchymal stem cells.

Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.

miR-155 inhibits mitophagy through suppression of BAG5, a partner protein of PINK1.

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.

Transforming Growth Factor ß-Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins.

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. These advantages make BMMSCs highly useful for cell transplantation therapy. However, the molecular network underlying BMMSC proliferation remains poorly understood. Here, we showed that TGFß-activated kinase 1 (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. Mechanistically, 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 intramuscular and 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. Stem Cells 2019;37:1595-1605.

Inflammation-associated miR-155 activates differentiation of muscular satellite cells.

Tissue renewal and muscle regeneration largely rely on the proliferation and differentiation of muscle stem cells called muscular satellite cells (MuSCs). MuSCs are normally quiescent, but they are activated in response to various stimuli, such as inflammation. Activated MuSCs proliferate, migrate, differentiate, and fuse to form multinucleate myofibers. Meanwhile, inappropriate cues for MuSC activation induce premature differentiation and bring about stem cell loss. Recent studies revealed that stem cell regulation is disrupted in various aged tissues. We found that the expression of microRNA (miR)-155, which is an inflammation-associated miR, is upregulated in MuSCs of aged muscles, and this upregulation activates the differentiation process through suppression of C/ebpß, which is an important molecule for maintaining MuSC self-renewal. We also found that Notch1 considerably repressed miR-155 expression, and loss of Notch1 induced miR-155 overexpression. Our findings suggest that miR-155 can act as an activator of muscular differentiation and might be responsible for accelerating aging-associated premature differentiation of MuSCs.

Laser-assisted cell removing (LACR) technology contributes to the purification process of the undifferentiated cell fraction during pluripotent stem cell culture.

Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine.