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.

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.

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.

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.

Involvement of ADAM12 in Chondrocyte Differentiation by Regulation of TGF-ß1-Induced IGF-1 and RUNX-2 Expressions.

A disintegrin and metalloproteinase 12 (ADAM12) is known to be involved in chondrocyte proliferation and maturation; however, the mechanisms are not fully understood. In this study, expression and localization of ADAM12 during chondrocyte differentiation were examined in the mouse growth plate by immunohistochemistry. Adam12 expression during ATDC5 chondrogenic differentiation was examined by real-time PCR and compared with the expression pattern of type X collagen. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to generate Adam12-knockout (KO) ATDC5 cells. Adam12-KO and Adam12 overexpressing cells were used for analyses of ADAM12 expression with or without TGF-ß1 stimulation. ADAM12 was identified predominantly in chondrocytes of the proliferative zone in mouse growth plates by immunohistochemistry. Adam12 was upregulated prior to Col10a1 during chondrogenic differentiation in wild-type ATDC5 cells. In Adam12-KO ATDC5 cells, following initiation of chondrogenic differentiation, we observed a reduction in Igf-1 expression along with an upregulation of hypertrophy-associated Runx2, Col10a1, and type X collagen protein expressions. In ATDC5 wild-type cells, stimulation with TGF-ß1 upregulated the expressions of Adam12 and Igf-1 and downregulated the expression of Runx2. In contrast, in Adam12-KO ATDC5 cells, these TGF-ß1-induced changes were suppressed. Adam12 overexpression resulted in an upregulation of Igf-1 and downregulation of Runx2 expression in ATDC5 cells. The findings suggest that ADAM12 has important role in the regulation of chondrocyte differentiation, potentially by regulation of TGF-ß1-dependent signaling and that targeting of ADAM12 may have a role in management of abnormal chondrocyte differentiation.

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.

miR-155 induces ROS generation through downregulation of antioxidation-related genes in mesenchymal stem cells.

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/ebpß. 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.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes.

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.

Inflammation-induced miRNA-155 inhibits self-renewal of neural stem cells via suppression of CCAAT/enhancer binding protein ß (C/EBPß) expression.

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/EBPß, 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.

Cdh2 stabilizes FGFR1 and contributes to primed-state pluripotency in mouse epiblast stem cells.

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 over-expression 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.

Hyaluronic acid regulates a key redox control factor Nrf2 via phosphorylation of Akt in bovine articular chondrocytes.

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.

Reactive oxygen species induce Cox-2 expression via TAK1 activation in synovial fibroblast cells.

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-κB 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.

Induction of functional mesenchymal stem cells from rabbit embryonic stem cells by exposure to severe hypoxic conditions.

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 severelow O(2) 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.