Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Down-Regulation of Transglutaminase 2 Stimulates Redifferentiation of Dedifferentiated Chondrocytes through Enhancing Glucose Metabolism.

Expansion of chondrocytes for repair of articular cartilage can lead to dedifferentiation, making it difficult to obtain a sufficient quantity of chondrocytes. Although previous studies have suggested that culture in a three-dimensional environment induces redifferentiation of dedifferentiated chondrocytes, its underlying mechanisms are still poorly understood in terms of metabolism compared with a two-dimensional environment. In this study, we demonstrate that attenuation of transglutaminase 2 (TG2), a multifunctional enzyme, stimulates redifferentiation of dedifferentiated chondrocytes. Fibroblast-like morphological changes increased as TG2 expression increased in passage-dependent manner. When dedifferentiated chondrocytes were cultured in a pellet culture system, TG2 expression was reduced and glycolytic enzyme expression up-regulated. Previous studies demonstrated that TG2 influences energy metabolism, and impaired glycolytic metabolism causes chondrocyte dedifferentiation. Interestingly, TG2 knockdown improved chondrogenic gene expression, glycolytic enzyme expression, and lactate production in a monolayer culture system. Taken together, down-regulation of TG2 is involved in redifferentiaton of dedifferentiated chondrocytes through enhancing glucose metabolism.

Enhancement of Matrix Metalloproteinase-2 (MMP-2) as a Potential Chondrogenic Marker during Chondrogenic Differentiation of Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hASCs) have a capacity to undergo adipogenic, chondrogenic, and osteogenic differentiation. Recently, hASCs were applied to various fields including cell therapy for tissue regeneration. However, it is hard to predict the direction of differentiation of hASCs in real-time. Matrix metalloproteinases (MMPs) are one family of proteolytic enzymes that plays a pivotal role in regulating the biology of stem cells. MMPs secreted by hASCs are expected to show different expression patterns depending on the differentiation state of hASCs because biological functions exhibit different patterns during the differentiation of stem cells. Here, we investigated proteolytic enzyme activity, especially MMP-2 activity, in hASCs during their differentiation. The activities of proteolytic enzymes and MMP-2 were higher during chondrogenic differentiation than during adipogenic and osteogenic differentiation. During chondrogenic differentiation, mRNA expression of MMP-2 and the level of the active form of MMP-2 were increased, which also correlated with Col II. It is concluded that proteolytic enzyme activity and the level of the active form of MMP-2 were increased during chondrogenic differentiation, which was accelerated in the presence of Col II protein. According to our findings, MMP-2 could be a candidate maker for real-time detection of chondrogenic differentiation of hASCs.

Ultrasound-triggered three dimensional hyaluronic acid hydrogel promotes in vitro and in vivo reprogramming into induced pluripotent stem cells.

Cellular reprogramming technologies have been developed with different physicochemical factors to improve the reprogramming efficiencies of induced pluripotent stem cells (iPSCs). Ultrasound is a clinically applied noncontact biophysical factor known for regulating various cellular behaviors but remains uninvestigated for cellular reprogramming. Here, we present a new reprogramming strategy using low-intensity ultrasound (LIUS) to improve cellular reprogramming of iPSCs in vitro and in vivo. Under 3D microenvironment conditions, increased LIUS stimulation shows enhanced cellular reprogramming of the iPSCs. The cellular reprogramming process facilitated by LIUS is accompanied by increased mesenchymal to epithelial transition and histone modification. LIUS stimulation transiently modulates the cytoskeletal rearrangement, along with increased membrane fluidity and mobility to increase HA/CD44 interactions. Furthermore, LIUS stimulation with HA hydrogel can be utilized in application of both human cells and in vivo environment, for enhanced reprogrammed cells into iPSCs. Thus, LIUS stimulation with a combinatorial 3D microenvironment system can improve cellular reprogramming in vitro and in vivo environments, which can be applied in various biomedical fields.

Renal sympathetic denervation suppresses de novo podocyte injury and albuminuria in rats with aortic regurgitation.

BACKGROUND: The presence of chronic kidney disease is a significant independent risk factor for poor prognosis in patients with chronic heart failure. However, the mechanisms and mediators underlying this interaction are poorly understood. In this study, we tested our hypothesis that chronic cardiac volume overload leads to de novo renal dysfunction by coactivating the sympathetic nervous system and renin-angiotensin system in the kidney. We also examined the therapeutic potential of renal denervation and renin-angiotensin system inhibition to suppress renal injury in chronic heart failure. METHODS AND RESULTS: Sprague-Dawley rats underwent aortic regurgitation and were treated for 6 months with vehicle, olmesartan (an angiotensin II receptor blocker), or hydralazine. At 6 months, albuminuria and glomerular podocyte injury were significantly increased in aortic regurgitation rats. These changes were associated with increased urinary angiotensinogen excretion, kidney angiotensin II and norepinephrine (NE) levels, and enhanced angiotensinogen and angiotensin type 1a receptor gene expression and oxidative stress in renal cortical tissues. Aortic regurgitation rats with renal denervation had decreased albuminuria and glomerular podocyte injury, which were associated with reduced kidney NE, angiotensinogen, angiotensin II, and oxidative stress. Renal denervation combined with olmesartan prevented podocyte injury and albuminuria induced by aortic regurgitation. CONCLUSIONS: In this chronic cardiac volume-overload animal model, activation of the sympathetic nervous system augments kidney renin-angiotensin system and oxidative stress, which act as crucial cardiorenal mediators. Renal denervation and olmesartan prevent the onset and progression of renal injury, providing new insight into the treatment of cardiorenal syndrome.

MMP13-Overexpressing Mesenchymal Stem Cells Enhance Bone Tissue Formation in the Presence of Collagen Hydrogel.

BACKGROUND: Matrix metalloproteinases (MMPs) are proteins involved in the repair and remodeling the extracellular matrix (ECM). MMP13 is essential for bone development and healing through the remodeling of type I collagen (COL1), the main component of the ECM in bone tissue. Mesenchymal stem cells (MSCs)-based cell therapy has been considered a promising approach for bone regeneration because of their osteogenic properties. However, the approaches using MSC to completely regenerate bone tissue have been limited. To overcome the limitation, genetic engineering of MSC could be a strategy for promoting regeneration efficacy. METHODS: We performed in vitro and in vivo experiments using MMP13-overexpressing MSCs in the presence of COL1. To examine MMP13-overexpressing MSCs in vivo, we prepared a fibrin/COL1-based hydrogel to encapsulate MSCs and subcutaneously implanted gel-encapsulated MSCs in nude mice. We found that the osteogenic marker genes, ALP and RUNX2, were upregulated in MMP13-overexpressing MSCs through p38 phosphorylation. In addition, MMP13 overexpression in MSCs stimulated the expression of integrin α3, which is up-stream receptor of p38, and substantially increased osteogenic differentiation potential of MSCs. Bone tissue formation in MMP13-overexpressing MSCs was significantly higher than that in control MSCs. Taken together, our findings demonstrate that MMP13 is not only an essential factor for bone development and bone healing but also has a pivotal role in promoting osteogenic differentiation of MSCs to induce bone formation. CONCLUSION: MSCs Genetically engineered to overexpress MMP13, which have a powerful potential to differentiate into the osteogenic cells, might be beneficial in bone disease therapy.

Generation of bioactive MSC-EVs for bone tissue regeneration by tauroursodeoxycholic acid treatment.

Extracellular vesicles (EVs) are nano-sized carriers that reflect the parent cell's information and are known to mediate cell-cell communication. In order to overcome the disadvantages of mesenchymal stem cells (MSCs) in cell therapy, such as unexpected differentiation leading to tumorization, immune rejection, and other side effects, EVs derived from MSCs (MSC-EVs) with the tissue regenerative function have been studied as new cell-free therapeutics. However, therapeutic applications of EVs require overcoming several challenges. First, the production efficiency of MSC-EVs should be increased at least as much as the quantity of them are required to their clinical application; second, MSC-EVs needs to show various functionality further, thereby increasing tissue regeneration efficiency. In this study, we treated tauroursodeoxycholic acid (TUDCA), a biological derivative known to regulate cholesterol, to MSCs and investigated whether TUDCA treatment would be able to increase EV production efficiency and tissue regenerative capacity of EVs. Indeed, it appears that TUDCA priming to MSC increases the yield of MSC-EVs >2 times by reducing the cellular cholesterol level in MSCs and increasing the exocytosis-related CAV1 expression. Interestingly, it was found that the EVs derived from TUDCA-primed MSCs (T-EV) contained higher amounts of anti-inflammatory cytokines (IL1RN, IL6, IL10, and IL11) and osteogenic proteins (ALP, RUNX2, BMP2, BMPR1, and BMPR2) than those in control MSC-EVs (C-EV). Besides, it was shown that T-EV not only regulated M1/M2 macrophages differentiation of monocytes, also effectively increased the osteogenic differentiation of MSCs as well as bone tissue regeneration in a bone defect rat model. Based on these results, it is concluded that TUDCA treatment to MSC as a new approach endows EV with high-yield production and functionality. Thus, we strongly believe T-EV would be a powerful therapeutic material for bone tissue regeneration and potentially could be expanded to other types of tissue regeneration for clinical applications.

Combinatorial Effect of Mesenchymal Stem Cells and Extracellular Vesicles in a Hydrogel on Cartilage Regeneration.

BACKGROUND: Mesenchymal stem cells (MSCs) are used for tissue regeneration due to their wide differentiation capacity and anti-inflammatory effects. Extracellular vesicles (EVs) derived from MSCs are also known for their regenerative effects as they contain nucleic acids, proteins, lipids, and cytokines similar to those of parental cells. There are several studies on the use of MSCs or EVs for tissue regeneration. However, the combinatorial effect of human MSCs (hMSCs) and EVs is not clear. In this study, we investigated the combinatorial effect of hMSCs and EVs on cartilage regeneration via co-encapsulation in a hyaluronic-acid (HA)-based hydrogel. METHODS: A methacrylic-acid-based HA hydrogel was prepared to encapsulate hMSCs and EVs in hydrogels. Through in vitro and in vivo analyses, we investigated the chondrogenic potential of the HA hydrogel-encapsulated with hMSCs and EVs. RESULTS: Co-encapsulation of hMSCs with EVs in the HA hydrogel increased the chondrogenic differentiation of hMSCs and regeneration of damaged cartilage tissue compared with that of the HA hydrogel loaded with hMSCs only. CONCLUSION: Co-encapsulation of hMSCs and EVs in the HA hydrogel effectively enhances cartilage tissue regeneration due to the combinatorial therapeutic effect of hMSCs and EVs. Thus, in addition to cartilage tissue regeneration for the treatment of osteoarthritis, this approach would be a useful strategy to improve other types of tissue regeneration.

Progress and emerging techniques for biomaterial-based derivation of mesenchymal stem cells (MSCs) from pluripotent stem cells (PSCs).

The use of mesenchymal stem cells (MSCs) for clinical purposes has skyrocketed in the past decade. Their multilineage differentiation potentials and immunomodulatory properties have facilitated the discovery of therapies for various illnesses. MSCs can be isolated from infant and adult tissue sources, which means they are easily available. However, this raises concerns because of the heterogeneity among the various MSC sources, which limits their effective use. Variabilities arise from donor- and tissue-specific differences, such as age, sex, and tissue source. Moreover, adult-sourced MSCs have limited proliferation potentials, which hinders their long-term therapeutic efficacy. These limitations of adult MSCs have prompted researchers to develop a new method for generating MSCs. Pluripotent stem cells (PSCs), such as embryonic stem cells and induced PSCs (iPSCs), can differentiate into various types of cells. Herein, a thorough review of the characteristics, functions, and clinical importance of MSCs is presented. The existing sources of MSCs, including adult- and infant-based sources, are compared. The most recent techniques for deriving MSCs from iPSCs, with a focus on biomaterial-assisted methods in both two- and three-dimensional culture systems, are listed and elaborated. Finally, several opportunities to develop improved methods for efficiently producing MSCs with the aim of advancing their various clinical applications are described.

Emerging nano-scale delivery systems for the treatment of osteoporosis.

Osteoporosis is a pathological condition characterized by an accelerated bone resorption rate, resulting in decreased bone density and increased susceptibility to fractures, particularly among the elderly population. While conventional treatments for osteoporosis have shown efficacy, they are associated with certain limitations, including limited drug bioavailability, non-specific administration, and the occurrence of adverse effects. In recent years, nanoparticle-based drug delivery systems have emerged as a promising approach for managing osteoporosis. Nanoparticles possess unique physicochemical properties, such as a small size, large surface area-to-volume ratio, and tunable surface characteristics, which enable them to overcome the limitations of conventional therapies. These nanoparticles offer several advantages, including enhanced drug stability, controlled release kinetics, targeted bone tissue delivery, and improved drug bioavailability. This comprehensive review aims to provide insights into the recent advancements in nanoparticle-based therapy for osteoporosis. It elucidates the various types of nanoparticles employed in this context, including silica, polymeric, solid lipid, and metallic nanoparticles, along with their specific processing techniques and inherent properties that render them suitable as potential drug carriers for osteoporosis treatment. Furthermore, this review discusses the challenges and future suggestions associated with the development and translation of nanoparticle drug delivery systems for clinical use. These challenges encompass issues such as scalability, safety assessment, and regulatory considerations. However, despite these challenges, the utilization of nanoparticle-based drug delivery systems holds immense promise in revolutionizing the field of osteoporosis management by enabling more effective and targeted therapies, ultimately leading to improved patient outcomes.

Acid-activated prorenin binds to (pro)renin receptor in vitro.

Binding properties of acid-activated prorenin to (pro)renin receptor [(P)RR] was investigated in vitro to discuss possible roles of such reversibly acid-activated prorenin in the renin angiotensin (RA) system. Prorenin was acidified at pH 3.3, 4.5, 5.5, 6.5, and its activation level was measured at 1, 2, 4, 8, 12, and 25 h. Prorenin, activated non-proteolytically in time- and pH-dependent manners, was verified by Western blot analyses. Acidification of prorenin for 25 h at pH 3.3, 4.5, 5.5, and 6.5 showed 78%, 54%, 34%, and 20% activities, respectively when compared with the renin activity of trypsinized prorenin as 100%. Additionally, the binding properties of acidified prorenin to (P)RR were elucidated both at the equilibrium state and in the kinetic state using BIAcore. BIAcore assay showed that acidified prorenin at pH 3.3, 4.5, 5.5, and 6.5 had apparent K(D) of 1.57 × 10(4), 14.1, 8.29, and 8.04 nM, respectively while native prorenin at pH 7.4 had a K(D) of 7.8 nM. At equilibrium state, K(D) of native prorenin was 1.42 nM whereas apparent K(D) varied from 1.25 to 5.0 nM for the prorenin acidified at pH 4.5, 5.5, and 6.5. The K(m) values of free forms of acidified prorenin at different pH (0.33-0.5 µM) was almost similar to those of (P)RR-bound forms of acidified prorenin (0.5-0.77 µM). These in vitro data indicate that prorenin acidified in vivo possibly modulate RA system in receptor-dependent and/or -independent manners which could ultimately lead to the pathogenesis of diseases.

Therapeutic potential of epiphyseal growth plate cells for bone regeneration in an osteoporosis model.

Bone growth occurs in the epiphyseal growth plate (EGP) and epiphyseal growth plate cells (EGPCs) exist in EGP. EGPCs, including skeletal stem cells (SSCs), are cells that induce bone growth and development through endochondral ossification. Recently, the superiority of bone regeneration through endochondral ossification has been reported. Our study compared EGPCs with bone marrow-derived mesenchymal stem cells (BM-MSCs) and suggested the therapeutic potential of new bone regeneration. In this study, we analyzed the characteristics between EGPCs and BM-MSCs based on morphological characteristics and molecular profiles. EGPCs expressed chondrogenic and osteogenic markers higher than BM-MSCs. Additionally, in co-culture with BM-MSCs, EGPCs induced an increase in chondrogenic, osteogenic, and hypertrophic markers of BM-MSCs. Finally, EGPCs induced higher bone regeneration than BM-MSCs in the osteoporosis model. Overall, we suggest the possibility of EGPCs as cell therapy for effective bone regeneration.

Functional Duality of Chondrocyte Hypertrophy and Biomedical Application Trends in Osteoarthritis.

Chondrocyte hypertrophy is one of the key indicators in the progression of osteoarthritis (OA). However, compared with other OA indications, such as cartilage collapse, sclerosis, inflammation, and protease activation, the mechanisms by which chondrocyte hypertrophy contributes to OA remain elusive. As the pathological processes in the OA cartilage microenvironment, such as the alterations in the extracellular matrix, are initiated and dictated by the physiological state of the chondrocytes, in-depth knowledge of chondrocyte hypertrophy is necessary to enhance our understanding of the disease pathology and develop therapeutic agents. Chondrocyte hypertrophy is a factor that induces OA progression; it is also a crucial factor in the endochondral ossification. This review elaborates on this dual functionality of chondrocyte hypertrophy in OA progression and endochondral ossification through a description of the characteristics of various genes and signaling, their mechanism, and their distinguishable physiological effects. Chondrocyte hypertrophy in OA progression leads to a decrease in chondrogenic genes and destruction of cartilage tissue. However, in endochondral ossification, it represents an intermediate stage at the process of differentiation of chondrocytes into osteogenic cells. In addition, this review describes the current therapeutic strategies and their mechanisms, involving genes, proteins, cytokines, small molecules, three-dimensional environments, or exosomes, against the OA induced by chondrocyte hypertrophy. Finally, this review proposes that the contrasting roles of chondrocyte hypertrophy are essential for both OA progression and endochondral ossification, and that this cellular process may be targeted to develop OA therapeutics.

SPRY4 acts as an indicator of osteoarthritis severity and regulates chondrocyte hypertrophy and ECM protease expression.

Osteoarthritis (OA) causes serious changes in the metabolic and signaling pathways of chondrocytes, including the mitogen-activated protein kinase (MAPK) pathway. However, the role of sprouty RTK signaling antagonist 4 (SPRY4), an inhibitor of MAPK, in the human cartilage tissues and chondrocytes remains to be understood. Here, using SPRY4 gene delivery into healthy and degenerated chondrocytes, we elucidated the role of SPRY4 in preventing chondrocyte hypertrophy. In addition to using the human cartilage tissues with the destabilization of the medial meniscus (DMM) model in Sprague-Dawley (SD) rats, the role of SPRY4 in cartilage tissues and chondrocytes was explored through their molecular and histological analyses. In order to determine the effects of SPRY4 on healthy human chondrocyte hypertrophy, small interfering RNA (siRNA) was used to knock down SPRY4. Lentiviral transduction of SPRY4 into degenerated human chondrocytes allowed us to investigate its ability to prevent hypertrophy. SPRY4 expression levels were higher in healthy human cartilage tissue and chondrocytes than in degenerated human cartilage tissues and hypertrophy-induced chondrocytes. The knockdown of SPRY4 in healthy chondrocytes caused an increase in hypertrophy, senescence, reactive oxygen species (ROS) production, and extracellular matrix (ECM) protease expression. However, all these factors decreased upon overexpression of SPRY4 in degenerated chondrocytes via regulation of the MAPK signaling pathway. We conclude that SPRY4 is a crucial indicator of osteoarthritis (OA) severity and could play an important role in preventing OA in the cartilage by inhibiting chondrocyte hypertrophy.

Cryptic ligand on collagen matrix unveiled by MMP13 accelerates bone tissue regeneration via MMP13/Integrin α3/RUNX2 feedback loop.

Extracellular matrix (ECM) remodeling is necessary for the development and self-healing of tissue, and the process is tissue specific. Matrix metalloproteinases (MMPs) play a role in ECM remodeling by unwinding and cleaving ECM. We hypothesized that ECM remodeling by MMPs is involved in the differentiation of stem cells into specific lineages during self-healing. To prove the hypothesis, we investigated which MMPs are involved in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) grown on a type I collagen (Col I) matrix, and we found that specifically high expression of MMP13 in hMSCs grown on a Col I matirx during osteogenic differentiation. Moreover, knocking down of MMP13 decreased the osteogenic differentiation of hMSCs grown on a Col I matrix. In addition, pre-treatment of recombinant human MMP13 lead to remodeling of Col I matrix and increased the osteogenic differentiation of hMSCs and in vivo bone formation following the upregulation of the expression of runt-related transcription factor 2 (RUNX2), integrin α3 (ITGA3), and focal adhesion kinase. Furthermore, the transcription factor RUNX2 bound to the MMP13 promoter. These results suggest that growth on a remodeled Col I matrix by MMP13 stimulates osteogenic differentiation of hMSCs and self-healing of bone tissue via an MMP13/ITGA3/RUNX2 positive feedback loop. STATEMENT OF SIGNIFICANCE: Self-healing of tissue could be the key to treating diseases that cannot be overcome by present technology. We investigated the mechanism underlying the self-healing of tissue and we found that the osteogenic differentiation was increased in hMSCs grown on a remodeled Col I matrix by the optimized concentration of MMP13 not in hMSCs grown on a Col I fragments cleaved by a high concentration of MMP13. In addition, we found the remodeled Col I matrix by MMP13 increased the osteogenic capacity through a MMP13/integrin α3/RUNX2 positive feedback loop. This result would be able to not only provide a strategy for bone tissue-specific functional materials following strong evidence about the self-healing mechanism of bone through the interaction between stem cells and the ECM matrix. As such, we strongly believe our finding will be of interest to researchers studying biomaterials, stem cell biology and matrix interaction for regenerative medicine and therapy.

Bile acid-based dual-functional prodrug nanoparticles for bone regeneration through hydrogen peroxide scavenging and osteogenic differentiation of mesenchymal stem cells.

A high level of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) upregulates pro-inflammatory cytokines and inhibits the osteogenic differentiation of mesenchymal stem cells (MSCs), which are key factors in bone regeneration. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has antioxidant and anti-inflammatory activities and also plays beneficial roles in bone regeneration by stimulating the osteogenic differentiation of MSCs while suppressing their adipogenic differentiation. Despite its remarkable capacity for bone regeneration, multiple injections of UDCA induce adverse side effects such as mechanical stress and contamination in bone defects. To fully exploit the beneficial roles of UDCA, a concept polymeric prodrug was developed based on the hypothesis that removal of overproduced H2O2 will potentiate the osteogenic functions of UDCA. In this work, we report bone regenerative nanoparticles (NPs) formulated from a polymeric prodrug of UDCA (PUDCA) with UDCA incorporated in its backbone through H2O2-responsive peroxalate linkages. The PUDCA NPs displayed potent antioxidant and anti-inflammatory activities in MSCs and induced osteogenic rather than adipogenic differentiation of the MSCs. In rat models of bone defect, the PUDCA NPs exhibited significantly better bone regeneration capacity and anti-inflammatory effects than equivalent amounts of UDCA. We anticipate that PUDCA NPs have tremendous translational potential as bone regenerative agents.

Efficient Isolation and Enrichment of Mesenchymal Stem Cells from Human Embryonic Stem Cells by Utilizing the Interaction between Integrin α5ß1 and Fibronectin.

Human pluripotent stem cells (hPSCs) are a potent source of clinically relevant mesenchymal stem cells (MSCs) that confer functional and structural benefits in cell therapy and tissue regeneration. Obtaining sufficient numbers of MSCs in a short period of time and enhancing the differentiation potential of MSCs can be offered the potential to improve the regenerative activity of MSCs therapy. In addition, the underlying processes in the isolation and derivation of MSCs from hPSCs are still poorly understood and controlled. To overcome these clinical needs, an efficient and simplified technique on the isolation of MSCs from spontaneously differentiated human embryonic stem cells (hESCs) via integrin α5ß1 (fibronectin (FN) receptor)-to-FN interactions (hESC-FN-MSCs) is successfully developed. It is demonstrated that hESC-FN-MSCs exhibit a typical MSC surface phenotype, cellular morphology, with the whole transcriptome similar to conventional adult MSCs; but show higher proliferative capacity, more efficient trilineage differentiation, enhanced cytokine secretion, and attenuated cellular senescence. In addition, the therapeutic potential and regenerative capacity of the isolated hESC-FN-MSCs are confirmed by in vitro and in vivo multilineage differentiation. This novel method will be useful in the generation of abundant amounts of clinically relevant MSCs for stem cell therapeutics and regenerative medicine.

Suppression of SPRY4 Promotes Osteogenic Differentiation and Bone Formation of Mesenchymal Stem Cell.

The directed differentiation of human adipose-derived stem cells (hASCs) into different cell types has shown great therapeutic potential in treating various diseases. To maximize the therapeutic potentials, researchers have tried manipulating master transcriptional genes that promote efficient differentiation of mesenchymal stem cells (MSCs) such as the MAPK/ERK signaling pathway. Sprouty (SPRY) is a family of proteins that are known to inhibit the MAPK/ERK signaling pathway. Although the role of some SPRY isoforms in MSC differentiation is known, the function of SPRY4 isoform has not been fully elucidated. In the present study, the role of SPRY4 in the multilineage differentiation of hASCs has been elucidated. To investigate the role of SPRY4 in hASC differentiation and tissue regeneration, we performed a transient knockdown of SPRY expression via a small interfering RNA (siSPRY4). Western blot and quantitative polymerase chain reaction results revealed that the treatment of siSPRY4 before induction of differentiation had no significant effect on adipogenic, but reduced chondrogenic, differentiation of hASCs. Interestingly, SPRY4 transient knockdown had a significant effect on the osteogenic differentiation as indicated by the increased messenger RNA (mRNA) and protein expression of osteogenic markers such as alkaline phosphatase (ALP; 2.3-fold) and osteopontin (OPN; 3.5-fold) and increased calcium deposition measured via Alizarin red staining (3.3-fold). Moreover, in vivo tissue regeneration of siSPRY4-treated hASCs in ectopic bone formation and calvarial defect mouse models showed higher bone volume (5.24-fold) and trabecular number (4.59-fold) assessed via histological and microcomputed tomography analyses. We also determined that the enhanced osteogenic differentiation in SPRY4-treated hASCs was due to the induction of ERK1/2 phosphorylation. Taken together, our results suggest that the regulation of SPRY4 through MAPK signaling is a potentially critical aspect on the osteogenic differentiation of hASCs and for bone tissue regeneration, and thus, may be utilized as a potent technique in the development of effective bone therapeutics. Impact Statement This study tried to expand our current understanding of the osteogenic differentiation of mesenchymal stem cells. The transient downregulation of the SPRY4 expression via small interfering RNA (siRNA) showed significant enhancement of the osteogenic differentiation of adipose-derived stem cells via the induction of ERK 1/2 phosphorylation. This suggests the possible mechanism to maximize the potential of stem cell as therapeutics and has a great potential in treating various bone-related diseases.

Tauroursodeoxycholic acid (TUDCA) counters osteoarthritis by regulating intracellular cholesterol levels and membrane fluidity of degenerated chondrocytes.

Cholesterol and lipid metabolism are associated with osteoarthritis (OA) in human cartilage. High cholesterol levels in OA chondrocytes leads to decreased membrane fluidity and blocks the signaling cascade associated with the expression of chondrogenic genes. It is known that bile acid plays a role in regulating cholesterol homeostasis and the digestion of fats in the human body. Tauroursodeoxycholic acid (TUDCA), as a member of the bile acid family, also aids in the transport of cellular cholesterol. In this study, we hypothesized that TUDCA might be able to promote the restoration of OA cartilage by reducing membrane cholesterol levels in OA chondrocytes and by stimulating the chondrogenic signaling cascade. To assess this hypothesis, we investigated the effects of TUDCA on degenerated chondrocytes isolated from patients with OA. Importantly, treatment with TUDCA at sub-micellar concentrations (2500 µM) significantly increased cell proliferation and Cyclin D1 expression compared with the controls. In addition, the expression of chondrogenic marker genes (SOX9, COL2, and ACAN), proteins (SOX9 and COL2), and glycosaminoglycan (Chondroitin sulfate) was much higher in the TUDCA-treated group compared to the controls. We also found that TUDCA treatment significantly reduced the intracellular cholesterol levels in the chondrocytes and increased membrane fluidity. Furthermore, the stability of TGF receptor 1 and activity of focal adhesion proteins were also increased following TUDCA treatment. Together, these results demonstrated that TUDCA could be used as an alternative treatment for the restoration of OA cartilage.

Alzheimer Aß Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.

We identified ∼30-mer amyloid-ß protein (Aß) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aß levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.