Therapeutic potential of mesenchymal stem cells from human iPSC-derived teratomas for osteochondral defect regeneration.

Human induced pluripotent stem cells (iPSCs) hold great promise for personalized medicine, as they can be differentiated into specific cell types, especially mesenchymal stem cells (MSCs). Therefore, our study sought to assess the feasibility of deriving MSCs from teratomas generated from human iPSCs. Teratomas serve as a model to mimic multilineage human development, thus enriching specific somatic progenitors and stem cells. Here, we discovered a small, condensed mass of MSCs within iPSC-generated teratomas. Afterward, we successfully isolated MSCs from this condensed mass, which was a byproduct of teratoma development. To evaluate the characteristics and cell behaviors of iPSC-derived MSCs (iPSC-MSCs), we conducted comprehensive assessments using qPCR, immunophenotype analysis, and cell proliferation-related assays. Remarkably, iPSC-MSCs exhibited an immunophenotype resembling that of conventional MSCs, and they displayed robust proliferative capabilities, similar to those of higher pluripotent stem cell-derived MSCs. Furthermore, iPSC-MSCs demonstrated the ability to differentiate into multiple lineages in vitro. Finally, we evaluated the therapeutic potential of iPSC-MSCs using an osteochondral defect model. Our findings demonstrated that teratomas are a promising source for the isolation of condensed MSCs. More importantly, our results suggest that iPSC-MSCs derived from teratomas possess the capacity for tissue regeneration, highlighting their promise for future therapeutic applications.

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.

AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo.

Cardiac hypertrophy is one of the most common genetic heart disorders and considered a risk factor for cardiac morbidity and mortality. The mammalian target of rapamycin (mTOR) pathway plays a key regulatory function in cardiovascular physiology and pathology in hypertrophy. AZD2014 is a small-molecule ATP competitive mTOR inhibitor working on both mTORC1 and mTORC2 complexes. Little is known about the therapeutic effects of AZD2014 in cardiac hypertrophy and its underlying mechanism. Here, AZD2014 is examined in in vitro model of phenylephrine (PE)-induced human cardiomyocyte hypertrophy and a myosin-binding protein-C (Mybpc3)-targeted knockout (KO) mouse model of cardiac hypertrophy. Our results demonstrate that cardiomyocytes treated with AZD2014 retain the normal phenotype and AZD2014 attenuates cardiac hypertrophy in the Mybpc3-KO mouse model through inhibition of dual mTORC1 and mTORC2, which in turn results in the down-regulation of the Akt/mTOR signaling pathway.

Peptide Adjuvant to Invigorate Cytolytic Activity of NK Cells in an Obese Mouse Cancer Model.

Cancer patients who are overweight compared to those with normal body weight have obesity-associated alterations of natural killer (NK) cells, characterized by poor cytotoxicity, slow proliferation, and inadequate anti-cancer activity. Concomitantly, prohibitin overexpressed by cancer cells elevates glucose metabolism, rendering the tumor microenvironment (TME) more tumor-favorable, and leading to malfunction of immune cells present in the TME. These changes cause vicious cycles of tumor growth. Adoptive immunotherapy has emerged as a promising option for cancer patients; however, obesity-related alterations in the TME allow the tumor to bypass immune surveillance and to down-regulate the activity of adoptively transferred NK cells. We hypothesized that inhibiting the prohibitin signaling pathway in an obese model would reduce glucose metabolism of cancer cells, thereby changing the TME to a pro-immune microenvironment and restoring the cytolytic activity of NK cells. Priming tumor cells with an inhibitory the prohibitin-binding peptide (PBP) enhances cytokine secretion and augments the cytolytic activity of adoptively transferred NK cells. NK cells harvested from the PBP-primed tumors exhibit multiple markers associated with the effector function of active NK cells. Our findings suggest that PBP has the potential as an adjuvant to enhance the cytolytic activity of adoptively transferred NK cells in cancer patients with obesity.

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.

Nanoparticle-Based Hybrid Scaffolds for Deciphering the Role of Multimodal Cues in Cardiac Tissue Engineering.

Myocardial microenvironment plays a decisive role in guiding the function and fate of cardiomyocytes, and engineering this extracellular niche holds great promise for cardiac tissue regeneration. Platforms utilizing hybrid hydrogels containing various types of conductive nanoparticles have been a critical tool for constructing engineered cardiac tissues with outstanding mechanical integrity and improved electrophysiological properties. However, there has been no attempt to directly compare the efficacy of these hybrid hydrogels and decipher the mechanisms behind how these platforms differentially regulate cardiomyocyte behavior. Here, we employed gelatin methacryloyl (GelMA) hydrogels containing three different types of carbon-based nanoparticles: carbon nanotubes (CNTs), graphene oxide (GO), and reduced GO (rGO), to investigate the influence of these hybrid scaffolds on the structural organization and functionality of cardiomyocytes. Using immunofluorescent staining for assessing cellular organization and proliferation, we showed that electrically conductive scaffolds (CNT- and rGO-GelMA compared to relatively nonconductive GO-GelMA) played a significant role in promoting desirable morphology of cardiomyocytes and elevated the expression of functional cardiac markers, while maintaining their viability. Electrophysiological analysis revealed that these engineered cardiac tissues showed distinct cardiomyocyte phenotypes and different levels of maturity based on the substrate (CNT-GelMA: ventricular-like, GO-GelMA: atrial-like, and rGO-GelMA: ventricular/atrial mixed phenotypes). Through analysis of gene-expression patterns, we uncovered that the engineered cardiac tissues matured on CNT-GelMA and native cardiac tissues showed comparable expression levels of maturation markers. Furthermore, we demonstrated that engineered cardiac tissues matured on CNT-GelMA have increased functionality through integrin-mediated mechanotransduction (via YAP/TAZ) in contrast to cardiomyocytes cultured on rGO-GelMA.

Biocompatible Carbon Nanotube-Based Hybrid Microfiber for Implantable Electrochemical Actuator and Flexible Electronic Applications.

Biocompatible, electrically conductive microfibers with superior mechanical properties have received a great attention due to their potential applications in various biomedical applications such as implantable medical devices, biosensors, artificial muscles, and microactuators. Here, we developed an electrically conductive and mechanically stable carbon nanotube-based microactuator with a low degradability that makes it usable for an implantable device in the body or biological environments. The microfiber was composed of hyaluronic acid (HA) hydrogel and single-wall carbon nanotubes (SWCNTs) (HA/SWCNT). HA hydrogel acts as biosurfactant and ion-conducting binder to improve the dispersion of SWCNTs resulting in enhanced electrical and mechanical properties of the hybrid microfiber. In addition, HA was crosslinked to prevent the leaking of the nanotubes from the composite. Crosslinking of HA hydrogel significantly enhances Young's modulus, the failure strain, the toughness, the stability of the electrical conductivity, and the resistance to biodegradation and creep of hybrid microfibers. The obtained crosslinked HA/SWCNT hybrid microfibers show an excellent capacitance and actuation behavior under mechanical loading with a low potential of ±1 V in a biological environment. Furthermore, the HA/SWCNT microfibers exhibit an excellent in vitro viability. Finally, the biocompatibility is shown through the resolution of an early inflammatory response in less than 3 weeks after the implantation of the microfibers in the subcutaneous tissue of mice.

A Foreign Body Response-on-a-Chip Platform.

Understanding the foreign body response (FBR) and desiging strategies to modulate such a response represent a grand challenge for implant devices and biomaterials. Here, the development of a microfluidic platform is reported, i.e., the FBR-on-a-chip (FBROC) for modeling the cascade of events during immune cell response to implants. The platform models the native implant microenvironment where the implants are interfaced directly with surrounding tissues, as well as vasculature with circulating immune cells. The study demonstrates that the release of cytokines such as monocyte chemoattractant protein 1 (MCP-1) from the extracellular matrix (ECM)-like hydrogels in the bottom tissue chamber induces trans-endothelial migration of circulating monocytes in the vascular channel toward the hydrogels, thus mimicking implant-induced inflammation. Data using patient-derived peripheral blood mononuclear cells further reveal inter-patient differences in FBR, highlighting the potential of this platform for monitoring FBR in a personalized manner. The prototype FBROC platform provides an enabling strategy to interrogate FBR on various implants, including biomaterials and engineered tissue constructs, in a physiologically relevant and individual-specific manner.

Cell surface engineering and application in cell delivery to heart diseases.

Cell-based therapy has expanded its influence in cancer immunotherapy, regenerative medicine, and tissue engineering. Due to their secretory functions, differentiation capabilities, specific homing effects through chemotaxis, distinctive therapeutic potentials, and ex vivo expandability, cells have become an attractive reagent for advanced therapeutic strategies. Therefore, the ability to modify cells and manipulate their functions according to intended therapeutic designs has been the central scientific interest in the field of biomedical research. Many innovative methods have been developed with genetic modification of cells being the most advanced cell surface engineering technique. Although genetic modification is a powerful tool, it has a limited applicability due to the permanent modifications made on cells. Alternatively, many endeavors have been made to develop surface engineering techniques that can circumvent the limitations of genetic modification. In this review, current methods of non-genetic cell surface modification, including chemical conjugations, polymeric encapsulation, hydrophobic insertion, enzymatic and metabolic addition, will be introduced. Moreover, cell surface engineering plausible for cardiac remodeling and the future prospective will be discussed at the end.

Integrin-Mediated Interactions Control Macrophage Polarization in 3D Hydrogels.

Adverse immune reactions prevent clinical translation of numerous implantable devices and materials. Although inflammation is an essential part of tissue regeneration, chronic inflammation ultimately leads to implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the induction of M1 and M2 macrophages, respectively. Here, this paper demonstrates that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and collagen-derived matrix. Surface marker, gene expression, biochemical, and cytokine profiling consistently indicate that THP-1 cells within a biomaterial lacking cell attachment motifs yield proinflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induce an anti-inflammatory M2-like phenotype and propagate the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2ß1 plays a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity is further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively. Thus, this study represents a novel, versatile, and effective strategy to steer macrophage polarity through integrin-mediated 3D microenvironment for biomaterial-based programming.

Bioprinted 3D vascularized tissue model for drug toxicity analysis.

To develop biomimetic three-dimensional (3D) tissue constructs for drug screening and biological studies, engineered blood vessels should be integrated into the constructs to mimic the drug administration process in vivo. The development of perfusable vascularized 3D tissue constructs for studying the drug administration process through an engineered endothelial layer remains an area of intensive research. Here, we report the development of a simple 3D vascularized liver tissue model to study drug toxicity through the incorporation of an engineered endothelial layer. Using a sacrificial bioprinting technique, a hollow microchannel was successfully fabricated in the 3D liver tissue construct created with HepG2/C3A cells encapsulated in a gelatin methacryloyl hydrogel. After seeding human umbilical vein endothelial cells (HUVECs) into the microchannel, we obtained a vascularized tissue construct containing a uniformly coated HUVEC layer within the hollow microchannel. The inclusion of the HUVEC layer into the scaffold resulted in delayed permeability of biomolecules into the 3D liver construct. In addition, the vascularized construct containing the HUVEC layer showed an increased viability of the HepG2/C3A cells within the 3D scaffold compared to that of the 3D liver constructs without the HUVEC layer, demonstrating a protective role of the introduced endothelial cell layer. The 3D vascularized liver model presented in this study is anticipated to provide a better and more accurate in vitro liver model system for future drug toxicity testing.

Engineering Photocrosslinkable Bicomponent Hydrogel Constructs for Creating 3D Vascularized Bone.

Engineering bone tissue requires the generation of a highly organized vasculature. Cellular behavior is affected by the respective niche. Directing cellular behavior and differentiation for creating mineralized regions surrounded by vasculature can be achieved by controlling the pattern of osteogenic and angiogenic niches. This manuscript reports on engineering vascularized bone tissues by incorporating osteogenic and angiogenic cell-laden niches in a photocrosslinkable hydrogel construct. Two-step photolithography process is used to control the stiffness of the hydrogel and distribution of cells in the patterned hydrogel. In addittion, osteoinductive nanoparticles are utilized to induce osteogenesis. The size of microfabricated constructs has a pronounced effect on cellular organization and function. It is shown that the simultaneous presence of both osteogenic and angiogenic niches in one construct results in formation of mineralized regions surrounded by organized vasculature. In addition, the presence of angiogenic niche improves bone formation. This approach can be used for engineered constructs that can be used for treatment of bone defects.

Unbiased Analysis of the Impact of Micropatterned Biomaterials on Macrophage Behavior Provides Insights beyond Predefined Polarization States.

Macrophages are master regulators of immune responses toward implanted biomaterials. The activation state adopted by macrophages in response to biomaterials determines their own phenotype and function as well as those of other resident and infiltrating immune and nonimmune cells in the area. A wide spectrum of macrophage activation states exists, with M1 (pro-inflammatory) and M2 (anti-inflammatory) representing either ends of the spectrum. In biomaterials research, cell-instructive surfaces that favor or induce M2 macrophages have been considered as beneficial due to the anti-inflammatory and pro-regenerative properties of these cells. In this study, we used a gelatin methacryloyl (GelMA) hydrogel platform to determine whether micropatterned surfaces can modulate the phenotype and function of human macrophages. The effect of microgrooves/ridges and micropillars on macrophage phenotype, function, and gene expression profile were assessed using conventional methods (morphology, cytokine profile, surface marker expression, phagocytosis) and gene microarrays. Our results demonstrated that micropatterns did induce distinct gene expression profiles in human macrophages cultured on microgrooves/ridges and micropillars. Significant changes were observed in genes related to primary metabolic processes such as transcription, translation, protein trafficking, DNA repair, and cell survival. However, interestingly conventional phenotyping methods, relying on surface marker expression and cytokine profile, were not able to distinguish between the different conditions, and indicated no clear shift in cell activation towards M1 or M2 phenotypes. This highlights the limitations of studying the effect of different physicochemical conditions on macrophages by solely relying on conventional markers that are primarily developed to differentiate between cytokine polarized M1 and M2 macrophages. We therefore propose the adoption of unbiased screening methods in determining macrophage responses to biomaterials. Our data clearly show that the exclusive use of conventional markers and methods for determining macrophage activation status could lead to missed opportunities for understanding and exploiting macrophage responses to biomaterials.

Delivery strategies to control inflammatory response: Modulating M1-M2 polarization in tissue engineering applications.

Macrophages are key players in many physiological scenarios including tissue homeostasis. In response to injury, typically the balance between macrophage sub-populations shifts from an M1 phenotype (pro-inflammatory) to an M2 phenotype (anti-inflammatory). In tissue engineering scenarios, after implantation of any device, it is desirable to exercise control on this M1-M2 progression and to ensure a timely and smooth transition from the inflammatory to the healing stage. In this review, we briefly introduce the current state of knowledge regarding macrophage function and nomenclature. Next, we discuss the use of controlled release strategies to tune the balance between the M1 and M2 phenotypes in the context of tissue engineering applications. We discuss recent literature related to the release of anti-inflammatory molecules (including nucleic acids) and the sequential release of cytokines to promote a timely M1-M2 shift. In addition, we describe the use of macrophages as controlled release agents upon stimulation by physical and/or mechanical cues provided by scaffolds. Moreover, we discuss current and future applications of "smart" implantable scaffolds capable of controlling the cascade of biochemical events related to healing and vascularization. Finally, we provide our opinion on the current challenges and the future research directions to improve our understanding of the M1-M2 macrophage balance and properly exploit it in tissue engineering and regenerative medicine applications.

Administration of tauroursodeoxycholic acid enhances osteogenic differentiation of bone marrow-derived mesenchymal stem cells and bone regeneration.

It is known that osteogenic differentiation of mesenchymal stem cells (MSCs) can be promoted by suppression of adipogenesis of MSCs. We have recently found that the chemical chaperone tauroursodeoxycholic acid (TUDCA) significantly reduces adipogenesis of MSCs. In the present study, we examined whether TUDCA can promote osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) by regulating Integrin 5 (ITGA5) associated with activation of ERK1/2 signal pathway and thereby enhance bone tissue regeneration by reducing apoptosis and the inflammatory response. TUDCA treatment promoted in vitro osteogenic differentiation of BMMSCs and in vivo bone tissue regeneration in a calvarial defect model, as confirmed by micro-computed tomography, histological staining, and immunohistochemistry for osteocalcin. In addition, TUDCA treatment significantly decreased apoptosis and the inflammatory response in vivo and in vitro, which is important to enhance bone tissue regeneration. These results indicate that TUDCA plays a critical role in enhancing osteogenesis of BMMSCs, and is therefore a potential alternative drug for bone tissue regeneration.

The Effect of Fetal Bovine Serum (FBS) on Efficacy of Cellular Reprogramming for Induced Pluripotent Stem Cell (iPSC) Generation.

Induced pluripotent stem cells (iPSCs) are pivotal to the advancement of regenerative medicine. However, the low efficacy of iPSC generation and insufficient knowledge about the reprogramming mechanisms involved in somatic cell/adult stem cell reversion to a pluripotent phenotype remain critical hurdles to the therapeutic application of iPSCs. The present study investigated whether the concentration of fetal bovine serum (FBS), a widely employed cell culture additive, can influence the cellular reprogramming efficacy (RE) of human adipose-derived stem cells (hADSCs) to generate iPSCs. Compared with the typically employed concentration of FBS (10%), high concentrations (20% and 30%) increased the RE of hADSCs by approximately twofold, whereas a low concentration (5%) decreased the RE by the same extent. Furthermore, cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, and fluorescence-activated cell sorting (FACS) assays showed that hADSC proliferation during reprogramming was significantly enhanced by FBS at 20% and 30%, whereas quantitative polymerase chain reaction (qPCR) and Western blotting assays revealed a concomitant decrease in p53, p51, and p21 expression. In addition, the efficacy of retrovirus-mediated transduction into hADSCs was increased by approximately 10% at high concentrations of FBS. It was confirmed that platelet-derived growth factor in the FBS enhanced proliferation and reprogramming efficacy. Finally, the generated iPSCs showed a normal karyotype, the same fingerprinting pattern as parental hADSCs, a genome-wide transcriptome pattern similar to that of human embryonic stem cells (hESCs), and in vivo pluripotency. In conclusion, the current investigation demonstrated that high concentrations of FBS can modulate molecular and cellular mechanisms underlying the reprogramming process in hADSCs, thereby augmenting the cellular RE for iPSC generation.

The role of tauroursodeoxycholic acid on adipogenesis of human adipose-derived stem cells by modulation of ER stress.

Obesity has become a serious public health problem in the developed world. Increased mass of adipose tissue in the obese is due to an increase in both the size (hypertrophy) and number (hyperplasia) of adipocytes. The chemical chaperone tauroursodeoxycholic acid (TUDCA) not only decreases endoplasmic reticulum (ER) stress, but also plays a role as a leptin-sensitizing agent for preadipocytes in mice and humans. In this study, we examine whether TUDCA has an effect on adipogenesis from human adipose-derived stem cells (hASCs). Therefore, the effect of TUDCA on ER stress, lipid accumulation, and adipogenic differentiation from hASCs was investigated using histological staining, reverse-transcriptase polymerase chain reaction (RT-PCR), and western blotting in vitro. It was found that TUDCA treatment of hASCs significantly decreases the representative ER stress marker (glucose-regulated protein 78 kDa (GRP78)), adipogenic markers (peroxisome proliferator-activated receptor gamma (PPARγ) and glycerol-3-phosphate dehydrogenase 1 (GPDH)), and lipid accumulation. Furthermore, we confirmed that TUDCA treatment of hASCs significantly decreased in vivo adipogenic tissue formation and ER stress comparing with PBS treatment of hASCs. The results indicate that TUDCA plays a critical role in adipogenesis from hASCs by modulating ER stress and, therefore, has potential pharmacologic and therapeutic applications as an anti-obesity agent.

ARS-interacting multi-functional protein 1 induces proliferation of human bone marrow-derived mesenchymal stem cells by accumulation of ß-catenin via fibroblast growth factor receptor 2-mediated activation of Akt.

ARS-Interacting Multi-functional Protein 1 (AIMP1) is a cytokine that is involved in the regulation of angiogenesis, immune activation, and fibroblast proliferation. In this study, fibroblast growth factor receptor 2 (FGFR2) was isolated as a binding partner of AIMP peptide (amino acids 6-46) in affinity purification using human bone marrow-derived mesenchymal stem cells (BMMSCs). AIMP1 peptide induced the proliferation of adult BMMSCs by activating Akt, inhibiting glycogen synthase kinase-3ß, and thereby increasing the level of ß-catenin. In addition, AIMP1 peptide induced the translocation of ß-catenin to the nucleus and increased the transcription of c-myc and cyclin D1 by activating the ß-catenin/T-cell factor (TCF) complex. By contrast, transfection of dominant negative TCF abolished the effect of AIMP1. The inhibition of Akt, using LY294002, abolished the accumulation and nuclear translocation of ß-catenin induced by AIMP1, leading to a decrease in c-myc and cyclin D1 expression, which decreased the proliferation of BMMSCs. An intraperitoneal injection of AIMP1 peptide into C57/BL6 mice increased the colony formation of fibroblast-like cells. Fluorescence activated cell sorting analysis showed that the colony-forming cells were CD29(+)/CD44(+)/CD90(+)/CD105(+)/CD34(-)/CD45(-), which is characteristic of MSCs. In addition, the fibroblast-like cells differentiated into adipocytes, chondrocytes, and osteocytes. Taken together, these data suggest that AIMP1 peptide promotes the proliferation of BMMSCs by activating the ß-catenin/TCF complex via FGFR2-mediated activation of Akt, which leads to an increase in MSCs in peripheral blood.

The modulation of the oxidative stress response in chondrocytes by Wip1 and its effect on senescence and dedifferentiation during in vitro expansion.

Obtaining a sufficient number of cells ex vivo for tissue regeneration, which are appropriate for cartilage repair, requires improved techniques for the continuous expansion of chondrocytes in a manner that does not change their innate characteristics. Rapid senescence or dedifferentiation during in vitro expansion results in loss of chondrocyte phenotype and the formation of fibrous cartilage replacement tissue, rather than hyaluronic cartilage, after transplantation. As demonstrated in the current study, wild-type p53-inducible phosphatase (Wip1), a well-established stress modulator, was highly expressed in early-passage chondrocytes, but declined rapidly during in vitro expansion. Stable Wip1-expressing chondrocytes generated by microporation were less susceptible to the onset of senescence and dedifferentiation, and were more resistant to oxidative stress. The increased resistance of Wip1 chondrocytes to oxidative stress was due to modulation of p38 mitogen-activated protein kinase (MAPK) activity. Importantly, chondrocytes expressing Wip1 maintained their innate chondrogenic properties for a longer period of time, resulting in improvements in cartilage regeneration after transplantation. Chondrocytes from Wip1 knockout (Wip1(-/-)) mice were defective in cartilage regeneration compared with those from wild-type mice. Thus, Wip1 expression represents a potentially useful mechanism by which a chondrocyte phenotype can be retained during in vitro expansion through modulation of cellular stress responses.

Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector.

Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.