Embryonic Stem Cell-derived Mesenchymal Progenitors Research Articles

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging

ObjectivesCurrently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence.Materials and methodsIn a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function.ResultsThe number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow.ConclusionsMultiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

Prevention of chemotherapy-induced premature ovarian insufficiency in mice by scaffold-based local delivery of human embryonic stem cell-derived mesenchymal progenitor cells

BackgroundPremature ovarian insufficiency (POI) is one of the most serious side effects of chemotherapy in young cancer survivors. It may not only reduce fecundity but also affect lifelong health. There is no standard therapy for preserving ovarian health after chemotherapy. Recently, administration of embryonic stem cell-derived mesenchymal progenitor cells (ESC-MPCs) has been considered a new therapeutic option for preventing POI. However, the previous method of directly injecting cells into the veins of patients exhibits low efficacy and safety. This study aimed to develop safe and effective local delivery methods for the prevention of POI using two types of bioinspired scaffolds.MethodsFemale mice received intraperitoneal cisplatin for 10 days. On day 11, human ESC-MPCs were delivered through systemic administration using intravenous injection or local administration using intradermal injection and intradermal transplantation with a PLGA/MH sponge or hyaluronic acid (HA) gel (GEL) type of scaffold. PBS was injected intravenously as a negative control. Ovarian function and fertility were evaluated 4 weeks after transplantation. Follicle development was observed using hematoxylin and eosin staining. The plasma levels of sex hormones were measured using ELISA. Expression levels of anti-Müllerian hormone (AMH) and ki-67 were detected using immunostaining, and the quality of oocytes and embryos was evaluated after in vitro fertilization. The estrous cycles were observed at 2 months after transplantation.ResultsThe local administration of human ESC-MPCs using the bioinspired scaffold to the backs of mice effectively prolonged the cell survival rate in vivo. The HA GEL group exhibited the best recovered ovarian functions, including a significantly increased number of ovarian reserves, estrogen levels, and AMH levels and decreased apoptotic levels. Furthermore, the HA GEL group showed improved quality of oocytes and embryos and estrous cycle regularity.ConclusionsHA GEL scaffolds can be used as new delivery platforms for ESC-MPC therapy, and this method may provide a novel option for the clinical treatment of chemotherapy-induced POI.

Design and Evaluation of an Osteogenesis-on-a-Chip Microfluidic Device Incorporating 3D Cell Culture.

Microfluidic-based tissue-on-a-chip devices have generated significant research interest for biomedical applications, such as pharmaceutical development, as they can be used for small volume, high throughput studies on the effects of therapeutics on tissue-mimics. Tissue-on-a-chip devices are evolving from basic 2D cell cultures incorporated into microfluidic devices to complex 3D approaches, with modern designs aimed at recapitulating the dynamic and mechanical environment of the native tissue. Thus far, most tissue-on-a-chip research has concentrated on organs involved with drug uptake, metabolism and removal (e.g., lung, skin, liver, and kidney); however, models of the drug metabolite target organs will be essential to provide information on therapeutic efficacy. Here, we develop an osteogenesis-on-a-chip device that comprises a 3D environment and fluid shear stresses, both important features of bone. This inexpensive, easy-to-fabricate system based on a polymerized High Internal Phase Emulsion (polyHIPE) supports proliferation, differentiation and extracellular matrix production of human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs) over extended time periods (up to 21 days). Cells respond positively to both chemical and mechanical stimulation of osteogenesis, with an intermittent flow profile containing rest periods strongly promoting differentiation and matrix formation in comparison to static and continuous flow. Flow and shear stresses were modeled using computational fluid dynamics. Primary cilia were detectable on cells within the device channels demonstrating that this mechanosensory organelle is present in the complex 3D culture environment. In summary, this device aids the development of ‘next-generation’ tools for investigating novel therapeutics for bone in comparison with standard laboratory and animal testing.

Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates.

Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL).

Gene expression profiling and genetic stability of human somatic cell nuclear transfer embryonic stem cell-derived mesenchymal progenitor cells produced by two different protocols

Background & Aim As therapeutic cells, ES-MPC has been studied as a good alternative source to overcoming the disadvantages of adult MSC such as a limiting proliferative capacity and different biological characteristics result from various donor. There are various methods of producing ES-MPC, but can be divided into two main ways: the 3D platform (EB formation) method and the 2D culture (Direct) method. All of the ES-MPC differentiated using each method is known to have MSC-specific characteristics and tri-lineage differentiation capacities. However, there are still differences in proliferative capacity, therapeutic efficacy, differentiation duration and cost of ES-MPC according to differentiation methods. We developed a simple, direct differentiation method of hES-MPC. In this study, we produced ES-EB-MPC and ES-Direct-MPC from the same SCNT-hESC, and analyzed their characters, gene expression profiles, and genetic stability in order to understand ES-MPC characteristics and find the best protocol. Methods, Results & Conclusion We used CHA-hES NT17 and NT18 for this study. For Direct method, SCNT-hESC were treated with SB431542 for 3 days, and then additionally treated with RS-1 and Y27632, simultaneously. After 24 hour, the cells were transferred with collagenase and cultured. We have got homogenous cell population by serial sub-passaging. For EB method, SCNT-hESC were differentiated as described in our previous report (Cell Prolif. 2019). The ES-Direct-MPC and ES-EB-MPC were expressed by typical MSC markers and undergone tri-lineage differentiation similar to each other. Although ES-Direct-MPC were established faster than ES-EB-MPC, the analysis of gene expression patterns using microarray analysis between ES-Direct-MPC and ES-EB-MPC showed very similar patterns (scatter plot, R=0.99). Moreover, gene ontology analysis showed the same gene expression pattern classified by cellular function. ArrayCGH results showed that both ES-Direct-MPC and ES-EB-MPC had high genetic stability. Based on these results, there are no differences in MSC characteristics and proliferation capacity between EB and Direct methods, if so, the direct differentiation method we developed can save time and cost to produce ES-MPC. This research was supported by grants (No.2017M3A9C6061284, 2017M3A9F8072235 and 2019R1A6A1A03032888) from the Bio & Medical Technology Development Program of the NRF funded by the Korean government (MSIP).

Effect of sterilization processes on nanostructured Ti6Al4V surfaces obtained by electropolishing

Abstract

Human Embryonic Stem Cell-Derived Mesenchymal Progenitor (hESCs-MP) Growth on Nanostructured Ti6Al4V Surfaces

Nanotexturing processes that focus on enhancing the bone-implant contact, such as electropolishing, have been proposed. The aim of this work was to evaluate the influence of Ti6Al4V surface morphology on human embryonic stem cell-derived mesenchymal progenitor (hESCs-MP) growth. Three surface treatments were used in this study: mechanically polished samples and two types of electropolished samples that were treated for 4 min and 12 min, respectively. The systems were characterized by atomic force microscopy, contact profilometry, X-ray diffraction, and wettability. Each system was submitted to a cell culture containing hESCs-MP cells for 14 days, and the resultant cell growth on each system was then evaluated. The results indicated that surfaces with higher nanometric and micrometric roughnesses experienced greater hESCs-MP cell growth in osteogenic media. The same behavior was not observed for cell growth in non-osteogenic media due to the absence of dexamethasone, which is responsible for controlling protein adsorption on the surface.

Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis.

Porous microspheres have the potential for use as injectable bone fillers to obviate the need for open surgery. Successful bone fillers must be able to support vascularisation since tissue engineering scaffolds often cease functioning soon after implantation due to a failure to vascularise rapidly. Here, we test the angiogenic potential of a tissue engineered bone filler based on a photocurable acrylate-based high internal phase emulsion (HIPE). Highly porous microspheres were fabricated via two processes, which were compared. One was taken forward and investigated for its ability to support human mesenchymal progenitor cells and angiogenesis in a chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow or broad size distribution were prepared via a T-junction microfluidic device or by a controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively. Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells showed proliferation over 11 days and formation of cell-microsphere aggregates. In-vitro, hES-MP cells were found to migrate into microspheres through their surface pores over time. The presence of osteoblasts, differentiated from the hES-MP cells, was evidenced through the presence of collagen and calcium after 30 days. Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared with control microspheres without pre-cultured cells. The hES-MP seeded microspheres supported greater angiogenesis, as measured by the number of blood vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth. This investigation shows that controlled fabrication of porous microspheres has the potential to create an angiogenic, bone filling material for use as a cell delivery vehicle.

Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors within Collagen Microspheres

Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells. However, mesenchymal stem cell fate determined by the effect of physical and mechanical cues is not yet fully understood. Understanding how mechanical cues in the microenvironment orchestrate stem cell differentiation provides valuable insight that can be used to improve current techniques in cell therapy. This study investigates the osteogenic effect of mechanical stimulations on soft cellular microspheres loaded with human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) when subjected to dynamic loading and in the absence of chemical stimulation. Microspheres were produced by gelation of bovine collagen type I with 1000 to 2000 hES-MP cells seeded per droplet. Four loading conditions were studied: (1) 10% constant strain was applied by a Bose biodynamic bioreactor for 15 min/day or 40 min/day for 5 or 10 days respectively; (2) 10% adjusted strain was applied (subtraction of polydimethylsiloxane (PDMS) plastic elongation from global strain) using Bose biodynamic bioreactor for the same 4 duration/conditions as in the constant strain protocol. The results indicate that applying mechanical stimulation to hES-MPs/collagen microspheres induced osteogenic differentiation of cells when the loading protocol was adjusted. Alkaline phosphatase activity of samples in the adjusted loading protocol increased significantly on day 14 whilst, the deposited minerals, matrix reorganisation and alignment of collagen fibres enhanced from day 21 post encapsulation onward. Application of cyclic loading to 3D culture of hES-MP cells can be used as a model to regulate mechano-stimulation and linage differentiation in vitro.

Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors within Collagen Microspheres

Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells. However, mesenchymal stem cell fate determined by the effect of physical and mechanical cues is not yet fully understood. Understanding how mechanical cues in the microenvironment orchestrate stem cell differentiation provides valuable insight that can be used to improve current techniques in cell therapy. This study investigates the osteogenic effect of mechanical stimulations on soft cellular microspheres loaded with human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) when subjected to dynamic loading and in the absence of chemical stimulation. Microspheres were produced by gelation of bovine collagen type I with 1000 to 2000 hES-MP cells seeded per droplet. Four loading conditions were studied: (1) 10% constant strain was applied by a Bose biodynamic bioreactor for 15 min/day or 40 min/day for 5 or 10 days respectively; (2) 10% adjusted strain was applied (subtraction of polydimethylsiloxane (PDMS) plastic elongation from global strain) using Bose biodynamic bioreactor for the same 4 duration/conditions as in the constant strain protocol. The results indicate that applying mechanical stimulation to hES-MPs/collagen microspheres induced osteogenic differentiation of cells when the loading protocol was adjusted. Alkaline phosphatase activity of samples in the adjusted loading protocol increased significantly on day 14 whilst, the deposited minerals, matrix reorganisation and alignment of collagen fibres enhanced from day 21 post encapsulation onward. Application of cyclic loading to 3D culture of hES-MP cells can be used as a model to regulate mechano-stimulation and linage differentiation in vitro.

Stiffness of Cell Micro-Environment Guides Long Term Cell Growth in Cell Seeded Collagen Microspheres

Mesenchymal stem cells are widely implicated as a cell source for tissue engineering of skeletal tissue in cell-based therapy. Physical and mechanical cues are potent controlling factors in cell differentiation and can be implemented as a guide to study cellular response, matrix production and tissue regeneration. Microspheres were produced by gelation of bovine collagen type I with concentration of 2 mg/mL and 1,000-2,000 cells per droplet. Short and long term cell viability of human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) and MG-63 osteoblastic cells as well as collagen microstructure and contraction were monitored during 28 days post encapsulation (pc). Results indicated that collagen concentration, hence mechanical properties of cell’s extracellular micro-environment are important in cell proliferation and differentiation. Contraction of cell-embedded microspheres was found to be vital in cell adaptation and the remodelling of their new environment. It was also found that collagen concentration of 2 mg/mL supports proliferation of hES-MPs while higher collagen concentration promoted the viability of MG-63s. Results of hES-MPs characterization in 3D soft environment and mechanically stimulated hES-MPs collagen microspheres can be used in cells/therapeutic carriers, implants in bone and cartilage healing applications. The microspheres developed in this study can also be used as a tool to build more optimised construct to transfer mechanically stimulated stem cells to the specific area of a defective bone which would add significant benefit to the field of bone regeneration.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30mm diameter × 5mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds wereeither maintained in static conditions or transferred to the bioreactor 3days post-seeding and a continuous flow rate of 3.47mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration

ObjectiveThe regeneration of periodontal tissues lost as a consequence of destructive periodontal disease remains a challenge for clinicians. Guided tissue regeneration (GTR) has emerged as the most widely practiced regenerative procedure. Aim of this study was to electrospin chitosan (CH) membranes with a low or high degree of fiber orientation and examines their suitability for use as a surface layer in GTR membranes, which can ease integration with the periodontal tissue by controlling the direction of cell growth. MethodsA solution of CH-doped with polyethylene oxide (PEO) (ratio 95:5) was prepared for electrospinning. Characterization was performed for biophysiochemical and mechanical properties by means of scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, swelling ratio, tensile testing and monitoring degradation using pH analysis, weight profile, ultraviolet–visible (UV–vis) spectroscopy and FTIR analysis. Obtained fibers were also assessed for viability and matrix deposition using human osteosarcoma (MG63) and human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells. ResultsRandom and aligned CH fibers were obtained. FTIR analysis showed neat CH spectral profile before and after electrospinning. Electropsun mats were conducive to cellular attachment and viability increased with time. The fibers supported matrix deposition by hES-MPs. Histological sections showed cellular infiltration as well. SignificanceThe surface layer would act as seal to prevent junctional epithelium from falling into the defect site and hence maintain space for bone regeneration.

Osteogenic Differentiation of Human Embryonic Stem Cell-Derived Mesenchymal Progenitor Cells as a Model for Assessing Developmental Bone ToxicityIn Vitro

Abstract The assessment of adverse effects of substances on human embryonic bone formation is an integral part of regulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to reduce such in vivo testing is the application of stem cell-based in vitro methods. In the present study, we aimed to establish an in vitro system for developmental bone toxicity testing using human embryonic stem cell-derived mesenchymal progenitors (hES-MPs). Cell-based and biochemical assays as well as cytochemical stainings and flow cytometry confirmed the presence of a differentiation pattern characteristic for osteogenesis. Nondifferentiating hES-MPs highly expressed the early protein markers Runx2 and Dlx5, while alkaline phosphatase expression and activity strongly increased during differentiation. Moreover, immunofluorescence staining revealed the formation of a compact collagen-rich extracellular matrix, which quickly mineralized. Based on these results, we established toxic...

Freeze gelated porous membranes for periodontal tissue regeneration

Guided tissue regeneration (GTR) membranes have been used for the management of destructive forms of periodontal disease as a means of aiding regeneration of lost supporting tissues, including the alveolar bone, cementum, gingiva and periodontal ligaments (PDL). Currently available GTR membranes are either non-biodegradable, requiring a second surgery for removal, or biodegradable. The mechanical and biofunctional limitations of currently available membranes result in a limited and unpredictable treatment outcome in terms of periodontal tissue regeneration. In this study, porous membranes of chitosan (CH) were fabricated with or without hydroxyapatite (HA) using the simple technique of freeze gelation (FG) via two different solvents systems, acetic acid (ACa) or ascorbic acid (ASa). The aim was to prepare porous membranes to be used for GTR to improve periodontal regeneration. FG membranes were characterized for ultra-structural morphology, physiochemical properties, water uptake, degradation, mechanical properties, and biocompatibility with mature and progenitor osteogenic cells. Fourier transform infrared (FTIR) spectroscopy confirmed the presence of hydroxyapatite and its interaction with chitosan. μCT analysis showed membranes had 85–77% porosity. Mechanical properties and degradation rate were affected by solvent type and the presence of hydroxyapatite. Culture of human osteosarcoma cells (MG63) and human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) showed that all membranes supported cell proliferation and long term matrix deposition was supported by HA incorporated membranes. These CH and HA composite membranes show their potential use for GTR applications in periodontal lesions and in addition FG membranes could be further tuned to achieve characteristics desirable of a GTR membrane for periodontal regeneration.

Derivation and Chondrogenic Commitment of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors.

The induction of human embryonic stem cells to a mesenchymal-like progenitor population constitutes a developmentally relevant approach for efficient directed differentiation of human embryonic stem (hES) cells to the chondrogenic lineage. The initial enrichment of a hemangioblast intermediate has been shown to yield a replenishable population of highly purified progenitor cells that exhibit the typical mesenchymal stem cell (MSC) surface markers as well as the capacity for multilineage differentiation to bone, fat, and cartilage. Herein, we provide detailed methodologies for the derivation and characterization of potent mesenchymal-like progenitors from hES cells and describe in vitro assays for bone morphogenetic protein (BMP)-2-mediated differentiation to the chondrogenic lineage.

Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus

Mesenchymal progenitor cells play a vital role in bone regenerative medicine and tissue engineering strategies. To be clinically useful osteoprogenitors should be readily available with the potential to form bone matrix. While mesenchymal stromal cells from bone marrow have shown promise for tissue engineering, they are obtained in small numbers and there is risk of donor site morbidity. Osteogenic progenitor cells derived from dermal tissue may provide a more abundant and easily expandable source of cells. Bone turnover in vivo is regulated by mechanical forces, particularly oscillatory fluid shear stresses (FSS), and in vitro osteogenic progenitors have been shown to be regulated by mechanical stimuli. The aim of this study was to assess what effect osteogenic media and FSS, generated by a simple rocking platform, had on cell behaviour and matrix production in human progenitor dermal fibroblasts (HDFs) and the embryonic stem cell-derived mesenchymal progenitor cell line (hES-MP). Osteogenic media stimulated alkaline phosphatase activity (ALP) and calcium deposition in HDFs. The addition of FSS further enhanced ALP activity and mineralised matrix deposition in both progenitor cells cultured in osteogenic media. Both types of progenitor cell subjected to FSS showed increases in collagen secretion and apparent collagen organisation as imaged by second harmonic generation.

Efficient Derivation and Concise Gene Expression Profiling of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors (EMPs)

New potential sources of stem cells for clinical application include bone marrow mesenchymal stem cells (BMMSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPS). However, each source is not without its own concerns. While research continues in an effort to overcome these problems, the generation of mesenchymal progenitors from existing hESC lines may circumvent many of these issues. We report here a simple and efficient method of generating hESC-derived mesenchymal progenitors (EMPs) and transcriptome profiling using a concise, custom-designed, oligomnucleotide gene expression microarray. Characterization of EMPs shows that these cells are similar to BMMSCs in terms of differentiation capacity as well as cell surface marker expression. In addition, EMPs express several ESC markers and HLA-G, a nonclassical MHC class I molecule with immunomodulatory properties. Morevoer, EMPs possess significantly enhanced proliferative ability over BMMSCs during which karyotypic stability was maintained. Although derived from hESCs, EMPs do not form any tumors in immunocompromised mice. To efficiently profile gene expression in multiple samples, we designed an oligoarray to probe just over 11,000 genes highly expressed in stem cells. We found that the transcriptome of EMPs is more similar to BMMSCs than hESCs. Both cell types highly express genes involved in processes related to the cytoskeleton, extracellular matrix, and cell adhesion, but EMPs show higher expression of genes involved in cell proliferation whereas BMMSCs showed higher expression of immune-related genes. Based on our data, EMPs may be an accessible source of mesenchymal progenitor for therapeutic use.

Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis

Reports of the bone-protective effects of resveratrol, a naturally occurring phytoestrogen and agonist for the longevity gene SIRT1, have highlighted this compound as a candidate for therapy of osteoporosis. Moreover, SIRT1 antagonism enhances adipogenesis. There has been speculation that resveratrol can promote osteogenesis through SIRT1, but the mechanism remains unclear. In this study we investigated the molecular mechanism of how resveratrol can modulate the lineage commitment of human mesenchymal stem cells to osteogenesis other than adipogenesis. We found that resveratrol promoted spontaneous osteogenesis but prevented adipogenesis in human embryonic stem cell-derived mesenchymal progenitors. Resveratrol upregulated the expression of osteo-lineage genes RUNX2 and osteocalcin while suppressing adipo-lineage genes PPARγ2 and LEPTIN in adipogenic medium. Furthermore, we found that the osteogenic effect of resveratrol was mediated mainly through SIRT1/FOXO3A with a smaller contribution from the estrogenic pathway. Resveratrol activated SIRT1 activity and enhanced FOXO3A protein expression, a known target of SIRT1, in an independent manner. As a result, resveratrol increased the amount of the SIRT1-FOXO3A complex and enhanced FOXO3A-dependent transcriptional activity. Ectopic overexpression or silencing of SIRT1/FOXO3A expression regulated RUNX2 promoter activity, suggesting an important role for SIRT1-FOXO3A complex in regulating resveratrol-induced RUNX2 gene transcription. Further mutational RUNX2 promoter analysis and chromatin immunoprecipitation assay revealed that resveratrol-induced SIRT1-FOXO3A complex bound to a distal FOXO response element (-1269/-1263), an action that transactivated RUNX2 promoter activity in vivo. Taken together, our results describe a novel mechanism of resveratrol in promoting osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis.

Human embryonic stem cell-derived mesenchymal progenitors—Potential in regenerative medicine

Tissue engineering and cell therapy require large-scale production of homogeneous populations of lineage-restricted progenitor cells that easily can be induced to differentiate into a specific tissue. We have developed straightforward protocols for the establishment of human embryonic stem (hES) cell-derived mesenchymal progenitor (hES-MP) cell lines. The reproducibility was proven by derivation of multiple hES-MP cell lines from 10 different hES cell lines. To illustrate clinical applicability, a xeno-free hES-MP cell line was also derived. None of the markers characteristic for undifferentiated hES cells were detected in the hES-MP cells. Instead, these cells were highly similar to mesenchymal stem cells with regard to morphology and expression of markers. The safety of hES-MP cells following transplantation was studied in severely combined immunodeficient (SCID) mice. The implanted hES-MP cells gave rise to homogeneous, well-differentiated tissues exclusively of mesenchymal origin and no teratoma formation was observed. These cells further have the potential to differentiate toward the osteogenic, adipogenic, and chondrogenic lineages in vitro. The possibility of easily and reproducibly generating highly expandable hES-MP cell lines from well-characterized hES cell lines with differentiation potential into several mesodermal tissues entails an enormous potential for the field of regenerative medicine.