MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.     

Effect of Inducible BMP-7 Expression on the Osteogenic Differentiation of Human Dental Pulp Stem Cells

BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.     

Enhanced Osteogenic Differentiation of Pluripotent Stem Cells via γ-Secretase Inhibition

Bone healing is a complex, well-organized process. Multiple factors regulate this process, including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the most important signaling pathways that affect bone healing is the Notch signaling pathway. It has a significant role in controlling the differentiation of bone mesenchymal stem cells and forming new bone. Interventions to enhance the healing of critical-sized bone defects are of great importance, and stem cell transplantations are eminent candidates for treating such defects. Understanding how Notch signaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis and improve the overall healing process upon transplantation. In Rancourt’s lab, mouse embryonic stem cells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigates the role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic and induced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced the differentiation of both mouse embryonic and induced pluripotent stem cells.     

Efficient Generation of Neural Stem Cells from Embryonic Stem Cells Using a Three-Dimensional Differentiation System

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).     

Receptor-Targeted,Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin α_νβ₃. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin α_νβ₃ and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation.     

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O₂) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.     

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.     

Epigenetic Regulation of Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells

With the intent to achieve the best modalities for myocardial cell therapy, different cell types are being evaluated as potent sources for differentiation into cardiomyocytes. Embryonic stem cells and induced pluripotent stem cells have great potential for future progress in the treatment of myocardial diseases. We reviewed aspects of epigenetic mechanisms that play a role in the differentiation of these cells into cardiomyocytes. Cardiomyocytes proliferate during fetal life, and after birth, they undergo permanent terminal differentiation. Upregulation of cardiac-specific genes in adults induces hypertrophy due to terminal differentiation. The repression or expression of these genes is controlled by chromatin structural and epigenetic changes. However, few studies have reviewed and analyzed the epigenetic aspects of the differentiation of embryonic stem cells and induced pluripotent stem cells into cardiac lineage cells. In this review, we focus on the current knowledge of epigenetic regulation of cardiomyocyte proliferation and differentiation from embryonic and induced pluripotent stem cells through histone modification and microRNAs, the maintenance of pluripotency, and its alteration during cardiac lineage differentiation.     

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.     

Development of Electrospun Composite Fibers in Multiscale Structure and Investigating the Performance on Proliferation and Osteogenic Differentiation of ADSCs

Electrospun composite membranes in multiscale structures are developed for bone tissue engineering. Aligned polycaprolactone (PCL) fibers entrapping CA‐HAp microparticles (containing CaCO₃, hydroxyapatite, and casein in a hierarchical organization) are electrospun to find whether synergistic effects of fiber alignment and CA‐HAp microparticles on improving osteogenic differentiation can be obtained. CA‐HAp microparticles are in a spherical morphology of 1.42 ± 0.26 µm. Their presence increases fiber diameter and does not significantly affect fiber alignment. On all membranes, adipose derived stem cells (ADSCs) from humans spread very well. On a random group, cells distribute randomly and the presence of CA‐HAp microparticles facilitates cell proliferation, especially for the one at CA‐HAp/PCL 50 wt%; the one at CA‐HAp/PCL 20 wt% shows significantly much higher alkaline phosphatase (ALP) activity (112.0% higher) than the pure PCL membrane. On aligned samples, cells align along fibers and expression of ALP is enhanced. However, at the same composition (CA‐HAp/PCL 20 wt%), the random sample has much higher ALP activity than the aligned sample. The expressions of osteogenic marker genes are also evaluated. Combining the results and the applicability of membranes together, the random membrane at CA‐HAp/PCL 20 wt% is the best candidate for bone tissue engineering.     

Electroactive Hydroxyapatite/Carbon Nanofiber Scaffolds for Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Traditional bone defect treatments are limited by an insufficient supply of autologous bone, the immune rejection of allogeneic bone grafts, and high medical costs. To address this medical need, bone tissue engineering has emerged as a promising option. Among the existing tissue engineering materials, the use of electroactive scaffolds has become a common strategy in bone repair. However, single-function electroactive scaffolds are not sufficient for scientific research or clinical application. On the other hand, multifunctional electroactive scaffolds are often complicated and expensive to prepare. Therefore, we propose a new tissue engineering strategy that optimizes the electrical properties and biocompatibility of carbon-based materials. Here, a hydroxyapatite/carbon nanofiber (HAp/CNF) scaffold with optimal electrical activity was prepared by electrospinning HAp nanoparticle-incorporated polyvinylidene fluoride (PVDF) and then carbonizing the fibers. Biochemical assessments of the markers of osteogenesis in human adipose-derived stem cells (h-ADSCs) cultured on HAp/CNF scaffolds demonstrate that the material promoted the osteogenic differentiation of h-ADSCs in the absence of an osteogenic factor. The results of this study show that electroactive carbon materials with a fibrous structure can promote the osteogenic differentiation of h-ADSCs, providing a new strategy for the preparation and application of carbon-based materials in bone tissue engineering.     

Electrical Stimulation of Human Adipose-Derived Mesenchymal Stem Cells on O2 Plasma-Treated ITO Glass Promotes Osteogenic Differentiation

Electrical signals represent an essential form of cellular communication. For decades, electrical stimulation has been used effectively in clinical practice to enhance bone healing. However, the detailed mechanisms between electrical stimulation and bone healing are not well understood. In addition, there have been many difficulties in setting up a stable and efficient electrical stimulation system within the in vitro environment. Therefore, various conductive materials and electrical stimulation methods have been tested to establish an effective electrical stimulation system. Through these systems, many studies have been conducted on the effects of electrical stimulation on bone healing and osteogenic differentiation. However, previous studies were limited by the use of opaque conductive materials that obscure the cells; fluorescent observations and staining are known to be two of the critical methods to confirm the states of the cells. Indium tin oxide (ITO) glass is known to have excellent transparency and conductivity, but it is challenging to cultivate cells due to low cell adhesion characteristics. Therefore, we used O₂ plasma treatment to increase the hydrophilicity and wettability of ITO glass. This enhanced cell affinity to the glass, providing a stable surface for the cells to attach. Then, electrical stimulation was applied with an amplitude range of 10 to 200 µA at a frequency of 10 Hz. Our results demonstrated that the osteogenic differentiation efficiency was maximized under the amplitude conditions of 10 µA and 50 µA. Accordingly, the results of our study suggest the development of an excellent platform in the field of biological research as a good tool to elucidate various mechanisms of cell bioactivity under electrical conditions.     

Glutamatergic Neurons Differentiated from Embryonic Stem Cells: An Investigation of Differentiation and Associated Diseases

Neurons that have been derived from various types of stem cells have recently undergone significant study due to their potential for use in various aspects of biomedicine. In particular, glutamatergic neurons differentiated from embryonic stem cells (ESCs) potentially have many applications in both basic research and regenerative medicine. This review summarized the literatures published thus far and focused on two areas related to these applications. Firstly, these neurons can be used to investigate neuronal signal transduction during differentiation and this means that the genes/proteins/markers involved in this process can be identified. In this way, the dynamic spatial and temporal changes associated with neuronal morphology can be investigated relatively easily. Such an in vitro system can also be used to study how neurons during neurogenesis integrate into normal tissue. At the same time, the integration, regulation and functions of extracellular matrix secretion, various molecular interactions, various ion channels, the neuronal microenvironment, etc., can be easily traced. Secondly, the disease-related aspects of ESC-derived glutamatergic neurons can also be studied and then applied therapeutically. In the future, greater efforts are needed to explore how ESC-differentiated glutamatergic neurons can be used as a neuronal model for the study of Alzheimer’s disease (AD) mechanistically, to identify possible therapeutic strategies for treating AD, including tissue replacement, and to screen for drugs that can be used to treat AD patients. With all of the modern technology that is available, translational medicine should begin to benefit patients soon.     

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.     

Chemically Induced Cardiomyogenesis of Mouse Embryonic Stem Cells

A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α‐myosine heavy chain (α‐MHC) promoter (pα‐MHC‐EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good ( IV‐5 , V‐4 ) to very good activities ( II‐16 , IV‐8 ), as determined by a 50 to 80 % increase in the EGFP fluorescence compared to untreated cells. Time‐dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets.     

Gelatin Meshes Enriched with Graphene Oxide and Magnetic Nanoparticles Support and Enhance the Proliferation and Neuronal Differentiation of Human Adipose-Derived Stem Cells

The field of tissue engineering is constantly evolving due to the fabrication of novel platforms that promise to stimulate tissue regeneration in the scenario of accidents. Here, we describe the fabrication of fibrous nanostructured substrates based on fish gelatin (FG) and enriched with graphene oxide (GO) and magnetic nanoparticles (MNPs) and demonstrate its biological properties in terms of cell viability and proliferation, cell adhesion, and differentiation. For this purpose, electrospun fibers were fabricated using aqueous precursors containing either only GO and only MNP nanospecies, or both of them within a fish gelatin solution. The obtained materials were investigated in terms of morphology, aqueous media affinity, tensile elasticity, and structural characteristics. The biological evaluation was assessed against adipose-derived stem cells by MTT, LDH, Live/Dead assay, cytoskeleton investigation, and neuronal trans-differentiation. The results indicate an overall good interaction and show that these materials offer a biofriendly environment. A higher concentration of both nanospecies types induced some toxic effects, thus 0.5% GO, MNPs, and GO/MNPs turned out to be the most suitable option for biological testing. Moreover, a successful neuronal differentiation has been shown on these materials, where cells presented a typical neuronal phenotype. This study demonstrates the potential of this scaffold to be further used in tissue engineering applications.