Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.     

Gadolinium-chelate nanoparticle entrapped human mesenchymal stem cell via photochemical internalization for cancer diagnosis

To improve the gadolinium (Gd) internalization efficiency in stem cells, gadolinium-chelate nanoparticles were prepared from a pullulan derivative (pullulan-deoxycholic acid (DOCA)-diethylene triamine pentaacetic (DTPA)-Gd conjugate; PDDG) and then the PDDG was entrapped into human mesenchymal stem cells (hMSCs) by the photochemical-internalization (PCI) method for cancer diagnosis via the cancer homing property of hMSCs. The internalization efficiency of Gd in hMSCs was significantly increased to 98 ± 4 pg Gd/cell from 32 ± 2 pg Gd/cell via the PCI method. Moreover, the Gd-entrapped hMSCs revealed a low exocytosis ratio of gadolinium-chelate nanoparticles during cell division in vitro and a high cellular labeling efficiency for at least 21 days in vivo. The cancer-targeting and diagnosis effect of the Gd-entrapped hMSCs were confirmed in a small CT26 tumor-bearing mice model. The stem cells detected an early tumor (∼3 mm³) within 2 h using 4.7-T MR and optical imaging. The results demonstrated that the PCI-mediated internalization of Gd-incorporated nanoparticles into hMSCs is a promising protocol for efficient cell labeling and tracking.     

A comparison of bone regeneration with human mesenchymal stem cells and muscle-derived stem cells and the critical role of BMP

Adult multipotent stem cells have been isolated from a variety of human tissues including human skeletal muscle, which represent an easily accessible source of stem cells. It has been shown that human skeletal muscle-derived stem cells (hMDSCs) are muscle-derived mesenchymal stem cells capable of multipotent differentiation. Although hMDSCs can undergo osteogenic differentiation and form bone when genetically modified to express BMP2; it is still unclear whether hMDSCs are as efficient as human bone marrow mesenchymal stem cells (hBMMSCs) for bone regeneration. The current study aimed to address this question by performing a parallel comparison between hMDSCs and hBMMSCs to evaluate their osteogenic and bone regeneration capacities. Our results demonstrated that hMDSCs and hBMMSCs had similar osteogenic-related gene expression profiles and had similar osteogenic differentiation capacities in vitro when transduced to express BMP2. Both the untransduced hMDSCs and hBMMSCs formed very negligible amounts of bone in the critical sized bone defect model when using a fibrin sealant scaffold; however, when genetically modified with lenti-BMP2, both populations successfully regenerated bone in the defect area. No significant differences were found in the newly formed bone volumes and bone defect coverage between the hMDSC and hBMMSC groups. Although both cell types formed mature bone tissue by 6 weeks post-implantation, the newly formed bone in the hMDSCs group underwent quicker remodelling than the hBMMSCs group. In conclusion, our results demonstrated that hMDSCs are as efficient as hBMMSCs in terms of their bone regeneration capacity; however, both cell types required genetic modification with BMP in order to regenerate bone in vivo.     

Nanoparticle delivery of stable miR-199a-5p agomir improves the osteogenesis of human mesenchymal stem cells via the HIF1a pathway

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.     

CPNE7, a preameloblast-derived factor,regulates odontoblastic differentiation of mesenchymal stem cells

Tooth development involves sequential interactions between dental epithelial and mesenchymal cells. Our previous studies demonstrated that preameloblast-conditioned medium (PA-CM) induces the odontogenic differentiation of human dental pulp cells (hDPCs), and the novel protein Cpne7 in PA-CM was suggested as a candidate signaling molecule. In the present study, we investigated biological function and mechanisms of Cpne7 in regulation of odontoblast differentiation. Cpne7 was expressed in preameloblasts and secreted extracellularly during ameloblast differentiation. After secretion, Cpne7 protein was translocated to differentiating odontoblasts. In odontoblasts, Cpne7 promoted odontoblastic markers and the expression of Dspp in vitro. Cpne7 also induced odontoblast differentiation and promoted dentin/pulp-like tissue formation in hDPCs in vivo. Moreover, Cpne7 induced differentiation into odontoblasts of non-dental mesenchymal stem cells in vitro, and promoted formation of dentin-like tissues including the structure of dentinal tubules in vivo. Mechanistically, Cpne7 interacted with Nucleolin and modulated odontoblast differentiation via the control of Dspp expression. These results suggest Cpne7 is a diffusible signaling molecule that is secreted by preameloblasts, and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts.     

Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation

Mesenchymal stem cells (MSCs) isolated from human postnatal dental pulp and periodontal ligament (PDL) tissues can give rise to multilineage differentiation in vitro and generate related dental tissues in vivo. However, the cell properties of human dental pulp stem cells (DPSCs) and PDL stem cells (PDLSCs) after in vivo implantation remain largely unidentified. In this study, cells were re-isolated from in vivo-generated dental pulp-like and PDL-like tissues (termed re-DPCs and re-PDLCs, respectively) as a result of ectopic transplantation of human DPSC and PDLSC sheets. The cell characteristics in terms of colony-forming ability, cell surface antigens and multi-differentiation potentials were all evaluated before and after implantation. It was found that re-DPCs and re-PDLCs were of human and mesenchymal origin and positive for MSC markers such as STRO-1, CD146, CD29, CD90 and CD105; and, to some extent, re-DPCs could maintain their colony forming abilities. Moreover, both cell types were able to form mineral deposits and differentiate into adipocytes and chondrocytes; however, quantitative analysis and related gene expression determination showed that the osteo-/chondro-differentiation capabilities of re-DPCs and re-PDLCs were significantly reduced compared to those of DPSCs and PDLSCs, respectively (P < 0.05); re-PDLCs showed a greater reduction potential than re-DPCs. We conclude that DPSCs and PDLSCs may maintain their MSC characteristics after in vivo implantation and, compared to PDLSCs, DPSCs appear much more stable under in vivo conditions. These findings provide additional cellular and molecular evidence that supports expanding the use of dental tissue-derived stem cells in cell therapy and tissue engineering.     

In vivo real-time visualization of mesenchymal stem cells tropism for cutaneous regeneration using NIR-II fluorescence imaging

Mesenchymal stem cells (MSCs) have shown great potential for cutaneous wound regeneration in clinical practice. However, the in vivo homing behavior of intravenously transplanted MSCs to the wounds is still poorly understood. In this work, fluorescence imaging with Ag₂S quantum dots (QDs) in the second near-infrared (NIR-II) window was performed to visualize the dynamic homing behavior of transplanted human mesenchymal stem cells (hMSCs) to a cutaneous wound in mice. Benefiting from the desirable spatial and temporal resolution of Ag₂S QDs-based NIR-II imaging, for the first time, the migration of hMSCs to the wound was dynamically visualized in vivo. By transplanting a blank collagen scaffold in the wound to help the healing, it was found that hMSCs were slowly recruited at the wound after intravenous injection and were predominantly accumulated around the edge of wound. This resulted in poor healing effects in terms of slow wound closure and thin thickness of the regenerated skin. In contrast, for the wound treated by the collagen scaffold loaded with stromal cell derived factor-1α (SDF-1α), more hMSCs were recruited at the wound within a much shorter time and were homogenously distributed across the whole wound area, which enhances the re-epithelialization, the neovascularization, and accelerates the wound healing.     

Availability of extracellular matrix biopolymers and differentiation state of human mesenchymal stem cells determine tissue-like growth in vitro

To explore the space-filling growth of adherent mesenchymal stem cells (MSC) into tissue-like structures in vitro, human bone marrow derived MSC were exposed to fibronectin-coated, millimeter-sized, triangular channels casted in poly(dimethyl siloxane) carriers. The results revealed that the three dimensional (3D) growth of MSC differs in dependence on differentiation status and availability of extracellular matrix (ECM) proteins: Massive 3D structure formation was observed for MSC under pro-osteogenic stimulation but not for undifferentiated MSC nor for MSC under pro-adipogenic stimulation; boosting cellular matrix secretion and addition of soluble ECM proteins caused extensive 3D tissue formation of undifferentiated MSC. The reported findings may contribute to bridge the gap between in vitro and in vivo analyses and guide the application of MSC in tissue replacement approaches.     

Controlling stem cell-mediated bone regeneration through tailored mechanical properties of collagen scaffolds

Mechanical properties of the extracellular matrix (ECM) play an essential role in cell fate determination. To study the role of mechanical properties of ECM in stem cell-mediated bone regeneration, we used a 3D in vivo ossicle model that recapitulates endochondral bone formation. Three-dimensional gelatin scaffolds with distinct stiffness were developed using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) mediated zero-length crosslinking. The mechanical strength of the scaffolds was significantly increased by EDC treatment, while the microstructure of the scaffold was preserved. Cell behavior on the scaffolds with different mechanical properties was evaluated in vitro and in vivo. EDC-treated scaffolds promoted early chondrogenic differentiation, while it promoted both chondrogenic and osteogenic differentiation at later time points. Both micro-computed tomography and histologic data demonstrated that EDC-treatment significantly increased trabecular bone formation by transplanted cells transduced with AdBMP. Moreover, significantly increased chondrogenesis was observed in the EDC-treated scaffolds. Based on both in vitro and in vivo data, we conclude that the high mechanical strength of 3D scaffolds promoted stem cell mediated bone regeneration by promoting endochondral ossification. These data suggest a new method for harnessing stem cells for bone regeneration in vivo by tailoring the mechanical properties of 3D scaffolds.     

A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration

Cartilage regeneration after trauma is still a great challenge for clinicians and researchers due to many reasons, such as joint load-bearing, synovial movement and the paucity of endogenous repair cells. To overcome these limitations, we constructed a functional biomaterial using a biphasic scaffold platform and a bone-derived mesenchymal stem cells (BMSCs)-specific affinity peptide. The biphasic scaffold platform retains more cells homogeneously within the sol–gel transition of chitosan and provides sufficient solid matrix strength. This biphasic scaffold platform is functionalized with an affinity peptide targeting a cell source of interest, BMSCs. The presence of conjugated peptide gives this system a biological functionality towards BMSC-specific homing both in vitro and in vivo. The functional biomaterial can stimulate stem cell proliferation and chondrogenic differentiation during in vitro culture. Six months after in vivo implantation, compared with routine surgery or control scaffolds, the functional biomaterials induced superior cartilage repair without complications, as indicated by histological observations, magnetic resonance imaging and biomechanical properties. Beyond cartilage repair, this functional biphasic scaffold may provide a biomaterial framework for one-step tissue engineering strategy by homing endogenous cells to stimulate tissue regeneration.     

Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration

Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue.     

A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion, differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded, that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10⁷ cells/ml); (ii) quick recovery of encapsulated cells (<10 min at 37 °C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with >17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype in vitro and the ability to form derivatives of the three germ layers both in vitro and in vivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1⁺) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1⁺ cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1⁺ cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.     

The nanoscale geometry of TiO2 nanotubes influences the osteogenic differentiation of human adipose-derived stem cells by modulating H3K4 trimethylation

Nanostructured materials can direct stem cell lineage commitment solely by their various, but controllable, geometric cues, which would be very important for their future application in bone tissue engineering and bone regeneration. However, the mechanisms by which nano-geometric cues dictate the osteogenic differentiation of stem cells remain unclear. Epigenetics is central to cellular differentiation, a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Here, we explored the varied osteogenic behaviors of human adipose-derived stem cells (hASCs) on titanium dioxide (TiO₂) nanotube arrays of different diameters. Both in vitro and in vivo studies demonstrated that the nanoscale geometry influenced cellular differentiation and TiO₂ nanotubes with a diameter of 70 nm was the optimal dimension for the osteogenic differentiation of hASCs. Moreover, we observed that TiO₂ nanotubes promoted the osteogenic differentiation of hASCs by upregulating methylation level of histone H3 at lysine 4 (H3K4) in the promoter regions of osteogenic genes Runx2 and osteocalcin, by inhibiting demethylase retinoblastoma binding protein 2 (RBP2). These results revealed, for the first time, the epigenetic mechanism by which nanotopography directs stem cell fate.     

Paracrine exchanges of molecular signals between alginate-encapsulated pericytes and freely suspended endothelial cells within a 3D protein gel

Paracrine signals, essential for the proper survival and functioning of tissues, may be mimicked by delivery of therapeutic proteins within engineered tissue constructs. Conventional delivery methods are of limited duration and are unresponsive to the local environment. We developed a system for sustained and regulated delivery of paracrine signals by encapsulating living cells of one type in alginate beads and co-suspending these cell-loaded particles along with unencapsulated cells of a second type within a 3D protein gel. This system was applied to vascular tissue engineering by placing human placental microvascular pericytes (PCs) in the particulate alginate phase and human umbilical vein endothelial cells (HUVECs) in the protein gel phase. Particle characteristics were optimized to keep the encapsulated PCs viable for at least two weeks. Encapsulated PCs were bioactive in vitro, secreting hepatocyte growth factor, an angiogenic protein, and responding to externally applied HUVEC-derived signals. Encapsulated PCs influenced HUVEC behavior in the surrounding gel by enhancing the formation of vessel-like structures when compared to empty alginate bead controls. In vivo, encapsulated PCs modulated the process of vascular self-assembly by HUVECs in 3D gels following implantation into immunodeficient mice. We conclude that alginate encapsulated cells can provide functional paracrine signals within engineered tissues.