Human Mesenchymal Stem Cell Sheets Research Articles

Sutureless transplantation of in vivo priming human mesenchymal stem cell sheet promotes the therapeutic potential for cardiac repair

The heart, contrary to its small size, vigorously pumps oxygen and nutrients to our entire body indeterminably; and thus, its dysfunction could be devastating. Until now, there ave been several major obstacles to applying a cardiac patch for the treatment for myocardial infarction, including poor integration and low engraftment rates, due to the highly-curved surface of the heart and its dynamic nature. Here, we demonstrate a novel way for a comprehensive cardiac repair achieved by the sutureless transplantation of a highly integrable in vivo priming bone marrow mesenchymal stem cell (BMSC) sheet based on the utilization of a highly aligned thermoresponsive nanofiber membrane. Moreover, we developed a BMSC sheet specialized for vascular regeneration through ‘in-vivo priming’ using human umbilical vein endothelial cells. A prolonged secretion of multiple angiogenic cytokines, such as vascular endothelial growth factor, angiopoietin-1, insulin-like growth factor-1, which was observed in vitro from the specialized BMSC sheet seemed to lead a significant improvement in the cardiac function, including intrinsic contractibility and remodeling. In this study, we provide strong evidence that in vivo priming of a human BMSC sheet develops the therapeutic potential for cardiac repair.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cell sheets co-expressing BMP2 and VEGF

Over-dependence on existing synthetic scaffolds and insufficient osteoinductive and vasculogenic growth factors have limited the development of bone regeneration. The study aimed to assess the feasibility of using marrow-derived mesenchymal stem cells (BMSCs) cell sheets co-expressing bone morphogenetic proteins 2 (BMP2) and vascular endothelial growth factor (VEGF) for repairing critical-sized calvarial defects. BMSCs cell sheets were genetically engineered to express BMP2/VEGF alone or together. Alterations in osteogenic markers were examined by quantitative real-time PCR (qRT-PCR) and western blotting. A critical-sized calvarial bone defect model was used to investigate the osteogenesis effects of BMP2/VEGF cell sheets alone or in combination. The efficacy was assessed with micro-computed tomography (micro-CT) and histology. Invitro, the expression of BMP2 and VEGF through lentiviral transduction was confirmed by qRT-PCR and western blotting against BMP2 and VEGF. Lentiviral delivery of BMP2 and VEGF resulted in the upregulation of osteogenic markers. Invivo, in a critical-sized calvarial bone defect model, 3D-reconstructed micro-CT images revealed that treatment of the calvarial defects with the BMP2/VEGF cell sheet resulted in significantly greater amounts of newly formed bone at 8 weeks after surgery than treatment with cell sheets with single gene transduction or vehicle controls. The results were confirmed by histological assessment by H&E staining and Masson staining. This study demonstrates that BMP2/VEGF co-expressing BMSCs sheets promote bone regeneration in critical-sized calvarial bone defects. The BMP2/VEGF cell sheets provide a functional bioactive scaffold for critical-size bone reconstruction.

Enhancement of Lymphangiogenesis by Human Mesenchymal Stem Cell Sheet.

Preparation of human mesenchymal stem cell (hMSC) suspension for lymphedema treatment relies on conventional enzymatic digestion methods, which severely disrupts cell-cell and cell-extracellular matrix (ECM) connections, and drastically impairs cell retention and engraftment after transplantation. The objective of the present study is to evaluate the ability of hMSC-secreted ECM to augment lymphangiogenesis by using an in vitro coculturing model of hMSC sheets with lymphatic endothelial cells (LECs) and an in vivo mouse tail lymphedema model. Results demonstrate that the hMSC-secreted ECM augments the formation of lymphatic capillary-like structure by a factor of 1.2-3.6 relative to the hMSC control group, by serving as a prolymphangiogenic growth factor reservoir and facilitating cell regenerative activities. hMSC-derived ECM enhances MMP-2 mediated matrix remodeling, increases the synthesis of collagen IV and laminin, and promotes lymphatic microvessel-like structure formation. The injection of rat MSC sheet fragments into a mouse tail lymphedema model confirms the benefits of the hMSC-derived ECM by stimulating lymphangiogenesis and wound closure.

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets.

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs' multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties.

Hypoxia Created Human Mesenchymal Stem Cell Sheet for Prevascularized 3D Tissue Construction.

3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O2 ) and then cocultured with ECs under normoxia (20% O2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force

An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.