Erella Livne

Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Engineering the Growth Factor Microenvironment with Fibronectin Domains to Promote Wound and Bone Tissue Healing

A multifunctional fibronectin fragment enhances the regenerative effects of growth factors in vivo in animal models of chronic wounds and critical-size bone defects. Sweet Synergy Engineers have long been interested in creating the perfect environment for repairing injured tissues, which range from broken blood vessels to shattered nerves. Such efforts have included both simple materials, like collagen, and complex ones comprising a polymeric labyrinth of biomolecules and cells. As described in this issue, Martino et al. have hit the sweet spot for engineering the cellular microenvironment: a combination of natural polymer and recombinant protein that recruits growth factors to wounds and convinces cells to repair the damage. Martino and colleagues sought to generate a matrix that would sequester growth factors. The authors started with a fibrin matrix, which is used clinically as a tissue substitute to promote healing. Next, they pieced together two fibronectin (FN) fragments—the 9th to 10th and the 12th to 14th type III repeats—to control integrin and growth factor binding, respectively. Finally, the resulting recombinant FN fragment, FN III9-10/12-14, was covalently immobilized on the fibrin scaffold. The FN III9-10/12-14 matrix was able to bind vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and bone morphogenetic protein (BMP)—three factors that are intricately involved in skin and bone repair. FN III9-10/12-14 in combination with VEGF and PDGF enhanced proliferation of endothelial cells, smooth muscle cells, and mesenchymal stem cells in vitro. The engineered FN fragment, when co-delivered with all three growth factors, also stimulated cell migration to a greater extent than control FN proteins, suggesting improved signaling synergy between growth factors and the recombinant FN. To see whether the material healed tissues in vivo, Martino and colleagues injected their designer scaffold into the wounds of diabetic mice and into the calvarial defects of skeletally mature rats. Enhanced reepithelialization, granulation tissue formation, and angiogenesis were noted for the wounds. For the bone defects, the authors reported increased bone tissue deposition and recruitment of bone progenitor cells. These preclinical demonstrations in rodent models show promise for the use of the FN III9-10/12-14–modified matrices in humans to heal chronic wounds and repair bones. Although testing in larger animal models might be necessary before translation, it is clear that these engineered microenvironments improve the synergy between endogenous growth factors and cells to restore tissue form and function to normal. Although growth factors naturally exert their morphogenetic influences within the context of the extracellular matrix microenvironment, the interactions among growth factors, their receptors, and other extracellular matrix components are typically ignored in clinical delivery of growth factors. We present an approach for engineering the cellular microenvironment to greatly accentuate the effects of vascular endothelial growth factor–A (VEGF-A) and platelet-derived growth factor–BB (PDGF-BB) for skin repair, and of bone morphogenetic protein–2 (BMP-2) and PDGF-BB for bone repair. A multifunctional recombinant fragment of fibronectin (FN) was engineered to comprise (i) a factor XIIIa substrate fibrin-binding sequence, (ii) the 9th to 10th type III FN repeat (FN III9-10) containing the major integrin-binding domain, and (iii) the 12th to 14th type III FN repeat (FN III12-14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2. We show potent synergistic signaling and morphogenesis between α5β1 integrin and the growth factor receptors, but only when FN III9-10 and FN III12-14 are proximally presented in the same polypeptide chain (FN III9-10/12-14). The multifunctional FN III9-10/12-14 greatly enhanced the regenerative effects of the growth factors in vivo in a diabetic mouse model of chronic wounds (primarily through an angiogenic mechanism) and in a rat model of critical-size bone defects (through a mesenchymal stem cell recruitment mechanism) at doses where the growth factors delivered within fibrin only had no significant effects.

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting.

Maxillary sinus membrane lifting is a common procedure aimed at increasing the volume of the maxillary sinus osseous floor prior to inserting dental implants. Clinical observations of bone formation in sinus lifting procedures without grafting bone substitutes were observed, but the biological nature of bone regeneration in sinus lifting procedures is unclear. This study tested whether this osteogenic activity relies on inherent osteogenic capacity residing in the sinus membrane by simulating the in vivo clinical condition of sinus lifting in an animal model. Maxillary sinus membrane cells were cultured in alpha-MEM medium containing osteogenic supplements (ascorbic acid, dexamethasone). Cultured cells revealed alkaline phosphatase activity and mRNA expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and osteonectin) verifying the osteogenic potential of the cells. Fresh tissue samples demonstrated positive alkaline phosphatase enzyme activity situated along the membrane-bone interface periosteum-like layer. To simulate the in vivo clinical conditions, the membranes were folded to form a pocket-like structure and were transplanted subcutaneously in immunodeficient mice for 8 weeks. New bone formation was observed in the transplants indicating the innate osteogenic potential within the maxillary Schneiderian sinus membrane and its possible contribution to bone regeneration in sinus lifting procedures.

Controlled release of BMP-2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model.

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP‐2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature‐sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP‐2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP‐2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP‐2 loaded into the scaffolds was released by the end of this time period. The released BMP‐2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP‐2‐loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP‐2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP‐2 delivery for bone‐regeneration applications. Copyright

The c-Jun Dimerization Protein 2 Inhibits Cell Transformation and Acts as a Tumor Suppressor Gene

The c-Jun dimerization protein, JDP2, is a member of the AP-1 (activating protein-1) family of the basic leucine zipper transcription factors. JDP2 can bind 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and cAMP-responsive element DNA response elements, resulting in the inhibition of transcription. Although the role of AP-1 in cell proliferation and malignant transformation is well established, the role of JDP2 in this process is of subject to debate. On the one hand, JDP2 was shown to inhibit cyclin D transcription and promote differentiation of skeletal muscle and osteoclast cells. On the other hand, JDP2 was shown to partially transform chicken embryo fibroblast and was identified in a screen for oncogenes able to collaborate with the loss of p27kip cyclin-dependent inhibitor to induce lymphomas. Using cell transformation assays in NIH3T3 cells and injection of prostate cancer cell lines overexpressing JDP2 into severe combined immuno-deficient (SCID) mice, we show for the first time the potential role of JDP2 in inhibition of cell transformation and tumor suppression. The mechanism of tumor suppressor action of JDP2 can be partially explained by the generation of inhibitory AP-1 complexes via the increase of JunB, JunD, and Fra2 expression and decrease of c-Jun expression.

Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix

Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

Enhancement of bone defect healing in old rats by TGF-β and IGF-1

Bone defects are often created in order to repair bone pathologies. In the aging population, the healing of such defects is very limited. Bone healing in aging depends on the availability of various hormone and growth factors. The ability of growth factors to enhance bone formation in femoral defects in old rats was tested. Bone defects were induced in femurs of old rats. A single dose of transforming growth factor-beta (TGF-beta), IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were tested after 2 and 4 weeks. Radiology revealed that mineralization appeared in the 2 weeks group in defects treated with TGF-beta and in defects treated with TGF-beta, TGF-beta+IGF-1 in the 4 weeks groups. Computerized tomography (CT) coronal and axial images revealed that 4 weeks after treatment with TGF-beta+IGF-1, a complete bone bridge was observed. Morphology revealed that these defects were filled with trabecular bone. A less pronounced bone healing was observed after TGF-beta or IGF-1, while control specimens revealed partial healing of the bone defect. Biomechanical tests indicated that treatment with TGF-beta, IGF-1 or TGF-beta+IGF-1 resulted in a significant increase of bone bending rigidity compared to control in the 4 weeks group and that TGF-beta+IGF-1 was the most inductive in this respect. The ability to induce bone healing in aging by TGF-beta+IGF-1 is of a great clinical importance for restoration of bone strength and biomechanical properties of bone defects in aging.

Bone marrow stem cells and biological scaffold for bone repair in aging and disease

The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute in the prevention of the complications resulting from long-term immobilization that are especially fatal in old age. Our aim was to test the ability of MSCs inserted into a biological scaffold to enhance bone defect repair. Osteoprogenitor cells were selected from rat bone marrow stem cells cultured in DMEM medium supplemented with FCS, antibiotics, ascorbic acid, beta-glycerophosphate, and dexamethasone. The selected osteogenic subpopulation was identified by osteocalcin immunohistochemistry as well as Alizarin red S and von Kossa staining which are specific for bone matrix and mineral deposition. Committed osteoprogenitor cells cultured on the hydrogel scaffold were transplanted into the area of a rat tibia segmental bone defect and examined after 6 weeks. Radiology images revealed that 6 weeks post-implantaion, calcified material was present in the site of the defect, indicating new bone formation. It is concluded that committed osteogenic MSCs contained in a biocompatible scaffold can provide a promising surgical tool for enhancement of bone defect healing that will minimize the complications of bone repair in aging and disease.

HUMAN BONE MARROW MESENCHYMAL STEM CELLS : A SYSTEMATIC REAPPRAISAL VIA THE GENOSTEM EXPERIENCE

Genostem (acronym for “Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side”) has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.