Transplantation of myocyte precursors derived from embryonic stem cells transfected with IGFII gene in a mouse model of muscle injury.

BACKGROUND: Reconstruction of skeletal muscle tissue is hampered by the lack of availability of functional substitution of the tissue. METHODS: Embryonic stem (ES) cells were transfected with the insulin-like growth factor (IGF) II gene and were selected with G418. The resultant cell clones were analyzed regarding their myogenic differentiation in vitro and in vivo. RESULTS: The cells expressed early and late myogenic differentiation markers, including myoD, myogenin, and dystrophin in vitro. They had phosphorylated Akt within the cells, suggesting their activation by the secreted IGFII. Transplantation of the cells to injured anterior tibial muscle of mice significantly improved their motor functions compared to injured mice transplanted with undifferentiated ES cells and injured mice given vehicle alone. The transfected cells adapted to the injured muscle, formed myofibers positive for dystrophin and negative for MyoD and myogenin. Trichrome staining and toluidine blue staining support myofiber formation in vivo. The enzymatic activity of acetylcholine esterase suggested the functional activity of the regenerated motor units. The evoked electromyogram of anterior tibial muscle transplanted with the transfected cells showed significantly higher potentials compared to that transplanted with undifferentiated ES cells and that injected with phosphate-buffered saline (control injury). Electron microscopic examination confirmed the myofiber formation in the cells in vivo. CONCLUSIONS: Transfection of IGFII gene into ES cells may be applicable for transplantation therapy of muscle damage due to injury and myopathies.

Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury.

ES cells transfected with the MASH1 gene yielded purified spinal motoneuron precursors expressing HB9 and Islet1. The cells lacked the expression of Nogo receptor that was of great advantage for axon growth after transplantation to an injured spinal cord. After transplantation, mice with the complete transection of spinal cord exhibited excellent improvement of the motor functions. Electrophysiological assessment confirmed the quantitative recovery of motor-evoked potential in the transplanted spinal cord. In the grafted spinal cord, gliosis was inhibited and Nogo receptor expression was scarcely detected. The transplanted cells labeled with GFP showed extensive outgrowth of axons positive for neurofilament middle chain, connected to each other and expressed Synaptophysin, Lim1/2 and Islet1. Thus, the in vivo differentiation into mature spinal motoneurons and the reconstitution of neuronal pathways were suggested. The grafted cell population was purified for neurons and was free from teratoma development. These therapeutic strategies may contribute to a potent treatment for spinal cord injury in future.

Transplantation of neural cells derived from retinoic acid-treated cynomolgus monkey embryonic stem cells successfully improved motor function of hemiplegic mice with experimental brain injury.

We induced neural cells by treating cynomolgus monkey embryonic stem (ES) cells with retinoic acid. The treated cells mainly expressed betaIIItubulin. They further differentiated into neurons expressing neurofilament middle chain (NFM) in elongated axons. Half of the cells differentiated into Islet1+ motoneurons in vitro. The monkey ES-derived neural cells were transplanted to hemiplegic mice with experimental brain injury mimicking stroke. The neural cells that had grafted into periventricular area of the mice distributed extensively over the injured cortex. Some of the transplanted cells expressed the neural stem/progenitor marker nestin 2 days after transplantation. The cells expressed markers characteristic of mature motoneurons 28 days after transplantation. Mice with the neural cell graft gradually recovered motor function, whereas control animals remained hemiplegic. This is the first demonstration that neural cells derived from nonhuman primate ES cells have the ability to restore motor function in an animal model of brain injury.

Noggin and basic FGF were implicated in forebrain fate and caudal fate, respectively, of the neural tube-like structures emerging in mouse ES cell culture.

We developed neural tube-like structures accompanying neural crest-like cells by treating embryonic stem (ES) cells with retinoic acid. The structures contained pseudostratified Nestin+Vimentin+ neuroepithelial cells surrounded by Masson staining+ basement membrane. betaIIItubulin+Synaptophysin+ mature neurons and glial fibrillary acidic protein (GFAP)+ glial cells dispersed outside of the membrane. Addition of Noggin to the culture induced prominent proliferation of the neuroepithelial cells, leading to epithelial hyperstratification of the structures. mRNAs of transcription factors essential for forebrain development such as Emx1/2 and Pax6 were specifically expressed and Islet1+Lim1/2- motoneurons appeared by the addition of Noggin. In contrast, basic fibroblast growth factor (bFGF) promoted enlargement of central lumen and elongation of the structures. mRNAs of caudal markers, Gbx2, Cdx2 and Hoxb4/9 were expressed and Lim1/2+ spinal motoneurons appeared by the addition of bFGF. Addition of BMP-4 similarly brought about mild enlargement of central lumen of the structures. Interestingly, the addition of BMP-4 induced Slug+ neural crest-like cells surrounding the tube-like structures. mRNAs of Snail and dHand, other markers for neural crest cells, were also expressed by the addition of BMP-4. These results suggest that Noggin lead the neural-tube like structures to forebrain fate, whereas bFGF was involved in the caudalization. BMP-4 was implicated in emergence of the neural crest-like cells. Differentiation of ES cells by the present methods may mimic neurulation and subsequent neural development of early embryos, and elucidates the opposite effects of Noggin and bFGF for the neural tube development.

Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice.

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

Anatomical and functional recovery by embryonic stem cell-derived neural tissue of a mouse model of brain damage.

We have treated undifferentiated mouse embryonic stem (ES) cells with all-trans retinoic acid (RA) to induce differentiation in vitro into neuron-like cells with good cell viability for use as a graft. Furthermore, we asked whether the RA-induced neuron-like cells restored neurological dysfunction. To this end, the cells were transplanted into right hemiplegia model of mice, developed by a cryogenic injury of motor cortex. Motor function of the recipients was gradually improved, whereas little improvement was observed in control mice. The lesion showed clustering of mature and almost mature neuron-like cells in mice transplanted with the RA-treated cells. The grafted cells had synaptic vesicles. This finding may suggest their maturation and synaptic connection in the recipient brain. Even though further study is necessary to elucidate molecular and cellular mechanisms responsible for the functional recovery, we consider that the ES cells may have advantage for use as a donor source in various neurological disorders including motor dysfunction.

Behçet's disease.

Behçet's disease (BD) is a systemic disorder characterized by recurrent attacks of acute inflammation. Major symptoms are oral aphthous ulcers, uveitis, skin lesions, and genital ulcerations. Involvement of vessels, gastrointestinal (GI) tract, and central nervous system (CNS) is less frequent but is associated with a poor prognosis. Pulmonary complications of BD include aneurysms of the aorta, great vessels, or pulmonary arteries; arterial or venous thrombosis; pulmonary parenchymal changes; pleurisy, and intracardiac thrombosis. Hemoptysis caused by pulmonary artery aneurysms may lead to lethal hemorrhage. Recent advances in therapeutic strategies have improved the prognosis. In this review, the salient clinical and histopathological features of BD and treatment strategies are discussed.

Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor 4-mediated lipopolysaccharide stimulation.

IL-3-dependent mucosal-like mast cells undergo apoptosis upon withdrawal of IL-3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan-caspase inhibitors z-VAD-FMK or BOC-D-FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z-VAD-FMK or BOC-D-FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase-dependent and -independent mechanisms. The caspase-independent apoptosis is mediated by the translocation of endonuclease G from mitochondria into nuclei. Withdrawal of IL-3 caused down-regulation of Bcl-xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome c and endonuclease G from mitochondria. However, stimulation of mast cells through Toll-like receptor 4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl-xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN-gamma, which up-regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play important roles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines.

Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes.

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.