A murine model of cerebral cavernous malformations with acute hemorrhage

Cavernomas are multi-lumen and blood-filled vascular malformations which form in the brain and the spinal cord. They lead to hemorrhage, epileptic seizures, neurological deficits, and paresthesia. An effective medical treatment is still lacking, and the available murine models for cavernomas have several limitations for preclinical studies. These include disease phenotypes that differ from human diseases, such as restriction of the lesions to the cerebellum, and absence of acute hemorrhage. Additional limitations of current murine models include rapid development of lesions, which are lethal before the first month of age. Here, we have characterized a murine model that recapitulates features of the human disease: lesions develop after weaning throughout the entire CNS, including the spinal cord, and undergo acute hemorrhage. This provides a preclinical model to develop new drugs for treatment of acute hemorrhage in the brain and spinal cord, as an unmet medical emergency for patients with cavernomas

Prep1 (pKnox1) transcription factor contributes to pubertal mammary gland branching morphogenesis

Prep1 (pKnox1) is a homeodomain transcription factor essential for in utero and post-natal development and an oncosuppressor gene in human and adult mice. We have analyzed its role in the development of the mouse mammary gland. We used Prep1i/i hypomorphic and Prep1F/F-Ker5CRE crosses to analyze the role of Prep1 in vivo in adult mouse mammary gland development. We also cultured mammary gland stem/progenitor cells in mammospheres to perform biochemical studies. Prep1 was expressed in mammary gland progenitors and fully differentiated mammary gland cells. Using different Prep1-deficient mouse models we show that in vivo Prep1 contributes to mammary gland branching since the branching efficiency of the mammary gland in Prep1-deleted or Prep1 hypomorphic mice was largely reduced. In-vitro, Prep1 sustained functions of the mammary stem/progenitor compartment. Prep1-deficient mammary stem/progenitor cells showed reduced ability to form mammospheres; they were not able to branch in a 3D assay, and exhibited reduced expression of Snail1, Snail2 and vimentin. The branching phenotype associated with increased Tp53-dependent apoptosis and inability to properly activate signals involved in branching morphogenesis. Finally, Prep1 formed complexes with Snail2, a transcription factor essential in branching morphogenesis, and its absence destabilizes and promotes Snail2 proteasome-mediated degradation. We conclude that Prep1 is required for normal adult mammary gland development, in particular at its branching morphogenesis step. By binding Snail2, Prep1 protects it from the proteasomal degradation

Alternative sources of neurons and glia from somatic stem cells.

Stem cell populations have been shown to be extremely versatile: they can generate differentiated cells specific to the tissue in which they reside and descendents that are of different germ layer origin. This raises the possibility of obtaining neuronal cells from new biological source of the same adult human subjects. In this study, we found that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cell-like population isolated from several newborn and adult mouse tissues: muscle and hematopoietic tissues. This population, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Brain engraftment of the somatic-derived neural stem cells generated neuronal phenotypes, demonstrating the great plasticity of these cells with potential clinical application

A critical role for regulatory T cells in driving cytokine profiles of Th17 cells and their modulation of glioma microenvironment.

IL-17A, produced by Th17 cells, may play a dual role in antitumor immunity. Using the GL261-glioma model, we investigated the effects of Th17 cells on tumor growth and microenvironment. Th17 cells infiltrate mouse gliomas, increase significantly in a time-dependent manner similarly to Treg and do not express Foxp3. To characterize the direct effects of Th17 cells on GL261 murine gliomas and on tumor microenvironment, we isolated IL-17-producing cells enriched from splenocytes derived from naïve (nTh17) or glioma-bearing mice (gTh17) and pre-stimulated in vitro with or without TGF-β. Spleen-derived Th17 cells co-expressing IL-17, IFN-γ and IL-10, but not Treg marker Foxp3, were co-injected intracranially with GL261 in immune-competent mice. Mice co-injected with GL261 and nTh17 survived significantly longer than gTh17 (P < 0.006) and gliomas expressed high level of IFN-γ and TNF-α, low levels of IL-10 and TGF-β. In vitro IL-17 per se did not exert effects on GL261 proliferation; in vivo gliomas grew equally well intracranially in IL-17 deficient and wild-type mice. We further analyzed relationship between Th17 cells and Treg. Treg were significantly higher in splenocytes from glioma-bearing than naïve mice (P = 0.01) and gTh17 produced more IL-10 than IFN-γ (P = 0.002). In vitro depletion of Treg using PC61 in splenocytes from glioma-bearing mice causes increased IL-17/IFN-γ cells (P = 0.007) and decreased IL-17/IL-10 cells (P = 0.03). These results suggest that Th17 polarization may be induced by Treg and that Th17 cells in gliomas modulate tumor growth depending on locally produced cytokines

Effect of human skin-derived stem cells on vessel architecture, tumor growth, and tumor invasion in brain tumor animal models

Glioblastomas represent an important cause of cancer-related mortality with poor survival. Despite many advances, the mean survival time has not significantly improved in the last decades. New experimental approaches have shown tumor regression after the grafting of neural stem cells and human mesenchymal stem cells into experimental intracranial gliomas of adult rodents. However, the cell source seems to be an important limitation for autologous transplantation in glioblastoma. In the present study, we evaluated the tumor targeting and antitumor activity of human skin-derived stem cells (hSDSCs) in human brain tumor models. The hSDSCs exhibit tumor targeting characteristics in vivo when injected into the controlateral hemisphere or into the tail vein of mice. When implanted directly into glioblastomas, hSDSCs distributed themselves extensively throughout the tumor mass, reduced tumor vessel density, and decreased angiogenic sprouts. In addition, transplanted hSDSCs differentiate into pericyte cell and release high amounts of human transforming growth factor-beta1 with low expression of vascular endothelial growth factor, which may contribute to the decreased tumor cell invasion and number of tumor vessels. In long-term experiments, the hSDSCs were also able to significantly inhibit tumor growth and to prolong animal survival. Similar behavior was seen when hSDSCs were implanted into two different tumor models, the chicken embryo experimental glioma model and the transgenic Tyrp1-Tag mice. Taken together, these data validate the use of hSDSCs for targeting human brain tumors. They may represent therapeutically effective cells for the treatment of intracranial tumors after autologous transplantation

Induction of neurotrophin expression via human adult mesenchymal stem cells: implication for cell therapy in neurodegenerative diseases.

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFRp75) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders

Formation of laser plasma channels in a stationary gas

The formation of plasma channels with nonuniformity of about +- 3.5% has been demonstrated. The channels had a density of 1.2x10^19 cm-3 with a radius of 15 um and with length >= 2.5 mm. The channels were formed by 0.3 J, 100 ps laser pulses in a nonflowing gas, contained in a cylindrical chamber. The laser beam passed through the chamber along its axis via pinholes in the chamber walls. A plasma channel with an electron density on the order of 10^18 - 10^19 cm-3 was formed in pure He, N2, Ar, and Xe. A uniform channel forms at proper time delays and in optimal pressure ranges, which depend on the sort of gas. The influence of the interaction of the laser beam with the gas leaking out of the chamber through the pinholes was found insignificant. However, the formation of an ablative plasma on the walls of the pinholes by the wings of the radial profile of the laser beam plays an important role in the plasma channel formation and its uniformity. A low current glow discharge initiated in the chamber slightly improves the uniformity of the plasma channel, while a high current arc discharge leads to the formation of overdense plasma near the front pinhole and further refraction of the laser beam. The obtained results show the feasibility of creating uniform plasma channels in non-flowing gas targets.Comment: 15 pages, 7 figures, submitted to Physics of Plasma

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

It is unclear whether the establishment of apical\u2013basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane

VCAM-1 expression on dystrophic muscle vessels has a critical role in the recruitment of human blood-derived CD133+ stem cells after intra-arterial transplantation.

Recently our group demonstrated the myogenic capacity of human CD133(+) cells isolated from peripheral blood when delivered in vivo through the arterial circulation into the muscle of dystrophic scid/mdx mice. CD133(+) stem cells express the adhesion molecules CD44, LFA-1, PSGL-1, alpha4-integrins, L-selectin, and chemokine receptor CCR7. Moreover these cells adhere in vitro to VCAM-1 spontaneously and after stimulation with CCL19. Importantly, after muscle exercise, we found that the expression of VCAM-1 is strongly up-regulated in dystrophic muscle vessels, whereas the number of rolling and firmly adhered CD133(+) stem cells significantly increased. Moreover, human dystrophin expression was significantly increased when muscle exercise was performed 24 hours before the intra-arterial injection of human CD133(+) cells. Finally, treatment of exercised dystrophic mice with anti-VCAM-1 antibodies led to a dramatic blockade of CD133(+) stem cell migration into the dystrophic muscle. Our results show for the first time that the expression of VCAM-1 on dystrophic muscle vessels induced by exercise controls muscle homing of human CD133(+) stem cells, opening new perspectives for a potential therapy of muscular dystrophy based on the intra-arterial delivery of CD133(+) stem cells