A mouse bone marrow stromal cell line with skeletal stem cell characteristics to study osteogenesis in vitro and in vivo.

Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy.

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels.

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.

Stimulation of axon regeneration in the mature optic nerve by intravitreal application of the toll-like receptor 2 agonist Pam3Cys.

PURPOSE: After injury of the optic nerve, mature retinal ganglion cells (RGCs) are normally unable to regenerate axons and undergo apoptosis. However, inflammatory stimulation in the eye induced by the release of beta/gamma-crystallins from the injured lens or intravitreal zymosan injection transforms RGCs into an active regenerative state, protecting these neurons from cell death and allowing them to regenerate axons back into the optic nerve. METHODS: The authors tested whether intravitreal application of the selective, water-soluble, toll-like receptor 2 agonist Pam(3)Cys can delay axotomized RGC cell death and stimulate the regeneration of axons using an in vitro and in vivo paradigm. RESULTS: Intravitreal injection of Pam(3)Cys, as lens injury (LI), induced the upregulation of ciliary neurotrophic factor and glial fibrillary acidic protein expression in retinal glia accompanied by the activation of the JAK/STAT3 pathway in RGCs. As a consequence, RGCs switched to a regenerative state, indicated by a significant upregulation of GAP43 expression and increased neurite outgrowth of RGCs in culture. Repeated intravitreal Pam(3)Cys application in vivo induced neuroprotective effects and caused stronger axon regeneration in the injured optic nerve than observed after LI. CONCLUSIONS: Pam(3)Cys may be a suitable agent for stimulating CNS regeneration.

Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor.

After optic nerve injury retinal ganglion cells (RGCs) normally fail to regenerate axons in the optic nerve and undergo apoptosis. However, lens injury (LI) or intravitreal application of zymosan switch RGCs into an active regenerative state, enabling these neurons to survive axotomy and to regenerate axons into the injured optic nerve. Several factors have been proposed to mediate the beneficial effects of LI. Here, we investigated the contribution of glial-derived ciliary neurotrophic factor (CNTF) to LI-mediated regeneration and neuroprotection using wild-type and CNTF-deficient mice. In wild-type mice, CNTF expression was strongly upregulated in retinal astrocytes, the JAK/STAT3 pathway was activated in RGCs, and RGCs were transformed into an active regenerative state after LI. Interestingly, retinal LIF expression was correlated with CNTF expression after LI. In CNTF-deficient mice, the neuroprotective and axon growth-promoting effects of LI were significantly reduced compared with wild-type animals, despite an observed compensatory upregulation of LIF expression in CNTF-deficient mice. The positive effects of LI and also zymosan were completely abolished in CNTF/LIF double knock-out mice, whereas LI-induced glial and macrophage activation was not compromised. In culture CNTF and LIF markedly stimulated neurite outgrowth of mature RGCs. These data confirm a key role for CNTF in directly mediating the neuroprotective and axon regenerative effects of inflammatory stimulation in the eye and identify LIF as an additional contributing factor.

Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF.

Intravitreal injections of exogenous CNTF stimulate axon regeneration of RGCs in vivo. Nevertheless, controversy exists over the ability of exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Here we demonstrate that CNTF potently stimulated axon outgrowth of mature RGCs in culture in a JAK/STAT3- and PI3K/AKT-signaling pathway-dependent fashion and stronger than oncomodulin. Additional cAMP elevation or inhibition of MAPK activity increased these effects. In vivo intravitreal injections of exogenous CNTF induced endogenous CNTF expression in astrocytes in a manner that depended on the MAPK/ERK-signaling pathway activation. Reduction of endogenous CNTF expression by MAPK/ERK pathway inhibitors or its absence in CNTF deficient mice markedly reduced the neurite growth-promoting effects of exogenous CNTF. These data demonstrate that CNTF is a potent axon growth-promoting factor for mature RGCs. However, exogenously applied CNTF stimulates RGCs in vivo partially indirectly via a mechanism that depends on astrocyte-derived CNTF.

Crystallins of the beta/gamma-superfamily mimic the effects of lens injury and promote axon regeneration.

Adult retinal ganglion cells (RGCs) can survive axotomy and regrow lengthy axons when exposed to lens injury (LI). The neuroprotective and axon-growth-promoting effects of LI have been attributed to an infiltration of activated macrophages into the inner eye and recently also to astrocyte-derived CNTF. The present work reveals that certain purified lens proteins (crystallins) cause the effects of LI. Intravitreal injections of beta- or gamma-crystallins, but not of alpha-crystallin, strongly enhanced axon regeneration from retinal explants in culture, within peripheral nerve grafts or the crushed optic nerve. Deposition of the effective crystallins within the vitreous body was also associated with an influx of circulating macrophages and an activation of retinal astrocytes, Müller cells, and resident microglia. Furthermore beta-crystallin induced CNTF expression in retinal astrocytes and activation of CNTF's major downstream signaling pathway (JAK/STAT3) when intravitreally injected or added to the culture medium ex vivo. Consistently, in culture the addition of beta- and gamma-crystallins to the medium also increased axon regeneration from retinal explants. These results demonstrate that crystallins of the beta/gamma-superfamily are the lens-derived activators of cascades, which lead to axonal regeneration and suggest that their effects might be mediated by astrocyte-derived CNTF.

Neuroprotective and axon growth promoting effects of intraocular inflammation do not depend on oncomodulin or the presence of large numbers of activated macrophages.

Retinal ganglion cells (RGCs) cannot regenerate their axons after injury and undergo apoptosis soon after an intraorbital injury of the optic nerve. However, RGCs reactivate their axonal growth program when inflammatory reactions occur in the eye, which enables them to survive axotomy and to regenerate lengthy axons into the lesioned optic nerve. Lens injury (LI) and zymosan injections can induce these beneficial processes and provoke also a strong accumulation of activated macrophages in the vitreous body. It has recently been suggested that macrophage-derived oncomodulin is the principal mediator of this phenomenon. We show here that oncomodulin is not significantly expressed in primary macrophages and that the intraocular levels of this protein do not increase after LI or zymosan treatment. Furthermore, greatly reducing the invasion of macrophages into the inner eye does not diminish the neuroprotective effects of LI, but rather increases axon regeneration into the optic nerve. Axon regeneration is correlated with the activation of retinal astrocytes and Müller cells. Our data suggest that intraocular inflammation mediates its main beneficial effects through factors other than oncomodulin and that the underlying mechanism might be independent of the presence of activated macrophages.

Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.

Retinal ganglion cells (RGCs) normally fail to regenerate injured axons and undergo apoptosis soon after injury. We have recently shown that lens injury (LI) or intravitreally applied zymosan allow RGCs to survive axotomy and regenerate axons in the injured optic nerve. Activated macrophages and oncomodulin have been suggested to be the principal mediators of this phenomenon. However, several lines of evidence show that macrophage-derived factors alone cannot account for all the beneficial effects of intraocular inflammation. We show here that LI or zymosan induce upregulation of ciliary neurotrophic factor (CNTF) in retinal astrocytes and release CNTF independent of macrophages and activate the transcription factor signal transducers and activators of transcription 3 (STAT3) in RGCs. Levels of CNTF expressed in retinal glia and STAT3 activation in RGC were correlated with the time course of RGCs switching to an active regenerative state. Intravitreal injections of antibodies against CNTF or a Janus-kinase inhibitor compromised the beneficial effects of LI, whereas an antiserum against oncomodulin was ineffective. Like the action of CNTF, the effects of LI were potentiated by drugs that increase intracellular cAMP levels, resulting in strong axon regeneration in vivo. These data indicate that astrocyte-derived CNTF is a major contributor to the neuroprotective and axon-growth-promoting effects of LI and zymosan. These findings could lead to the development of a therapeutic principle for promoting axon regeneration in the CNS as a whole.