CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells

Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-alpha, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. in the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site. (C) 2013 the Authors. Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Neurobiol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Neurobiol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilFAPESP: 2005/04061-8FAPESP: 2012/00652-5CNPq: 573909/2008-3]Web of Scienc

Long-lasting anxiolytic effect of neural precursor cells freshly prepared but not neurosphere-derived cell transplantation in newborn rats

Background: The GABAergic system plays an important role in modulating levels of anxiety. When transplanted into the brain, precursor cells from the medial ganglionic eminence (MGE) have the ability to differentiate into GABAergic interneurons and modify the inhibitory tone in the host brain. Currently, two methods have been reported for obtaining MGE precursor cells for transplantation: fresh and neurosphere dissociated cells. Here, we investigated the effects generated by transplantation of the two types of cell preparations on anxiety behavior in rats. Results: We transplanted freshly dissociated or neurosphere dissociated cells into the neonate brain of male rats on postnatal (PN) day 2–3. At early adulthood (PN 62–63), transplanted animals were tested in the Elevated Plus Maze (EPM). To verify the differentiation and migration pattern of the transplanted cells in vitro and in vivo, we performed immunohistochemistry for GFP and several interneuron-specific markers: neuropeptide Y (NPY), parvalbumin (PV) and calretinin (CR). Cells from both types of preparations expressed these interneuronal markers. However, an anxiolytic effect on behavior in the EPM was observed in animals that received the MGE-derived freshly dissociated cells but not in those that received the neurosphere dissociated cells. Conclusion: Our results suggest a long-lasting anxiolytic effect of transplanted freshly dissociated cells that reinforces the inhibitory function of the GABAergic neuronal circuitry in the hippocampus related to anxiety-like behavior in rats

Effects of FGF-2 and EGF removal on the differentiation of mouse neural precursor cells

Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.As terapias celulares para doenças neurológicas têm avançado e células precursoras neurais (NPC) surgem como candidatas ideais para o transplante de células neurais em muitas doenças. No entanto, trabalhos adicionais devem ser feitos para determinar o ambiente de cultivo ideal para a expansão in vitro das NPC, sem alterar sua plasticidade, e os mecanismos de sinalização celular do fator de crescimento epidérmico (EGF) e fator de crescimento de fibroblasto 2 (FGF-2) no crescimento, sobrevivência e diferenciação da neuroesfera. Nesse trabalho avaliamosNPCcultivadas na presença e na ausência de FGF-2 e EGF e mostramos que esses fatores de crescimento são responsáveis pela proliferação das NPC. Também foi demonstrado que a produção endógena de fatores de crescimento alterna de FGF-2 a fator de crescimento de insulina 1 (IGF-1) e fator de crescimento derivado de plaquetas b (PDGFb) após remoção de EGF e FGF-2. NPC de camundongo cultivadas em suspensão mostraram padrões de localização neuronal distintos (centro versus borda) tanto no grupo controle como no grupo sem EGF e FGF-2. Juntos, esses resultados mostram que a remoção de EGF e FGF-2 exerce importante ação na diferenciação de NPC e possivelmente contribui para melhor compreensão dos mecanismos envolvidos na diferenciação. Nossos achados sugerem que, privando as NPC de fatores de crescimento antes do transplante, talvez aumente as chances de que as células efetivamente se integrem ao hospedeiro.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Departamento de FisiologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de BiofísicaUNIFESP, Depto. de FisiologiaUNIFESP, Depto. de BiofísicaSciEL

Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells

Background: Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.Principal Findings: We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. in this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.Conclusions: These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institutos do Milenio de Bioengenharia TecidualUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilFAPESP: fellowCNPq: fellowWeb of Scienc

Comparative assessment of stem cell therapy in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons, to which no efficient treatment is currently available able of increasing life span. Because ALS is a multifactorial disease, it is supposed that the most promising therapies may be the ones capable of playing multiple roles. Despite some aspects remained unknown, stem cells (SC) may be able to modify the lesion microenvironment, modulating multiple systems and, under this aspect, they show up as candidates for neurodegenerative disease therapies. In this work we used a model of transgenic rats that superexpress a mutaded form of human superoxide dismutase I (SOD1) as a tool to study ALS. In the first step of this work we revealed some of the cellular and molecular events that took place during the neuronal degeneration process in ALS rat model. We noticed that motor neuron death was accompanied by gliosis, increased expression of inflammatory cytokines and some growth factors, besides the mobilization of endogenous neural precursor cells. Accordingly to the data acquired in the first step, two disease stages were chosen to study cell therapy in SOD1 rats: the pre-symptomatic stage and the initial symptomatic stage. The second step experimental design has been organized accordingly to two distinct questions: I) Can SC therapies retard disease progression? II) How SC behave to neurodegenerative environment in different stages? The effects of various types of SC were compared: neural, mesenchymal and the combination of both. SC transplantation did not promoted any motor function improvement, possibly due to the great variability of ALS rat model and to the fact that SC did not went far from the transplantation site. Each type of SC used here resulted in distinct effects over ALS degenerative microenvironment, highlighting gliosis modulation, mobilization of endogenous neural precursors and differential expression of chemokines and cytokines in the spinal cord. These effects depends on disease stage when cell transplantation happened, illustrating a complex interaction between donor and host cells. Finally, neurospheres derived from SOD1 transgenic fetal spinal cord, that consists in an ALS in vitro study model, indicated that some alterations promoted by mutated SOD1 can be early noticed in the development. A main contribution of this work is in the conception that the development of cell therapies for ALS must consider a large comprehension of the degenerative mechanisms and the biological properties of the several sources of SC.A esclerose lateral amiotrófica (ELA) é uma síndrome neurodegenerativa caracterizada por degeneração progressiva dos neurônios motores superiores e inferiores, para a qual, atualmente, não há um tratamento eficiente capaz de aumentar a sobrevida dos pacientes. Por tratar-se de uma doença multifatorial, acredita-se que terapias que consigam atuar em diversas causas simultaneamente sejam mais efetivas. Apesar de ainda não ser totalmente compreendida, as células-tronco (CT) aparentemente são capazes de modificar o microambiente da lesão e, sob este aspecto, apresentam-se como boas candidatas às terapias para doenças neurodegenerativas. No presente trabalho utilizamos o modelo de ratos transgênicos que superexpressam a enzima superóxido dismutase dependente de cobre e zinco (SOD1) humana mutada como ferramenta de estudo sobre a ELA. Na primeira etapa do trabalho esclarecemos a sucessão de eventos celulares e moleculares que ocorrem ao longo do processo de degeneração neuronal no modelo de ELA em ratos. Observamos que a morte dos neurônios motores foi acompanhada por gliose, aumento da expressão de citocinas inflamatórias e de alguns fatores tróficos, além de mobilização das células precursoras neurais. De acordo com os dados obtidos na primeira etapa, duas fases distintas da doença foram selecionadas para estudar a terapia celular nos ratos SOD1: a fase pré-sintomática e a fase sintomática inicial. O desenho experimental da segunda etapa foi elaborado de acordo com duas questões distintas: I) A progressão da doença pode ser retardada por terapias com CT? II) Como as CT reagem ao ambiente neurodegenerativo em diferentes estágios? Foram comparados os efeitos de diversos tipos de CT: neurais, mesenquimais ou a associação de ambas. O transplante celular não promoveu qualquer melhora no quadro motor, possivelmente devido à grande variabilidade do modelo e pelo fato de que as CT não se distanciaram muito do local do transplante. Vimos que os diferentes tipos de CT utilizados resultaram em efeitos distintos no microambiente degenerativo da ELA, destacando-se a modulação da gliose, a mobilização dos precursores neurais endógenos e a expressão diferencial de quimocinas e citocinas na medula espinal. Esses efeitos dependeram da fase da doença em que ocorreu o transplante celular, caracterizando uma complexa interação entre as células transplantadas e do hospedeiro. Por fim, as neuroesferas derivadas da medula espinal fetal de transgênicos para SOD1, que consiste em um modelo in vitro para estudo da ELA, indicam que, em fases precoces do desenvolvimento, algumas alterações produzidas pela SOD1 mutada já podem ser detectadas. Uma das maiores contribuições deste trabalho está na concepção de que o desenvolvimento de terapias celulares para ELA deve envolver uma vasta compreensão dos mecanismos degenerativos e das propriedades biológicas das inúmeras fontes de CT.TED