A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

Background: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. Methods: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. Results: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. Conclusions: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Respuesta a la sequía de Pinus radiata D. Don y su implicación en los procesos de tolerancia

160 p. (Bibliogr. 141-160)

Polilaktida oinarri duten inplante polimeriko biodegradagarrien diseinu, fabrikazio eta propietateak

[EU]3D inprimaketa gaur egun, biomedikuntzaren garapenerako aukera ezberdinak ematen dituen teknologia iraultzaile gisa aurkezten da; bai medikuntza arloan formazio eta ikerkuntzarako erreminta gisa, baita dispositibo berrien diseinu eta fabrikaziorako. Bere abantailarik aipagarriena prototipaketa azkarra da. Gainera, teknologia honek barne egitura eta forma ezberdineko objektuak fabrikatzea ahalbidetzen du koste lehiakor batean. Lan honen helburua 3D inprimagailuen bidezko prototipoen fabrikazioan zentratzen da, horretarako lehengai bezala polimero biodegradagarri eta biobateragarria erabiliz. Horrez gain, metodo tradizionalarekin konparatuz teknologia honek izan ditzakeen abantailak ere aztertu nahi dira, ez bakarrik alde ekonomikoari edo denborari begira, baita fabrikatutako objektuen propietateei begira ere. Dena dela, horrekin hasi aurretik ezaugarritze fisiko eta termikoa burutu beharko zaie lehengaiei, 3D inprimagailuaren parametroen aukeraketa egokia egiteko eta parametro horien eta amaierako produktuaren kalitate, egitura eta propietateen artean erlazio egokia ezartzeko. Lan hau aurrera eramateko poli(L-laktida)-rekin (PLLA) egingo da lan, bai ehun ingeniaritzan baita hezurren apurketen finkapenerako dispositiboetan oso erabilia izan den polimeroa.

Structural studies of the hipersaline adaptation of proteins belonging to halophilic archaea

183 p.

Inplante kirurgiko erradiopakoen bideragarritasuna: plla/bario sulfato sistemaren ``in vitro´´ degradazioa.

[EU]Polimero biobateragarri eta biodegradagarrien erabilera medikuntza arloan aurrerapauso handia suposatu du. Inplanteen arloan (torlojuak, iltzeak, plakak, eta abar…) eta ehun ingeniaritzan garrantzia handia dute. Hala ere, polimero hauen inplanteek desabantaila nabaria aurkezten dute ohiko material metalikoekin alderatuz: erradiopazitate eza. Ikerketa lan honetan karga erradiopako baten adizioak polimero biobateragarri eta biodegradagarri baten giza gorputzaren baldintzetan (pH=7,2 eta 37ºC) burututako in vitro degradazioan duen eragina aztertu da, %70 poli(L-laktida) (PLLA) eta %30 bario sulfato (BaSO4) sistemaren degradazioa, hain zuzen ere. Aztertutako karga erradiopakoak PLLAren degradazioan eragin handirik ez duela ondorioztatu da, beraz, sistema bideragarria dela.

Influence of Extracellular Matrix Components on the Expression of Integrins and Regeneration of Adult Retinal Ganglion Cells

Purpose Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites? Methods Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against beta III-Tubulin to identify the RGCs, and antibodies against the integrin subunits: alpha V, alpha 1, alpha 3, alpha 5, beta 1 or beta 3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed. Results PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly alpha 1 beta 1 or alpha 3 beta 1 on L, alpha 1 beta 1 on CI and CIV, and alpha 5 beta 3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK). Conclusions Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.