Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin(+) Sox2(+) neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). -- Results: Here we report the isolation and long term propagation of another population of Nestin(+) cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. -- Conclusion: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Aprendizaje colaborativo de fundamentos de tecnología de computadores en base a problemas y proyectos

Duración (en horas): Más de 50 horas. Destinatario: Estudiante y Docente

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.

The COMT Val158 Met polymorphism as an associated risk factor for Alzheimer disease and mild cognitive impairment in APOE 4 carriers

Background: The aim of this study is to examine the influence of the catechol-O-methyltranferase (COMT) gene (polymorphism Val158 Met) as a risk factor for Alzheimer's disease (AD) and mild cognitive impairment of amnesic type (MCI), and its synergistic effect with the apolipoprotein E gene (APOE). A total of 223 MCI patients, 345 AD and 253 healthy controls were analyzed. Clinical criteria and neuropsychological tests were used to establish diagnostic groups. The DNA Bank of the University of the Basque Country (UPV-EHU) (Spain) determined COMT Val158 Met and APOE genotypes using real time polymerase chain reaction (rtPCR) and polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLPs), respectively. Multinomial logistic regression models were used to determine the risk of AD and MCI. Results: Neither COMT alleles nor genotypes were independent risk factors for AD or MCI. The high activity genotypes (GG and AG) showed a synergistic effect with APOE epsilon 4 allele, increasing the risk of AD (OR = 5.96, 95% CI 2.74-12.94, p < 0.001 and OR = 6.71, 95% CI 3.36-13.41, p < 0.001 respectivily). In AD patients this effect was greater in women. In MCI patients such as synergistic effect was only found between AG and APOE epsilon 4 allele (OR = 3.21 95% CI 1.56-6.63, p = 0.02) and was greater in men (OR = 5.88 95% CI 1.69-20.42, p < 0.01). Conclusion: COMT (Val158 Met) polymorphism is not an independent risk factor for AD or MCI, but shows a synergistic effect with APOE epsilon 4 allele that proves greater in women with AD.