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The generation of induced pluripotent stem (iPS) cells will potentially allow increasing the probability of success of therapies for neurodegenerative diseases and others in the future. The following experimental work is focused on the analysis of the influence of different culture conditions, either using feeder layers or feeder-free coating substrates, such as the composition of the respective culture media and the oxygen tension, in order to achieve a serum-free, xeno-free culture system. The most promising system consisted in a Matrigel™ matrix with TeSR™2 medium, under 20% oxygen, which allowed a higher cell expansion in combination with the expression of the pluripotency extracellular markers TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4, as well as the intracellular marker Sox2. Under those culture conditions, more than 95% of Oct4 and Nanog positive cells were obtained within the population.