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This work was supported by grants from the Ministerio de Ciencia e Innovación (Spain) SAF2011 –22624 (to J.M.) and PI080566 (to C.L-L.) and from the CSIC (Spain) 200920I117 (to J.M.) and by Biogen-Idec (to J.M). P.O-N was supported by Biogen-Idec. B.M-R was supported by a FPI fellowship from the Ministerio de Ciencia e Innovación. E.S-P. was supported by a Severo Ochoa Fellowship (FICYT, Consejería de Educación y Ciencia, Spain). E.C-M. was supported by the Juan de la Cierva program (Ministerio de Ciencia e Innovación, Spain). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

We thank Cristina B. Mendoza for excellent technical assistance and Dr.Peter Andrews for providing us reagents. Conceived and designed the experiments: BS MFF CLL. Performed the experiments: BS VC STS. Analyzed the data: BS RMR MABG CLL. Contributed reagents/materials/analysis tools: RMR FO JO JC HM. Wrote the paper: BS HM MFF CLL.

Human embryonic stem cells (hESCs) are especially resistant to several cellular stresses, but the existence and induction of Endoplasmic Reticulum (ER) stress by culture conditions are unknown. Using qPCR, here, we investigated the behavior of the principal sensors of ER stress and their relation with the feeder layer, the type of conditioned media used in feeder free systems and the upregulation of several differentiation markers. We observed the preservation of pluripotency, and detected differential expression of differentiation markers in HS181 and SHEF1 hESCs growing on Adipose-derived mesenchymal stem cells (ASCs) and feeder-free system with different conditioned media (HEF-CM and ASC-CM). Taken together, these results demonstrate evidence of ER stress events that cells must resolve to survive and maintenance of markers of pluripotency. The early differentiation status defined could progress into a more differentiated state, and may be influenced by culture conditions.