Isolation and Characterization of hMSC

Bone marrow (BM) human mesenchymal stromal cells (hMSC) were isolated from 5 healthy donors (three males/two females) with a median age of 48 years (range 21–49 years). In all cases, written informed consent was previously obtained according to institutional guidelines and approved by the Comite Etico de Investigacion Clinica del Area de Salud de Salamanca (located in Salamanca, Spain) with reference number 70/07/2015. Mononuclear cells (MNC) were isolated from fresh BM aspirates and separated by density gradient centrifugation (Ficoll-Paque, GE Healthcare Bio-Sciences, AB, Uppsala, Sweden) and cultured in standard culture medium, as previously described [7 (link)]. Cells were cultured at 37 °C in a humidified atmosphere with 5 % of carbon dioxide. At passage 3, hMSC characterization was performed according to the recommendations of the International Society for Cellular Therapy (ISCT) [28 (link)]. Isolation of hMSC-EV were performed until passage number 6.