SummaryAimsThis open‐label prospective phase I/IIa clinical study used autologous bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) followed by mesenchymal stromal cells conditioned media (MSC‐CM) for the first time to treat multiple sclerosis (MS) patients. The primary goal was to assess the safety and feasibility and the secondary was efficacy. The correlation between the MSC‐CM content and treatment outcome was investigated.MethodsTen MS patients who failed conventional therapy were enrolled. Adverse events were recorded to assess safety. The Expanded Disability Status Scale (EDSS) was the primary efficacy measurement, the secondary included clinical (25WFT, 9‐PHT), cognitive (MMS), ophthalmology (OCT, VEP), and radiological (MRI lesion and volume) tests. The MSCs‐CM concentration of 27 inflammatory biomarkers was investigated.ResultsThe treatment protocol was well tolerated by patients. There was an overall trend of improvement in all the tests, except the lesion volume which increased significantly. A decrease of 4 and 3.5 points on the EDSS was achieved in two patients. We report a correlation between a decreased lesion number at baseline and higher IL‐6, IL‐8, and VEGF MSC‐CM content.ConclusionThe used protocol was safe and feasible with possible efficacy. The addition of MSC‐CM could be related to the magnitude of EDSS improvement observed.