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Mesenchymal stromal cells (MSCs) transiently transfected with  notch1 intracellular domain  ( NICD ) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from  NICD -transfected MSCs has been previously shown to support  in vitro  neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which  NICD -transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in  NICD -transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and  NICD -transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from  NICD -transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing  in vitro  cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy.

Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism