Abstract
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.
Highlights
SB623 is a cell therapy product comprised of human bone marrow-derived mesenchymal stromal cells (MSCs) transiently transfected with a notch1 intracellular domain (NICD)-expressingplasmid
The use of 5% sodium dodecyl sulfate (SDS) buffer with a subsequent treatment in 8 M urea buffer for protein quantification and ensuing gel-based analysis was chosen because of its ability to solubilize the highest amount of Extracellular matrix (ECM)
When all four donor comparisons are taken into consideration, SB623 and MSC express similar amounts of ECM protein
Summary
SB623 is a cell therapy product comprised of human bone marrow-derived mesenchymal stromal cells (MSCs) transiently transfected with a notch intracellular domain (NICD)-expressingplasmid. SB623 has been shown to improve functional behavior deficits and reduce neural cell loss in stroked rats, governing biological mechanisms remain to be elucidated [1,2]. Previous reports have demonstrated that MSCs promote neuroprotection/regeneration without replacing damaged/dead neural cells, but instead offer indirect supporting mechanisms for survival and regeneration in the central nervous system [3,4]. The ability of MSC- and SB623-derived ECM to support the growth of rat primary cortical cells has previously been demonstrated [7]. SB623-derived ECM was shown to have a greater ability to promote neural cell growth compared to un-transfected MSCs, suggesting differences between these matrices. We are comparing quantities and composition of ECM produced by MSC and SB623
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