Proteomic Characteristics of ex vivo-Enriched Adult Human Bone Marrow Mononuclear Cells in Continuous Perfusion Cultures

Abstract

A major challenge in developing cell therapies is reliable characterization of the cell product at the molecular level. Fresh autologous and passaged human bone marrow enriched for stem and mesenchymal stromal stem cells have been used to regenerate bone. We report the proteome of an innovative autologous human bone marrow-derived mixed cell product (BMMCP), cultured ex vivo for 12 days, in automated continuous media perfusion system to avoid passaging, and discuss reproducibility of protein composition. Each BMMCP is compared to its originating human adult bone marrow mononuclear cells (BMMNC). With the use of 2-D LC-MS/MS approach, 638 (BMMNC) and 867 (BMMCP) distinct proteins were identified including cell adhesion molecules, extracellular matrix and growth factors. Overlap of protein identifications revealed that 67% of the BMMNC proteome was retained in the BMMCP, and protein expression of selected cell lineages was enhanced. Isotope-coded affinity tags (ICAT) and MS/MS were used to identify and quantify relative changes in the proteome of BMMNC and their related BMMCP, obtained from 3 separate donors. In 3 separate ICAT experiments, 57% of proteome identified was shared between donors. Measurable and definable proteomic characterization of BMMCP will facilitate their use in clinical trials and provide insight into cell functionality needed to support multiple therapeutic indications.