Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix

Abstract

Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration.

Excitation–contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells

“…We obtained comparable results with human and mouse MSCs that were selected under different constraints. However, that does not exclude that factors secreted by these cells are part of both secretomes, where analysis of conditioned media by mass spectroscopy shows significant overlap [53–55]. Stem cell specificity of the effects is suggested, given that the effect is not simply reproduced by other mesenchyme derived cell types.…”

Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI

“…Proteins were solved in Laemmli sample buffer, separated by gradient SDS-PAGE (8.5%-18%) and stained with Coomassie brilliant blue. The gel lane was cut into 20-25 pieces and in-gel digestion was performed; proteins were destained with 50 mM NH 4 HCO 3 in 50% (v/v) acetonitrile (ACN) [50]. Then the gel pieces were dried with 100% ACN, vacuum evaporated and rehydrated with 20 μL of 5 ng/μL modified trypsin (Promega) in 20 mM NH 4 HCO 3 , 10% ACN.…”

Vimentin Mediates Uptake of C3 Exoenzyme

“…Peptides of 1D gels, 2D gel spots and the HT22 cell lysate probed membranes were analyzed using MALDI-MS/MS as described previously [24]. Internal calibration on autolytic porcine trypsin peptides was applied for precursor MS spectra and external calibration with Glu-Fib fragments was used for MS/MS spectra.…”