iTRAQ-Based Quantitative Proteomic Comparison of Early- and Late-Passage Human Dermal Papilla Cell Secretome in Relation to Inducing Hair Follicle Regeneration

Abstract

Alopecia is an exceedingly prevalent problem that lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential, we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs, and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM did not. In order to identify the key factors responsible for hair induction, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1. Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results indicate DPC-secreted proteins play important roles in HF regeneration, with SDF1, MMP3, biglycan, and LTBP1 being potential key inductive factors secreted by dermal papilla cells in the regeneration of hair follicles.

Real-time iTRAQ-based proteome profiling revealed the central metabolism involved in nitrogen starvation induced lipid accumulation in microalgae

“…4). The reason for this discrepancy might be the low abundance of peptides, smaller sample size, and complicated experimental procedures and data analysis lacking suitable internal standards3940. Despite several disadvantages, iTRAQ is the most amenable technique to orthogonal separation due to multiplexing that ensures its value for many analysis schemes and the reduction of costly LC–MS runtime414243.…”

A computational modelling framework to quantify the effects of passaging cell lines

“…Although problems associated with high passage numbers are widely acknowledged, the mechanism of passage-induced changes is not well understood [4,10,13,18,26,29,32,34,45]. For example, standard experimental protocols suggest avoiding cells at high passage numbers, whereas the definition of a 'high passage number' is rather vague [4,29].…”

Investigating the Reproducibility of In Vitro Cell Biology Assays Using Mathematical Models

“…Problems associated with high passage numbers, such as the change of cell proliferation, are widely acknowledged. However, the mechanisms are not well understood [31,36,45,73,90,95,99,129]. Therefore, the aim of this work is to use a computational approach to provide insight into the putative mechanisms that could possibly lead to the problems.…”

“…Although there are many properties of cells that are affected by the passaging process [21,31,36,45,88,95,99,129], we choose to focus on how passaging affects the cell proliferation rate. In our model, when a cell proliferates, the daughter cells directly inherit the same proliferation rate as the mother cell.…”

“…Although problems associated with high passage numbers are widely acknowledged, the mechanism of passage-induced changes is not well understood [9,31,36,45,73,90,95,99,129]. For example, standard experimental protocols suggest avoiding cells at high passage numbers, whereas the definition of a 'high passage number' is rather vague [9,90].…”