Culturing on Wharton's Jelly Extract Delays Mesenchymal Stem Cell Senescence through p53 and p16INK4a/pRb Pathways

Haojie Hao,Xiaobing Fu,Guanghui Chen,Dongdong Ti,Weidong Han,Yali Zhao,Jiejie Liu,Shenjun Xu,Pranela Rameshwar

doi:10.1371/journal.pone.0058314

Abstract

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

Highlights

Mesenchymal stem cells (MSCs) possess self-renewal capacity and have the ability to differentiate into mesoderm, endoderm, and even ectoderm lineages [1]
Of 11 bands, 8 were verified by comparison to their theoretical masses (Table 1). These results confirmed that the Wharton’s jelly extract (WJE) contained collagen a-1(I), fibronectin and other extracellular matrix (ECM) components, as well as cytokines such as IGF-1 and bFGF
We developed a new strategy for MSC culture expansion in vitro

Summary

Introduction

Mesenchymal stem cells (MSCs) possess self-renewal capacity and have the ability to differentiate into mesoderm, endoderm, and even ectoderm lineages [1] They cause only minimal immunoreactivity [2] and secrete bioactive factors with antiinflammatory and immunomodulatory effects [3]. MSCs can be derived from various human tissues such as bone marrow, umbilical cord, and placenta [4,5] They are extremely rare in primary tissues, and in vitro expansion by passaging is needed to obtain sufficient cells for clinical purposes. Studies show that human MSCs exhibit reduced differentiation potential in vitro [6], and replicative senescence of MSC preparations begins with the first passage [7] These problems have hindered the expansion of MSCs for therapeutic uses, causing a major bottleneck in clinical applications [8]

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Conclusion