Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Cornelia Tolg,Tyler Kirwan,Karen Aitken,Jeffery Yu,Darius Jehan Bägli,Alya Ahsan,Shaalee Dworski,Shree Ram Singh

doi:10.1371/journal.pone.0059413

Abstract

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.

Highlights

Bladder outlet obstruction, the result of congenital or acquired abnormalities such as posterior urethral valves, spina bifida, prostate hypertrophy, or neurogenic bladder leads to increased bladder pressure that over time induces bladder wall thickening and loss of bladder function
Steinbach et al [21] demonstrated that rat skin derived progenitor cells (SKPs) differentiate into cells resembling vascular smooth muscle cells (SMC) when exposed to FBS
SMC Differentiation of SKPs is Regulated by mTOR We recently demonstrated that the mammalian target of rapamycin signalling cascade is activated in bladder SMC exposed to strain, denatured collagen and/or hypoxia, three canonical stimuli that play key roles in pathobiology of bladder obstruction [29]

Summary

Introduction

The result of congenital or acquired abnormalities such as posterior urethral valves, spina bifida, prostate hypertrophy, or neurogenic bladder leads to increased bladder pressure that over time induces bladder wall thickening and loss of bladder function. The specific physiology of gastrointestinal tissue, which is specialized for uptake of nutrients, results in complications such as acidosis and bacteriuria and possibly leads to an increased bladder cancer risk [1] [2]. Bladder smooth muscle cells (SMC) and urothelial cells have been isolated from bladder biopsies and expanded in culture. These cells were used to seed scaffolds, creating engineered bladder tissue for augmentation surgery [3]. This expansion strategy may be counterintuitive as native bladder muscle cells continue to exhibit fixed phenopathology [4,5]

Methods

Results

Conclusion