Inhibition of Transforming Growth Factor-β Receptor signaling promotes culture expansion of undifferentiated human Endometrial Mesenchymal Stem/stromal Cells.

Shanti Gurung,Caroline E Gargett,Jerome A Werkmeister

doi:10.1038/srep15042

Abstract

Human endometrial MSC (eMSC) are a novel source of MSC easily harvested from the highly regenerative uterine lining. We have developed protocols for eMSC isolation from single cell suspensions using magnetic bead-sorting using a perivascular marker antibody to SUSD2 and culture expansion in serum free medium (SFM). Similar to other MSC, eMSC spontaneously differentiate into fibroblasts during culture expansion decreasing their purity and efficacy. The aim of this study was to determine if A83-01, a TGF-β receptor inhibitor prevents eMSC differentiation in culture. SUSD2+ eMSC were cultured in SFM with bFGF/EGF in 5% O2/5% CO2. At passage 6, eMSC were incubated with or without A83-01 for 7 days, then analysed for MSC properties. A83-01 dose dependently promoted SUSD2+ eMSC proliferation and blocked apoptosis via the SMAD 2/3 pathway. Fewer A83-01 treated cells were autofluorescent or stained with β-galactosidase, indicating reduced senescence. A83-01-treated cells had higher cloning efficiency, differentiated into mesodermal lineages and expressed MSC phenotypic markers. These data suggest that A83-01 maintains SUSD2+ eMSC stemness, promoting proliferation by blocking senescence and apoptosis in late passage cultures through binding to TGF-β receptors. Small molecules such as A83-01 may enable the expansion of undifferentiated MSC for use in tissue engineering and cell-based therapies.

Highlights

Trials for a variety of diseases, including graft versus host disease[12], cardio-vascular disease as a cell-based therapy[13] or in tissue-engineered constructs for bone
We showed that A83-01 promotes proliferation of late passage SUSD2+ cells in serum free medium (SFM), an effect mediated by SMAD2/3 signaling
Means for triplicates were obtained for each sample at each concentration, normalised to vehicle control DMSO (100%) and plotted as mean ± S EM of n = 6 patient samples. (B) Passage 6 endometrial mesenchymal stem/stromal cells (eMSC) lysates with or without 1μ M A83-01 were immunoblotted with anti-SMAD 2/3 or anti-pSMAD 2/3 antibodies

Summary

Introduction

Trials for a variety of diseases, including graft versus host disease[12], cardio-vascular disease as a cell-based therapy[13] or in tissue-engineered constructs for bone (www.clinicaltrials.gov). The use of eMSC for cell-based therapy requires their expansion in culture conditions that supports homogenous growth and maintains self-renewal and multipotency. A83-01 prevented senescence and apoptosis of cultured eMSC, suggesting that TGF-β has a role in regulating SUSD2 expression and eMSC growth, apoptosis and senescence and may have a role in spontaneous MSC differentiation during culture expansion. Small molecules such as A83-01 may provide an approach for the expansion of undifferentiated MSC for use in tissue engineering and cell-based therapy

Objectives

Methods

Results

Discussion

Conclusion