Stem Cell Microvesicles Transfer Cystinosin to Human Cystinotic Cells and Reduce Cystine Accumulation In Vitro

Abstract

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation

“…Other groups have reported that microvesicles/exosomes shed by CTNS-expressing cells could lead to significant cystine decrease in cystinotic cells in vitro. 45,46 This mechanism also has been described in co-culture assays of mucopolysaccharidosis VII fibroblasts with umbilical MSCs and as the mechanism responsible for the rescue of the corneal defects in mice after direct corneal transplantation of the MSCs. 47 However, we previously demonstrated using coculture assays of wild-type MSCs or macrophages with Ctns-deficient fibroblasts that, while microvesicle transfer could lead to a substantial decrease of cystine in the fibroblasts (approximately 19% clearance), TNT-mediated lysosomal transfer was a more efficient route (approximately 75% clearance).…”

Wilms Tumor Suppressor, WT1, Suppresses Epigenetic Silencing of the β-Catenin Gene

“…We used a mesenchymal stem cell (MSC) cell line derived from human amniotic fluid to model the effects of WT1 on EZH2-mediated silencing of CTNNB1. amMSC express the transcription factor, OSR1, a marker of the intermediate mesoderm that gives rise to the kidneys (14,15). We saw that repression of EZH2 by WT1B or siRNA led to a dramatic increase in both the expression of CTNNB1 and the activity of a ␤-catenin pathway reporter (TOPFlash) in response to WNT9b.…”

“…Cells growing in monolayer attached to plastic culture vessels after 6 -8 passages in the presence of DMEM culture medium with 15% fetal bovine serum and 1% penicillin/streptomycin were further characterized (14) and used in this study.…”

“…primary mesenchymal stem cell line from human amniotic fluid with many characteristics of the intermediate mesoderm lineage prior to onset of WT1 expression (14). These cells (amMSC) offer a convenient model system because they have high levels of EZH2 expression (Fig.…”

“…The amMSC express typical mesenchymal stem cell surface markers (CD90, CD105, CD73, and CD44) and the transcription factor, OSR1, characteristic of lineages derived from the mesoderm (15). The cells lack surface markers for endothelial (CD31) and hematopoietic (CD34, CD45 and HLA-DR) cell lineages (14). To select a WT1 isoform for our studies, we first transfected amMSC with each of the four primary WT1 isoforms and assessed the level of EZH2 expression in each cell line.…”