Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

Antonella Conforti,Simone Biagini,Anna Taranta,Angela Pitisci,Valentina Cirillo,Francesco Bellomo,Franco Locatelli,Nadia Starc,Francesco Emma,Maria Ester Bernardo

doi:10.1186/s12967-015-0494-0

Abstract

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Highlights

Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter
Cys-mesenchymal stromal cells (MSCs) were successfully isolated from the bone marrow (BM) of a patient with nephropathic cystinosis and were propagated in vitro; their biological and functional properties were compared with those of MSCs isolated from BM obtained from three healthy donors (HDs)
Cys-MSCs showed the same immunophenotype as HD-MSCs; in particular, more than 95% of Cys-MSCs were positive for CD13, Figure 1 Biological characterization of HD- and Cys-MSCs

Summary

Introduction

Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. Cystinosis is an autosomal recessive lysosomal disorder with a reported incidence of 0.5-1.0 per 100,000 live births [1,2] It is caused by mutations in the CTNS gene (17p13.2), which encodes a lysosomal cysteine-proton symporter, termed cystinosin [1,2,3]. 95% of patients have severe bi-allelic mutations/deletions of CTNS, causing the most severe form of the disease, termed “nephropathic cystinosis” (OMIM 219800) These subjects develop renal Fanconi syndrome during infancy and progress to end-stage renal failure during childhood, if not treated with cysteamine. Cystinosis is currently treated with cysteamine, a sulfhydryl compound that reduces accumulation of cystine in lysosomes [1] This drug delays dialysis and kidney transplantation by approximately 5–10 years, but has no significant effect on renal Fanconi syndrome and cannot prevent progression to end-stage renal failure [1,2]. It delays most of other disease-related long-term complications, but it is not yet clear if it completely prevents them [4,5]

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