RESUMO
BACKGROUND: Regenerative medicine is strictly dependent on stem cells as a source for a high diversity of somatic cells. However, the isolation of such from individuals suffering from severe genetic skin blistering diseases like epidermolysis bullosa (EB) is often associated with further organ damage. METHODS: Stem cells were isolated from 112 urine samples from 21 different healthy donors, as well as from 33 urine samples from 25 donors with EB. The cultivation of these cells was optimized by testing different media formulations and pre-coating of culture vessels with collagen. The identity of cells was confirmed by testing marker expression, differentiation potential and immune-modulatory properties. RESULTS: We provide here an optimized protocol for the reproducible isolation of mesenchymal stem cells from urine, even from small volumes as obtained from patients with EB. Furthermore, we offer a basic characterization of those urine-derived stem cells (USCs) from healthy donors, as well as from patients with EB, and demonstrate their potential to differentiate into chondrocytes, osteoblasts and adipocytes, as well as their immune-modulatory properties. CONCLUSIONS: Thus, USCs provide a novel and non-invasive source of stem cells, which might be applied for gene-therapeutic approaches to improve medical conditions of patients with EB.
Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Epidermólise Bolhosa/urina , Células-Tronco Mesenquimais/citologia , Adipogenia/genética , Agrecanas/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Condrogênese/genética , Colágeno Tipo X/genética , Feminino , Citometria de Fluxo , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Imunofenotipagem/métodos , Ativação Linfocitária/imunologia , Masculino , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Osteocalcina/genética , Osteogênese/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
4 patients suffering from epidermolysis bullosa, 11 persons with genetically determined ichthyosis, as well as 3 cases of x-linked recessive ectodermal dysplasia were investigated with regard to glycosaminoglycane(GAG)uria; the GAG fractions were analysed by means of GAG thin-layer chromatography. All three groups of patients showed increased GAG-uria. The GAG fractions proved to be chondroitin-6-sulphate, chondroitin-4-sulphate, and heparan-sulphate. The pathomechanism of the increased GAG-uria is supposed to be an increased GAG degradation process.
Assuntos
Displasia Ectodérmica/urina , Epidermólise Bolhosa/urina , Glicosaminoglicanos/urina , Ictiose/urina , Adulto , Criança , Displasia Ectodérmica/genética , Epidermólise Bolhosa/genética , Feminino , Humanos , Ictiose/genética , MasculinoRESUMO
Urine samples of five patients with epidermolysis bullosa hereditaria were examined for the presence of acid glycosaminoglycans (GAG) and compared to 10 healthy subjects. A significant increase (p less than 0.0005) could be detected in patients with epidermolysis (t = 6.66, means: 49.7 mg/day, standard deviation: +/- 18.3). Additionally, acid glycosaminoglycan concentration in sweat of patients and healthy controls was determined but statistical calculations showed no significant difference (p less than 0.15). On grounds of the increased mucopolysaccharide excretion and the previous studies of collagen-glycosaminoglycan interactions, we strongly suggest that acid GAG are involved in the pathogenesis of that disease with the molecular defect of disturbed fibril formation due to the altered skin collagen-glycosaminoglycan equilibrium.
Assuntos
Epidermólise Bolhosa/urina , Glicosaminoglicanos/urina , Adolescente , Adulto , Criança , Pré-Escolar , Epidermólise Bolhosa/genética , Feminino , Humanos , Lactente , Masculino , Suor/análiseRESUMO
A biochemical study was carried out on the urine of an 18-year-old woman with epidermolysis bullosa dystrophica recessive type E.b.d.r.). The urinary acid glycosaminoglycans were separated and subjected to quantitative and qualitative analyses by gel filtration and column chromatography. A hyaluronic acid and the 0.5 M NaCl elution fraction were increased characteristically in the acid glycosaminoglycan.