RESUMO
Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear.
Assuntos
Doenças do Desenvolvimento Ósseo/prevenção & controle , Transplante de Medula Óssea/métodos , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Fatores Etários , Animais , Animais Recém-Nascidos , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Modelos Animais de Doenças , Feminino , Glicosaminoglicanos/sangue , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/genéticaRESUMO
Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs). The increasing interest in newborn screening procedures for LSDs underlines the need for alternative cellular and gene therapy approaches to be developed during the perinatal period, supporting the treatment of MPS patients before the onset of clinical signs and symptoms. The rationale for considering these early therapies results from the clinical experience in the treatment of MPSs and other genetic disorders. The normal or gene-corrected hematopoiesis transplanted in patients can produce the missing protein at levels sufficient to improve and/or halt the disease-related abnormalities. However, these current therapies are only partially successful, probably due to the limited efficacy of the protein provided through the hematopoiesis. An alternative explanation is that the time at which the cellular or gene therapy procedures are performed could be too late to prevent pre-existing or progressive organ damage. Considering these aspects, in the last several years, novel cellular and gene therapy approaches have been tested in different animal models at birth, a highly early stage, showing that precocious treatment is critical to prevent long-term pathological consequences. This review provides insights into the state-of-art accomplishments made with neonatal cellular and gene-based therapies and the major barriers that need to be overcome before they can be implemented in the medical community.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Diagnóstico Precoce , Terapia Genética , Mucopolissacaridoses/terapia , Animais , Humanos , Recém-Nascido , Mucopolissacaridoses/genética , Triagem Neonatal/métodosRESUMO
BACKGROUND AIMS: Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated. METHODS: MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. RESULTS: We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. CONCLUSIONS: Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.
Assuntos
Diferenciação Celular/genética , Condrogênese/genética , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Linhagem da Célula/genética , Feminino , Feto , Humanos , Hibridização in Situ Fluorescente , Gravidez , Engenharia TecidualRESUMO
The use of bacterial cellulose (BC) in food systems is still limited due to production costs. Nine clones belonging to Komagataeibacter hansenii, Komagataeibacter nataicola, Komagataeibacter rhaeticus, Komagataeibacter swingsii, and Komagataeibacter xylinus species were screened for cellulose productivity in growth tests with five different carbon sources and three nitrogen sources. The water-holding and rehydration capacities of the purified cellulose were determined. The structure of the polymer was investigated through nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and X-ray diffraction (XRD) analysis, and observed by scanning electron microscope (SEM). Natural mutants of K. rhaeticus LMG 22126T and K. swingsii LMG 22125T showed different productivity. The factors "bacterial isolate" and "nitrogen source" significantly affected the production of cellulose (p < 0.01) rather than the factor "carbon source" (p = 0.15). However, on average, the best conditions for increasing yield were found in medium containing glucose and peptone. Water-holding capacity (WHC) values ranged from 10.7 to 42.3 (g water/g cellulose) with significant differences among strains (p < 0.01), while the rehydration capacity varied from 4.2 to 9.3 (g water/g cellulose). A high crystallinity (64-80%) was detected in all samples with Iα fractions corresponding to 67-93%. The ATR-FT-IR spectra and the XRD patterns confirmed the expected structure. BC made by GVP isolate of K. rhaeticus LMG 22126T, which was the strain with the highest yield, was added to a gluten-free bread formulation. Results obtained from measurements of technological parameters in dough leavening and baking trials were promising for implementation in potential novel foods.
RESUMO
Umbilical cord blood (UCB) is a promising source of stem cells to use in early haematopoietic stem cell transplantation (HSCT) approaches for several genetic diseases that can be diagnosed at birth. Mucopolysaccharidosis type I (MPS-I) is a progressive multi-system disorder caused by deficiency of lysosomal enzyme α-L-iduronidase, and patients treated with allogeneic HSCT at the onset have improved outcome, suggesting to administer such therapy as early as possible. Given that the best characterized MPS-I murine model is an immunocompetent mouse, we here developed a transplantation system based on murine UCB. With the final aim of testing the therapeutic efficacy of UCB in MPS-I mice transplanted at birth, we first defined the features of murine UCB cells and demonstrated that they are capable of multi-lineage haematopoietic repopulation of myeloablated adult mice similarly to bone marrow cells. We then assessed the effectiveness of murine UCB cells transplantation in busulfan-conditioned newborn MPS-I mice. Twenty weeks after treatment, iduronidase activity was increased in visceral organs of MPS-I animals, glycosaminoglycans storage was reduced, and skeletal phenotype was ameliorated. This study explores a potential therapy for MPS-I at a very early stage in life and represents a novel model to test UCB-based transplantation approaches for various diseases.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Animais , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Modelos Animais de Doenças , Disostoses/diagnóstico por imagem , Disostoses/etiologia , Disostoses/patologia , Disostoses/terapia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Mucopolissacaridose I/terapia , Gravidez , Resultado do Tratamento , Microtomografia por Raio-XRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance-associated gene (UVRAG), reducing the activity of the associated Beclin 1-Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1-Vps34-UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.