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1.
Sci Rep ; 11(1): 21722, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34741076

RESUMO

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Epotilonas/administração & dosagem , Neurônios Motores/citologia , Moduladores de Tubulina/administração & dosagem , Células-Tronco Adultas/ultraestrutura , Técnicas de Cultura de Células em Três Dimensões , Colágeno/química , Colágeno/farmacologia , Endométrio/citologia , Feminino , Proteínas Hedgehog/administração & dosagem , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Microesferas , Poliésteres , Cultura Primária de Células , Tretinoína/administração & dosagem
2.
Cell Death Dis ; 11(12): 1075, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323934

RESUMO

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6-/-) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6-/--BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6-/--BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6-/--BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Senescência Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , NADH Desidrogenase/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
3.
Sci Rep ; 9(1): 16381, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31705037

RESUMO

Stem cells undergo drastic morphological alterations during differentiation. While extensive studies have been performed to examine the cytoskeletal remodeling, there is a growing interest to determine the morphological, structural and functional changes of the nucleus. The current study is therefore aimed at quantifying the extent of remodeling of the nuclear morphology of human mesenchymal stem cells during biochemically-induced adipogenic differentiation. Results show the size of nuclei decreased exponentially over time as the lipid accumulation is up-regulated. Increases in the lipid accumulation appear to lag the nuclear reorganization, suggesting the nuclear deformation is a prerequisite to adipocyte maturation. Furthermore, the lamin A/C expression was increased and redistributed to the nuclear periphery along with a subsequent increase in the nuclear aspect ratio. To further assess the role of the nucleus, a nuclear morphology with a high aspect ratio was achieved using microcontact-printed substrate. The cells with an elongated nuclear shape did not efficiently undergo adipogenesis, suggesting the cellular and nuclear processes associated with stem cell differentiation at the early stage of adipogenesis cause a change in the nuclear morphology and cannot be abrogated by the morphological cues. In addition, a novel computational biomechanical model was generated to simulate the nuclear shape change during differentiation and predict the forces acting upon the nucleus. This effort led to the development of computational scaling approach to simulate the experimentally observed adipogenic differentiation processes over 15 days in less than 1.5 hours.


Assuntos
Adipócitos/citologia , Núcleo Celular/ultraestrutura , Células-Tronco Mesenquimais/citologia , Adipócitos/metabolismo , Adipócitos/ultraestrutura , Adipogenia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Simulação por Computador , Humanos , Lamina Tipo A/metabolismo , Metabolismo dos Lipídeos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia de Fluorescência , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura
4.
Nat Commun ; 10(1): 2943, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270333

RESUMO

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Células-Tronco Adultas/ultraestrutura , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Autofagia , Transporte Biológico , Diferenciação Celular , Autorrenovação Celular , Metabolismo Energético , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Ribonucleotídeos/farmacologia , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
5.
Front Immunol ; 9: 645, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29740426

RESUMO

Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC®) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients.


Assuntos
Células-Tronco Adultas/imunologia , Rejeição de Enxerto/prevenção & controle , Imunoterapia/métodos , Células-Tronco Pluripotentes/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Transplante , Células-Tronco Adultas/ultraestrutura , Animais , Proliferação de Células , Células Cultivadas , Dinoprostona/metabolismo , Modelos Animais de Doenças , Homeostase , Humanos , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/transplante
6.
Endocr J ; 65(1): 63-73, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29057768

RESUMO

Ghrelin functions as a neuroprotective agent and saves neurons from various insults include ischemic injury. However, it remains to be elucidated whether ghrelin protects neuronal cells against ischemic injury-induced excessive autophagy. Autophagy is required for the maintenance of neural stem cell homeostasis. However, regarding autophagic cell death, it is commonly assumed that excessive autophagy leads to self-elimination of mammalian cells. The purpose of this study was to investigate the potential neuroprotection effects of ghrelin from excessive autophagy in adult rat hippocampal neural stem cells (NSCs). Oxygen-Glucose Deprivation (OGD) strongly induces autophagy in adult rat hippocampal NSCs. Ghrelin treatment inhibited OGD-induced cell death of adult rat hippocampal NSCs assessed by cell-counting-kit-8 assay. Ghrelin also suppressed OGD-induced excessive autophagy activity. The protective effect of ghrelin was accompanied by an increased expression levels of Bcl-2, p-62 and decreased expression level of LC3-II, Beclin-1 by Western blot. Furthermore, ghrelin reduced autophagosome formation and number of GFP-LC3 transfected puncta. In conclusion, our data suggest that ghrelin protects adult rat hippocampal NSCs from excessive autophagy in experimental stroke (oxygen-glucose deprivation) model. Regulating autophagic activity may be a potential optimizing target for promoting adult rat hippocampal NSCs based therapy for stroke.


Assuntos
Autofagia , Grelina/metabolismo , Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neuroproteção , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteína Sequestossoma-1/agonistas , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Células-Tronco Adultas/ultraestrutura , Animais , Apoptose , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/metabolismo , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/patologia , Hipocampo/ultraestrutura , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Sequestossoma-1/metabolismo
7.
J Microbiol ; 55(1): 75-79, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28035603

RESUMO

Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells' integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.


Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/virologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/virologia , Reoviridae/fisiologia , Adipogenia , Tecido Adiposo/fisiologia , Tecido Adiposo/virologia , Células-Tronco Adultas/fisiologia , Células-Tronco Adultas/ultraestrutura , Proliferação de Células , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Neoplasias/terapia , Osteogênese , Tropismo Viral/fisiologia
8.
Am J Physiol Endocrinol Metab ; 311(2): E367-79, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27302752

RESUMO

Extensive evidence has revealed variations in the number of hormone-producing cells in the pituitary gland, which occur under physiological conditions such as gestation and lactancy. It has been proposed that new hormone-producing cells differentiate from stem cells. However, exactly how and when this takes place is not clear. In this work, we used immunoelectron microscopy to identify adult pituitary stem/progenitor cells (SC/P) localized in the marginal zone (MZ), and additionally, we detected GFRa2-, Sox2-, and Sox9-positive cells in the adenoparenchyma (AP) by fluorescence microscopy. Then, we evaluated fluctuations of SC/P mRNA and protein level markers in MZ and AP during gestation and lactancy. An upregulation in stemness markers was shown at term of gestation (AT) in MZ, whereas there were more progenitor cell markers in the middle of gestation and active lactancy. Concerning committed cell markers, we detected a rise in AP at beginning of lactancy (d1L). We performed a BrdU uptake analysis in MZ and AP cells. The highest level of BrdU uptake was observed in MZ AT cells, whereas in AP this was detected in d1L, followed by a decrease in both the MZ and AP. Finally, we detected double immunostaining for BrdU-GFRa2 in MZ AT cells and BrdU-Sox9 in the AP d1L cells. Taken together, we hypothesize that the expansion of the SC/P niche took place mainly in MZ from pituitary rats in AT and d1L. These results suggest that the SC niche actively participates in pituitary plasticity during these reproductive states, contributing to the origin of hormone cell populations.


Assuntos
Células-Tronco Adultas/citologia , Lactação/metabolismo , Hipófise/citologia , Prenhez/metabolismo , RNA Mensageiro/metabolismo , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Plasticidade Celular , Feminino , Perfilação da Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Microdissecção e Captura a Laser , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Hipófise/metabolismo , Hipófise/ultraestrutura , Gravidez , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição Pit-1/genética
9.
Cell Stem Cell ; 15(5): 605-18, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25312494

RESUMO

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.


Assuntos
Células-Tronco Adultas/citologia , Hepatócitos/patologia , Fígado/patologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Separação Celular , Microambiente Celular , Células Clonais , Regulação da Expressão Gênica , Ducto Hepático Comum/citologia , Hepatócitos/ultraestrutura , Humanos , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Transcrição SOX9/metabolismo
10.
Invest Ophthalmol Vis Sci ; 55(9): 5967-78, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25139734

RESUMO

PURPOSE: To evaluate whether mouse adipose tissue mesothelial cells (ATMCs) share morphologic and biochemical characteristics with mouse corneal endothelial cells (CECs) and to evaluate their capacity to adhere to the decellularized basal membrane of human anterior lens capsules (HALCs) as a potential tissue-engineered surrogate for corneal endothelium replacement. METHODS: Adipose tissue mesothelial cells were isolated from the visceral adipose tissue of adult mice, and their expression of several corneal endothelium markers was determined with quantitative RT-PCR, immunofluorescence, and Western blotting. Adipose tissue mesothelial cells were cultured in a mesothelial retaining phenotype medium (MRPM) and further seeded and cultured on top of the decellularized basal membrane of HALCs. ATMC-HALC composites were evaluated by optical microscopy, immunofluorescence, and transmission electron microscopy. RESULTS: Mesothelial retaining phenotype medium-cultured ATMCs express the corneal endothelium markers COL4A2, COL8A2, SLC4A4, CAR2, sodium- and potassium-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase), ß-catenin, zona occludens-1, and N-cadherin in a pattern similar to that in mouse CECs. Furthermore, ATMCs displayed strong adhesion capacity onto the basal membrane of HALCs and formed a confluent monolayer within 72 hours of culture in MRPM. Ultrastructural morphologic and marker characteristics displayed by ATMC monolayer on HALCs clearly indicated that ATMCs retained their original phenotype of squamous epithelial-like cells. CONCLUSIONS: Corneal endothelial cells and ATMCs share morphologic (structural) and marker (functional) similarities [corrected]. The ATMCs adhered and formed structures mimicking focal adhesion complexes with the HALC basal membrane. Monolayer structure and achieved density of ATMCs support the proposal to use adult human mesothelial cells (MCs) as a possible surrogate for damaged corneal endothelium.


Assuntos
Doenças da Córnea/terapia , Endotélio Corneano/citologia , Células Epiteliais/citologia , Gordura Intra-Abdominal/citologia , Cápsula do Cristalino/citologia , Engenharia Tecidual/métodos , Células-Tronco Adultas/citologia , Células-Tronco Adultas/ultraestrutura , Animais , Biomarcadores/metabolismo , Doenças da Córnea/patologia , Células Epiteliais/ultraestrutura , Epitélio , Feminino , Humanos , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Cultura Primária de Células
11.
Clin Exp Pharmacol Physiol ; 40(11): 724-34, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24033507

RESUMO

We found a group of non-platelet RNA-containing particles (NPRCPs) in human umbilical cord blood. These particles can aggregate, fuse and become non-nucleated cells when cocultured with nucleated cells in vitro. The non-nucleated cells further differentiate into nucleated cells expressing octamer binding transcription factor 4 (OCT4). The NPRCPs are approximately 1-5 µm in diameter, have a thin bilayer membrane, contain short RNAs and microRNAs and express OCT4, sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). To confirm the function of NPRCPs in vivo, we examined the effects of tail vein-injected green fluorescent protein (GFP)-labelled NPRCPs on mouse kidneys damaged by prior ischaemia and reperfusion from Day 1 to Week 6. Within 1 day of injection of NPRCPs, immunofluorescence and immunohistochemistry revealed a large number of extravasated NPRCPs in the renal calyces, damaged glomeruli and duct tubules. During the course of regeneration, NPRCPs fused into large, non-nucleated cellular structures that further became large nucleated cells to regenerate multicellular kidney tubules. In addition, many NPRCPs became tiny nucleated cellular structures that further differentiated into interstitial cells in connective tissue. The extravasated NPRCPs also arranged themselves into non-cell glomerular structures before further regenerating into nucleated cells of the glomerulus. In conclusion, the results demonstrate that, via different patterns of differentiation, NPRCP-derived cells can regenerate mouse kidney tissue damaged by ischaemia.


Assuntos
Células-Tronco Adultas/citologia , Transdiferenciação Celular , Micropartículas Derivadas de Células/transplante , Rim/fisiologia , RNA/metabolismo , Regeneração , Traumatismo por Reperfusão/terapia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isquemia/fisiopatologia , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , MicroRNAs/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Recombinantes/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Transcrição SOXB1/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 33(7): 4009-20, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23910308

RESUMO

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 wt.% or 3.0 wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73(+), CD90(+), CD105(+), CD34(-) and CD45(-) onto the 3 substrates for 30 days. Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5 wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.


Assuntos
Células-Tronco Adultas/citologia , Compostos de Cálcio/farmacologia , Fosfatos de Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cerâmica/farmacologia , Células-Tronco Mesenquimais/citologia , Silicatos/farmacologia , Adulto , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/enzimologia , Células-Tronco Adultas/ultraestrutura , Fosfatase Alcalina/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/análise , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fenômenos Químicos/efeitos dos fármacos , Meios de Cultura/química , Humanos , Teste de Materiais , Fenômenos Mecânicos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/ultraestrutura , Osteocalcina/metabolismo , Fósforo/análise , Silício/análise
13.
Mater Sci Eng C Mater Biol Appl ; 33(4): 1935-44, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23498215

RESUMO

The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.


Assuntos
Bactérias/química , Diferenciação Celular/efeitos dos fármacos , Celulose/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais/química , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/enzimologia , Células-Tronco Adultas/ultraestrutura , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Celulose/ultraestrutura , Condrogênese/efeitos dos fármacos , Liofilização , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/ultraestrutura , Osteogênese/efeitos dos fármacos , Coloração e Rotulagem
14.
J Nucl Med ; 54(3): 447-54, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23353687

RESUMO

UNLABELLED: Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). METHODS: Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. RESULTS: The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. CONCLUSION: MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.


Assuntos
Células-Tronco Adultas/diagnóstico por imagem , Fluordesoxiglucose F18 , Células-Tronco Mesenquimais/diagnóstico por imagem , Células-Tronco Multipotentes/diagnóstico por imagem , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Diferenciação Celular , Células Cultivadas , Radioisótopos de Flúor , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/ultraestrutura , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Ratos , Medicina Regenerativa , Engenharia Tecidual
15.
J Cell Physiol ; 228(6): 1229-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23129455

RESUMO

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase.


Assuntos
Células-Tronco Adultas/metabolismo , Materiais Biocompatíveis , Regeneração Óssea , Substitutos Ósseos , Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/metabolismo , Alicerces Teciduais , Células-Tronco Adultas/transplante , Células-Tronco Adultas/ultraestrutura , Biomarcadores/metabolismo , Transplante Ósseo/métodos , Técnicas de Cultura de Células , Forma Celular , Sobrevivência Celular , Células Cultivadas , Citoesqueleto/metabolismo , Durapatita/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Magnésio/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanopartículas , Fosforilação , Pós , Fatores de Tempo , Tirosina
16.
Acta Cir Bras ; 27(3): 201-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22460249

RESUMO

PURPOSE: To evaluate the effects of mesenchymal stem cells (MSC) from eight mice C57BL/6 gfp(+) bone marrows expanded in cultures associated with platelets rich plasma (PRP) deriving from another eight mice, in the repair of critical defects in calvarial bone produced in twenty-four adult isogenic mice C57BL/6. METHODS: The animals were submitted to a cranial defect of 6.0mm in diameter and divided into two equal experimental groups. Control group did not receive treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL associated with 50.0 µL of plasma gel containing 1.0 x 10(9) autologous platelets within the defect. RESULTS: In the treated group was observed process of angiogenesis and bone repair better than control group. CONCLUSION: Mesenchymal stem cells derived from bone marrow of C57BL/6 gfp(+) mice associated with PRP gel applied in bone critical defects produced in calvarial contributes positively to the process of bone repair.


Assuntos
Células-Tronco Adultas/transplante , Células da Medula Óssea/fisiologia , Regeneração Óssea/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Plasma Rico em Plaquetas/fisiologia , Crânio/cirurgia , Células-Tronco Adultas/ultraestrutura , Animais , Células da Medula Óssea/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Masculino , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese/fisiologia , Distribuição Aleatória , Crânio/lesões , Crânio/ultraestrutura , Engenharia Tecidual/métodos , Transplante Homólogo
17.
Arch Med Res ; 43(1): 83-8, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22374243

RESUMO

BACKGROUND AND AIMS: Tissue engineering strategy has been considered as an alternative treatment for diabetes mellitus due to lack of permanent pharmaceutical treatment and islet donors for transplantation. Various cell lines have been used to generate functional insulin-producing cells (IPCs) including progenitor pancreatic cell lines, embryonic stem cells (ESCs), umbilical cord blood stem cells (UCB-SCs), adult bone marrow stem cells (BMSCs), and adipose tissue-derived stem cells (ADSCs). METHODS: Human ADSCs from lipoaspirated abdominal fat tissue was differentiated into IPCs following a two-step induction protocol based on a combination of alternating high and low glucose, nicotinamide, activin A and glucagon-like peptide 1 (GLP-1) for a duration of 3 weeks. During differentiation, histomorphological changes of the stem cells towards pancreatic ß-islet characteristics were observed via light microscope and transmission electron microscope (TEM). Dithizone (DTZ) staining, which is selective towards IPCs, was used to stain the new islet-like cells. Production of insulin hormone by the cells was analyzed via enzyme-linked immunosorbent assay (ELISA), whereas its hormonal regulation was tested via a glucose challenge test. RESULTS: Histomorphological changes of the differentiated cells were noted to resemble pancreatic ß-cells, whereas DTZ staining positively stained the cells. The differentiated cells significantly produced human insulin as compared to the undifferentiated ADSCs, and its production was increased with an increase of glucose concentration in the culture medium. CONCLUSIONS: These initial data indicate that human lipoaspirated ADSCs have the potential to differentiate into functional IPCs, and could be used as a therapy to treat diabetes mellitus in the future.


Assuntos
Gordura Abdominal/citologia , Células-Tronco Adultas/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Insulina/metabolismo , Células-Tronco Adultas/ultraestrutura , Proteínas Aviárias/farmacologia , Proteínas Aviárias/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Meios de Cultura , Citocinas/farmacologia , Citocinas/fisiologia , Glucose/farmacologia , Glucose/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Vesículas Secretórias/ultraestrutura , Engenharia Tecidual
18.
Acta cir. bras ; 27(3): 201-209, Mar. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-617958

RESUMO

PURPOSE: To evaluate the effects of mesenchymal stem cells (MSC) from eight mice C57BL/6 gfp+ bone marrows expanded in cultures associated with platelets rich plasma (PRP) deriving from another eight mice, in the repair of critical defects in calvarial bone produced in twenty-four adult isogenic mice C57BL/6. METHODS: The animals were submitted to a cranial defect of 6.0mm in diameter and divided into two equal experimental groups. Control group did not receive treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL associated with 50.0µL of plasma gel containing 1.0 x 10(9) autologous platelets within the defect. RESULTS: In the treated group was observed process of angiogenesis and bone repair better than control group. CONCLUSION: Mesenchymal stem cells derived from bone marrow of C57BL/6 gfp+ mice associated with PRP gel applied in bone critical defects produced in calvarial contributes positively to the process of bone repair.


OBJETIVO: Avaliar os efeitos da associação das células-tronco mesenquimais (MSC) oriundas da medula óssea de oito camundongos jovens C57BL/6 gfp+ e expandidas em culturas, com Plasma Rico em Plaquetas (PRP) provenientes de outros oito camundongos, na reparação de defeitos críticos confeccionados em calvária de 24 camundongos adultos C57BL/6. MÉTODOS: Os animais foram submetidos a um defeito craniano de 6,0mm de diâmetro e separados em dois grupos experimentais iguais. O grupo controle não recebeu tratamento e no grupo tratado foi administrado, no interior do defeito, pellet de MSC contendo 1,0 x 10(7) células/mL associado com 50,0µL de plasma em gel autólogo contendo 1,0 x 10(9) plaquetas. RESULTADOS: No grupo tratado verificou-se processo de angiogênese e reparação óssea superior ao grupo controle. CONCLUSÃO: A associação das células-tronco mesenquimais (MSC) derivadas da medula óssea de camundongos C57BL/6 gfp+ com gel de PRP aplicadas em defeitos ósseos críticos confeccionadas em calvária de camundongos C57BL/6 jovens, contribuiu positivamente para o processo de reparação óssea.


Assuntos
Animais , Masculino , Camundongos , Células-Tronco Adultas/transplante , Células da Medula Óssea/fisiologia , Regeneração Óssea/fisiologia , Células-Tronco Mesenquimais , Transplante de Células-Tronco Mesenquimais/métodos , Plasma Rico em Plaquetas/fisiologia , Crânio/cirurgia , Células-Tronco Adultas/ultraestrutura , Células da Medula Óssea/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Células-Tronco Mesenquimais , Camundongos Transgênicos , Osteogênese/fisiologia , Distribuição Aleatória , Crânio/lesões , Crânio/ultraestrutura , Transplante Homólogo , Engenharia Tecidual/métodos
19.
J Comp Neurol ; 520(10): 2275-316, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22318736

RESUMO

A central goal of adult neurogenesis research is to characterize the cellular constituents of a neurogenic niche and to understand how these cells regulate the production of new neurons. Because the generation of adult-born neurons may be tightly coupled to their functional requirement, the organization and output of neurogenic niches may vary across different regions of the brain or between species. We have undertaken a comparative study of six (D, Vd, Vv, Dm, Dl, Ppa) periventricular zones (PVZs) harboring proliferative cells present in the adult forebrain of the zebrafish (Danio rerio), a species known to possess widespread neurogenesis throughout life. Using electron microscopy, we have documented for the first time the detailed cytoarchitecture of these zones, and propose a model of the cellular composition of pallial and subpallial PVZs, as well as a classification scheme for identifying morphologically distinct cell types. Immunolabeling of resin-embedded tissue confirmed the phenotype of three constitutively proliferating (bromodeoxyuridine [BrdU]+) cell populations, including a radial glial-like (type IIa) cell immunopositive for both S100ß and glutamine synthetase (GS). Our data revealed rostrocaudal differences in the density of distinct proliferative populations, and cumulative labeling studies suggested that the cell cycle kinetics of these populations are not uniform between PVZs. Although the peak numbers of differentiated neurons were generated after ~2 weeks among most PVZs, niche-specific decline in the number of newborn neurons in some regions occurred after 4 weeks. Our data suggest that the cytoarchitecture of neurogenic niches and the tempo of neuronal production are regionally distinct in the adult zebrafish forebrain.


Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Ventrículos Cerebrais/citologia , Neurogênese/fisiologia , Prosencéfalo/anatomia & histologia , Células-Tronco Adultas/ultraestrutura , Análise de Variância , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Ciclo Celular , Movimento Celular/fisiologia , Proliferação de Células , Proteínas ELAV/metabolismo , Proteínas do Olho/metabolismo , Glutamato-Amônia Ligase/metabolismo , Proteínas de Homeodomínio/metabolismo , Microscopia Eletrônica , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Ácidos Siálicos/metabolismo , Peixe-Zebra/anatomia & histologia
20.
J Control Release ; 152(3): 382-92, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21397644

RESUMO

Self-assembly driven processes can be utilized to produce a variety of nanostructures useful for various in vitro and in vivo applications. Characteristics such as size, stability, biocompatibility, high therapeutic loading and controlled delivery of these nanostructures are particularly crucial in relation to in vivo applications. In this study, we report the fabrication of tunable monodispersed elastin-like polypeptide (ELP) hollow spheres of 100, 300, 500 and 1000 nm by exploiting the self-assembly property and net positive charge of ELP. The microbial transglutaminase (mTGase) cross-linking provided robustness and stability to the hollow spheres while maintaining surface functional groups for further modifications. The resulting hollow spheres showed a higher loading efficiency of plasmid DNA (pDNA) by using polyplex (~70 µg pDNA/mg of hollow sphere) than that of self-assembled ELP particles and demonstrated controlled release triggered by protease and elastase. Moreover, polyplex-loaded hollow spheres showed better cell viability than polyplex alone and yielded higher luciferase expression by providing protection against endosomal degradation. Overall, the monodispersed, tunable hollow spheres with a capability of post-functionalization can provide an exciting new opportunity for use in a range of therapeutic and diagnostic applications.


Assuntos
Preparações de Ação Retardada , Elastina/genética , Técnicas de Transferência de Genes , Nanocápsulas/química , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Disponibilidade Biológica , Sobrevivência Celular , Células Cultivadas , Endocitose/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Furanos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisossomos/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microesferas , Elastase Pancreática/metabolismo , Tamanho da Partícula , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmídeos/farmacocinética , Ácidos Polimetacrílicos/química , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Propriedades de Superfície , Transfecção , Transglutaminases/química
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