RESUMO
Regenerative therapies offer attractive alternatives for the treatment of osteochondral defects. Adipose-derived stromal vascular fraction (SVF) cells allow the development of one-step surgical procedures by their abundant availability and high frequency. In this pilot study we evaluated the in vivo safety, feasibility, and efficacy of this concept using scaffolds seeded with freshly isolated (SVF) or cultured adipose stem cells (ASCs), and compared these to their acellular counterparts. Osteochondral defects were created in medial condyles and trochlear grooves in knees of eight goats. Defects were filled with acellular collagen I/III scaffolds or scaffolds seeded with SVF cells or cultured ASCs. Osteochondral regeneration was evaluated after 1 and 4 months by macroscopy, immunohistochemistry, biomechanical analysis, microCT analysis, and biochemistry. After 1 month, no adverse effects were noted. Microscopic, but not macroscopic evaluation showed considerable yet not significant differences, with cell-loaded constructs showing more extensive regeneration. After 4 months, acellular constructs displayed increased regeneration, however, to a lesser degree than cell-treated constructs. The latter exhibited more extensive collagen type II, hyaline-like cartilage, and higher elastic moduli, and their glycosaminoglycan content in the cartilaginous layer better approached native tissue values. Moreover, their defect regions contained higher levels of regenerated, mature subchondral bone with more intense collagen type I staining. SVF cells tended to perform best on all parameters. In summary, this pilot study demonstrated the preclinical safety and feasibility of a one-step surgical procedure for osteochondral defect regeneration. Similar regeneration was found between freshly isolated SVF cells and cultured ASCs. Larger studies with longer follow-up are required to substantiate these findings.
RESUMO
This study compares mesenchymal cells isolated from excised burn wound eschar with adipose-derived stem cells (ASCs) and dermal fibroblasts in their ability to conform to the requirements for multipotent mesenchymal stem cells (MSCs). A population of multipotent stem cells in burn eschar could be an interesting resource for tissue engineering approaches to heal burn wounds. Cells from burn eschar, dermis, and adipose tissue were assessed for relevant CD marker profiles using flow cytometry and for their trilineage differentiation ability in adipogenic, osteogenic, and chondrogenic conditions. Although the different cell types did not differ significantly in their CD marker expression, the eschar-derived cells and ASCs readily differentiated into adipocytes, osteoblasts, and chondrocytes, while dermal fibroblasts only exhibited some chondrogenic potential. We conclude that the eschar-derived mesenchymal cells represent a population of multipotent stem cells. The origin of the cells from burn eschar remains unclear, but it is likely they represent a population of adult stem cells mobilized from other parts of the body in response to the burn injury. Their resemblance to ASCs could also be cause for speculation that in deep burns the subcutaneous adipose tissue might be an important stem cell source for the healing wound.
Assuntos
Queimaduras/patologia , Células-Tronco Mesenquimais/citologia , Pele/citologia , Actinas/metabolismo , Adipócitos/citologia , Tecido Adiposo/citologia , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Diferenciação Celular , Criança , Pré-Escolar , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Humanos , Lactente , Transplante de Células-Tronco Mesenquimais , Pessoa de Meia-Idade , Osteoblastos/citologia , Engenharia Tecidual , Cicatrização , Adulto JovemRESUMO
BACKGROUND: Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. METHODS: Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n=15) or vehicle (n=13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. RESULTS: Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. CONCLUSIONS: Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.
Assuntos
Clusterina/uso terapêutico , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Animais , Imuno-Histoquímica , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/uso terapêutico , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
BACKGROUND AIMS: Stem cell therapies are being evaluated as promising alternatives for cartilage regeneration. We investigated whether stromal vascular fraction cells (SVF) from the infrapatellar (Hoffa) fat pad are suitable for a one-step surgical procedure to treat focal cartilage defects. METHODS: SVF was harvested from patients undergoing knee arthroplasty (n = 53). Colony-forming unit (CFU) assays, growth kinetics and surface marker profiles were determined, and the chondrogenic differentiation capacity of freshly isolated SVF was assessed after seeding in three-dimensional poly (L-lactic-co-epsilon-caprolactone) scaffolds. RESULTS: SVF yield per fat pad varied between 0.55 and 16 x 10(6) cells. CFU frequency and population doubling time were 2.6 +/- 0.6% and +/-2 days, respectively. Surface marker profiles matched those of subcutaneous-derived adipose-derived stem cells (ASC). CFU from Hoffa SVF showed differentiation toward osteogenic and adipogenic lineages. Cartilage differentiation was confirmed by up-regulation of the cartilage genes sox9, aggrecan, collagen type II and cartilage oligomeric matrix protein (COMP), collagen II immunostaining, Alcian Blue staining and glycosaminoglycan production. Compared with passaged cells, SVF showed at least similar chondrogenic potential. CONCLUSIONS: This study demonstrates that SVF cells from the infrapatellar fat pad are suitable for future application in a one-step surgical procedure to regenerate cartilage tissue. SVF shows similar favorable characteristics as cultured ASC, and chondrogenic differentiation even appears to be slightly better. However, because of variable harvesting volumes and yields, SVF from the infrapatellar fat pad might only be applicable for treatment of small focal cartilage defects, whereas for larger osteoarthritic defects subcutaneous adipose tissue depot would be preferable.
Assuntos
Tecido Adiposo/citologia , Cartilagem/fisiologia , Regeneração , Medicina Regenerativa/métodos , Células Estromais/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Condrogênese/genética , Ensaio de Unidades Formadoras de Colônias , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Glicosaminoglicanos/biossíntese , Humanos , Pessoa de Meia-Idade , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Estromais/metabolismo , Regulação para Cima/genéticaRESUMO
During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A(2) (sPLA(2)-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA(2)-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA(2)-IIA activity and reduced infarct size (reduction 73 +/- 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA(2)-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA(2)-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA(2)-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA(2)-IIA independent manner.
Assuntos
Apoptose , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Testes de Função Cardíaca , Imuno-Histoquímica , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Ratos , Ratos Wistar , Sinvastatina/farmacologia , Solubilidade/efeitos dos fármacosRESUMO
The ability of stem cells to self-renew as well as their multilineage differentiation potential makes them ideal candidates for skin regeneration strategies. Mesenchymal stem cells residing in human adult dermis, in contrast to adipose tissue, have not yet been described. The objective of this study was to determine the stemness and chemokine-mediated homing potential of dermal stromal cells (DSC) and to compare this with adipose stem cells (ASC). DSC have a less stellate form than ASC, confirming that DSC and ASC are two different types of mesenchymal cell populations. However, DSC display a mesenchymal stem cell phenotype (CD31(-), CD34(+), CD45(-), CD54(+), CD90(+), CD105(+), and CD166(+) similar to ASC and are also multipotent in their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Both ASC and DSC display a similar set of chemokine receptors (CCR3, CCR4, CCR6, CCR10, CXCR1, and CXCR2). Several ligands for these receptors, with CCL5/RANTES being the most potent, can induce migration of ASC and DSC in an in vitro wound-healing assay. Taken together, these results show that a population of mesenchymal stem cells resides in the dermis of human adult skin and these dermal-derived stem cells have a phenotypic and chemokine-mediated homing potential similar to adipose stem cells, which to our knowledge is previously unreported.
Assuntos
Quimiocina CCL5/fisiologia , Quimiocinas/metabolismo , Pele/citologia , Células-Tronco/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Quimiocina CCL5/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Fenótipo , Receptores de Quimiocinas/metabolismo , Pele/metabolismo , CicatrizaçãoRESUMO
The inevitable clinical recurrence of high grade gliomas after standard treatment is due to the highly diffuse infiltrating parts of these tumors, which remain after surgery and respond poorly to radiation and chemotherapy. It has been proposed to employ the homing capacity of neural stem cells (NSCs) to different types of intracerebral pathology for selective targeting of glioma cells, and delivery of transgenic expressed therapeutics. This approach has been successful in a number of preclinical experimental studies, however, a major drawback for clinical translation has been the limitation of harvesting and ex vivo expansion of NSCs in patients. Here we demonstrate that adipose derived stem cells (ASCs), which are easily harvested in relatively large quantities in humans, display the same tropism for gliomas as NSCs in vitro and in vivo. Both ipsilateral as well as contralateral injection of these cells in brains of glioma-bearing mice, led to extensive homing to the tumor by the ASCs. The potential of loading these cellular vehicles with transgenes was assessed using adenoviral vectors. ASCs could be infected with adenoviral vectors, albeit at very high MOI. Insertion of the arg-gly-asp (RGD) motif into the adenovirus fiber knob, thereby redirecting primary attachment of the virus to integrins, resulted in a striking 7000-fold increase in infection efficiency. However, in vivo migration of adenovirus-infected ASCs was not observed, most likely due to an inflammatory response to these cells which was not observed with control non-infected ASCs. These results indicate that ASCs are an interesting candidate for further development for cell-based therapy of gliomas, however adenoviruses are not appropriate vectors for delivery of transgenes in this context.
Assuntos
Adenoviridae/genética , Tecido Adiposo/citologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Células-Tronco Mesenquimais/fisiologia , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/virologia , Movimento Celular , Feminino , Fibroblastos/citologia , Vetores Genéticos , Glioma/imunologia , Glioma/virologia , Humanos , Camundongos , Camundongos Nus , Transfecção , Transgenes/fisiologia , Tropismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 +/- 1.1%, mean +/- SEM) and hip/thigh region (1.2 +/- 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Multipotentes/citologia , Coleta de Tecidos e Órgãos , Adulto , Diferenciação Celular , Células Cultivadas , Condrogênese , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Cinética , Pessoa de Meia-Idade , Osteogênese , Fenótipo , Transplante de Células-TroncoRESUMO
Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue engineering purposes, however, it would be advantageous to use the whole SVF, which can be transplanted without further in vitro selection or expansion steps. Because little is known about the freshly isolated ASCs in the SVF, we phenotypically characterized human freshly isolated ASCs, using flow cytometry. In addition, we investigated whether freshly isolated ASCs have functional properties comparable to cultured ASCs. For this, the differentiation potential of both freshly isolated ASCs and cultured ASCs into the osteogenic pathway was analyzed. Freshly isolated ASCs slightly differed in immunophenotype from cultured ASCs. Contrary to cultured ASCs, freshly isolated ASCs were shown to be highly positive for CD34, and positive for CD117 and HLA-DR. On the other hand, expression of CD105 and especially CD166 on the freshly isolated ASCs was relatively low. After osteogenic stimulation of freshly isolated ASCs, both Runx-2 and CollaI gene expression were significantly increased (p < 0.05). However, there was a difference in the kinetics of gene expression between freshly isolated and cultured ASCs and also between the different SVF isolates tested. There was no difference in alkaline phosphatase activity between freshly isolated ASCs and cultured ASCs. In addition, freshly isolated ASCs stained positive for osteonectin and showed matrix mineralization. We conclude that although there are minor differences in phenotype and kinetics of differentiation between freshly isolated ASCs and cultured ASCs, the use of freshly isolated ASCs for tissue engineering purposes involving bone repair is potentially applicable.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Separação Celular/métodos , Imunofenotipagem , Células-Tronco Multipotentes/citologia , Células Estromais/citologia , Adulto , Antígenos CD34/metabolismo , Células Cultivadas , Feminino , Humanos , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P < 0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection.