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1.
Trials ; 22(1): 595, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488845

RESUMO

BACKGROUND: Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. METHODS: A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. DISCUSSION: Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. TRIAL REGISTRATION: ClinicalTrials.gov NCT04466007 . Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.


Assuntos
COVID-19 , Diabetes Mellitus Tipo 2 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Noma , Tecido Adiposo , Animais , Ensaios Clínicos Fase II como Assunto , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/terapia , Método Duplo-Cego , Humanos , Isquemia/diagnóstico , Isquemia/terapia , Estudos Multicêntricos como Assunto , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Resultado do Tratamento
2.
J Transl Med ; 18(1): 356, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948200

RESUMO

BACKGROUND: Mesenchymal stromal cells are a safe and promising option to treat knee osteoarthritis as previously demonstrated in different clinical trials. However, their efficacy, optimal dose and addition of adjuvants must be determined. Here, we evaluated the clinical effects of a dose of 100 × 106 bone marrow mesenchymal stromal cells (BM-MSCs) in combination with Platelet Rich Plasma (PRGF®) as adjuvant in a randomized clinical trial. METHODS: A phase II, multicenter, randomized clinical trial with active control was conducted. Sixty patients diagnosed with knee OA were randomly assigned to 3 weekly doses of PRGF® or intraarticular administration of 100 × 106 cultured autologous BM-MSCs plus PRGF®. Patients were followed up for 12 months, and pain and function were assessed using VAS and WOMAC and by measuring the knee range of motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. RESULTS: No adverse effects were reported after BM-MSC administration or during follow-up. According to VAS, the mean value (SD) for PRGF® and BM-MSC with PRGF® went from 5 (1.8) to 4.5 (2.2) (p = 0.389) and from 5.3 (1.9) to 3.5 (2.5) (p = 0.01), respectively at 12 months. In WOMAC, the mean (SD) baseline and 12-month overall WOMAC scores in patients treated with PRGF® was 31.9 (16.2) and 22.3 (15.8) respectively (p = 0.002) while that for patients treated with BM-MSC plus PRGF® was 33.4 (18.7) and 23.0 (16.6) (p = 0.053). Although statistical significances between groups have been not detected, only patients being treated with BM-MSC plus PRGF® could be considered as a OA treatment responders following OARSI criteria. X-ray and MRI (WORMS protocol) revealed no changes in knee joint space width or joint damage. CONCLUSIONS: Treatment with BM-MSC associated with PRGF® was shown to be a viable therapeutic option for osteoarthritis of the knee, with clinical improvement at the end of follow-up. Further phase III clinical trials would be necessary to confirm the efficacy. Trial registration Clinical Trials.gov identifier NCT02365142. Nº EudraCT: 2011-006036-23.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteoartrite do Joelho , Plasma Rico em Plaquetas , Humanos , Injeções Intra-Articulares , Osteoartrite do Joelho/terapia , Resultado do Tratamento
3.
EClinicalMedicine ; 25: 100454, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32838232

RESUMO

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None.

4.
Invest Ophthalmol Vis Sci ; 58(2): 745-754, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28146239

RESUMO

Purpose: The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2. Methods: Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas. Results: Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na+/K+-ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction. Conclusions: We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.


Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Endotélio Corneano/citologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Smad2/fisiologia , Ativinas/farmacologia , Análise de Variância , Animais , Movimento Celular/efeitos dos fármacos , Transplante de Células/métodos , Células Cultivadas , Conexina 43/metabolismo , Lesões da Córnea/terapia , Modelos Animais de Doenças , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Neuregulina-1/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Coelhos , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
5.
J Transl Med ; 14(1): 246, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27565858

RESUMO

BACKGROUND: Mesenchymal stromal cells are a promising option to treat knee osteoarthritis. Their safety and usefulness must be confirmed and the optimal dose established. We tested increasing doses of bone marrow mesenchymal stromal cells (BM-MSCs) in combination with hyaluronic acid in a randomized clinical trial. MATERIALS: A phase I/II multicenter randomized clinical trial with active control was conducted. Thirty patients diagnosed with knee OA were randomly assigned to intraarticularly administered hyaluronic acid alone (control), or together with 10 × 10(6) or 100 × 10(6) cultured autologous BM-MSCs, and followed up for 12 months. Pain and function were assessed using VAS and WOMAC and by measuring the knee motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. RESULTS: No adverse effects were reported after BM-MSC administration or during follow-up. BM-MSC-administered patients improved according to VAS during all follow-up evaluations and median value (IQR) for control, low-dose and high-dose groups change from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 4 (3, 5), 2 (1, 3) and 2 (0,4) respectively at 12 months (low-dose vs control group p = 0.005 and high-dose vs control group p < 0.009). BM-MSC-administered patients were also superior according to WOMAC, although improvement in control and low-dose patients could not be significantly sustained beyond 6 months. On the other hand, the BM-MSC high-dose group exhibited an improvement of 16.5 (12, 19) points at 12 months (p < 0.01). Consistent with WOMAC and VAS values, motion ranges remained unaltered in the control group but improved at 12 months with BM-MSCs. X-ray revealed a reduction of the knee joint space width in the control group that was not seen in BM-MSCs high-dose group. MRI (WORMS protocol) showed that joint damage decreased only in the BM-MSC high-dose group, albeit slightly. CONCLUSIONS: The single intraarticular injection of in vitro expanded autologous BM-MSCs together with HA is a safe and feasible procedure that results in a clinical and functional improvement of knee OA, especially when 100 × 10(6) cells are administered. These results pave the way for a future phase III clinical trial. CLINICAL TRIALS: gov identifier NCT02123368. Nº EudraCT: 2009-017624-72.


Assuntos
Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/farmacologia , Transplante de Células-Tronco Mesenquimais , Osteoartrite do Joelho/terapia , Idoso , Terapia Combinada , Demografia , Feminino , Humanos , Ácido Hialurônico/efeitos adversos , Injeções Intra-Articulares , Imageamento por Ressonância Magnética , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/fisiopatologia , Amplitude de Movimento Articular/efeitos dos fármacos , Resultado do Tratamento , Escala Visual Analógica
6.
Stem Cell Res ; 16(1): 20-3, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27345779

RESUMO

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers.


Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Linhagem Celular , Hibridização Genômica Comparativa , Humanos , Cariotipagem
8.
PLoS One ; 9(12): e113936, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25436769

RESUMO

OBJECTIVE: Uncontrolled studies of mesenchymal stem cells (MSCs) in multiple sclerosis suggested some beneficial effect. In this randomized, double-blind, placebo-controlled, crossover phase II study we investigated their safety and efficacy in relapsing-remitting multiple sclerosis patients. Efficacy was evaluated in terms of cumulative number of gadolinium-enhancing lesions (GEL) on magnetic resonance imaging (MRI) at 6 months and at the end of the study. METHODS: Patients unresponsive to conventional therapy, defined by at least 1 relapse and/or GEL on MRI scan in past 12 months, disease duration 2 to 10 years and Expanded Disability Status Scale (EDSS) 3.0-6.5 were randomized to receive IV 1-2×10(6) bone-marrow-derived-MSCs/Kg or placebo. After 6 months, the treatment was reversed and patients were followed-up for another 6 months. Secondary endpoints were clinical outcomes (relapses and disability by EDSS and MS Functional Composite), and several brain MRI and optical coherence tomography measures. Immunological tests were explored to assess the immunomodulatory effects. RESULTS: At baseline 9 patients were randomized to receive MSCs (n = 5) or placebo (n = 4). One patient on placebo withdrew after having 3 relapses in the first 5 months. We did not identify any serious adverse events. At 6 months, patients treated with MSCs had a trend to lower mean cumulative number of GEL (3.1, 95% CI = 1.1-8.8 vs 12.3, 95% CI = 4.4-34.5, p = 0.064), and at the end of study to reduced mean GEL (-2.8±5.9 vs 3±5.4, p = 0.075). No significant treatment differences were detected in the secondary endpoints. We observed a non-significant decrease of the frequency of Th1 (CD4+ IFN-γ+) cells in blood of MSCs treated patients. CONCLUSION: Bone-marrow-MSCs are safe and may reduce inflammatory MRI parameters supporting their immunomodulatory properties. ClinicalTrials.gov NCT01228266.


Assuntos
Transplante de Células-Tronco Mesenquimais , Esclerose Múltipla Recidivante-Remitente/terapia , Adulto , Linfócitos B/imunologia , Linfócitos B/patologia , Método Duplo-Cego , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/imunologia , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Resultado do Tratamento , Adulto Jovem
9.
Int J Ophthalmol ; 7(6): 988-95, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25540752

RESUMO

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization. METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured. RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up. CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.

10.
Knee Surg Sports Traumatol Arthrosc ; 21(8): 1737-50, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22864678

RESUMO

PURPOSE: Due to the attractive properties of poly(L-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. METHODS: MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. RESULTS: MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. CONCLUSIONS: Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component.


Assuntos
Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Tecidos Suporte , Adulto , Agrecanas/metabolismo , Apoptose , Doenças das Cartilagens/terapia , Adesão Celular , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo X/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ácido Láctico , Microscopia Eletrônica de Varredura , Poliésteres , Polímeros
11.
Haematologica ; 96(7): 1072-6, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21393326

RESUMO

This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. Twenty-eight expansions were started. In 22, a minimum of more than 1 x 106 mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier NCT00447460.


Assuntos
Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Soro , Adulto , Idoso , Proliferação de Células , Células Cultivadas , Técnicas de Cultura , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Transplante Homólogo , Resultado do Tratamento , Adulto Jovem
12.
Cell Transplant ; 20(2): 259-69, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20719064

RESUMO

There is a need for comparative studies to determine which cell types are better candidates to remedy ischemia. Here, we compared human AC133(+) cells and multipotent adult progenitor cells (hMAPC) in a mouse model reminiscent of critical limb ischemia. hMAPC or hAC133(+) cell transplantation induced a significant improvement in tissue perfusion (measured by microPET) 15 days posttransplantation compared to controls. This improvement persisted for 30 days in hMAPC-treated but not in hAC133(+)-injected animals. While transplantation of hAC133(+) cells promoted capillary growth, hMAPC transplantation also induced collateral expansion, decreased muscle necrosis/fibrosis, and improved muscle regeneration. Incorporation of differentiated hAC133(+) or hMAPC progeny into new vessels was limited; however, a paracrine angio/arteriogenic effect was demonstrated in animals treated with hMAPC. Accordingly, hMAPC-conditioned, but not hAC133(+)-conditioned, media stimulated vascular cell proliferation and prevented myoblast, endothelial, and smooth muscle cell apoptosis in vitro. Our study suggests that although hAC133(+) cell and hMAPC transplantation both contribute to vascular regeneration in ischemic limbs, hMAPC exert a more robust effect through trophic mechanisms, which translated into collateral and muscle fiber regeneration. This, in turn, conferred tissue protection and regeneration with longer term functional improvement.


Assuntos
Células-Tronco Adultas/citologia , Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Células-Tronco Multipotentes/citologia , Peptídeos/metabolismo , Transplante de Células-Tronco , Antígeno AC133 , Animais , Apoptose , Vasos Sanguíneos/crescimento & desenvolvimento , Proliferação de Células , Citoproteção , Membro Posterior/patologia , Humanos , Isquemia/patologia , Masculino , Camundongos , Camundongos Nus , Músculos/fisiopatologia , Neovascularização Fisiológica , Regeneração , Reperfusão , Sobrevivência de Tecidos
13.
J Biomed Mater Res A ; 96(2): 341-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21171153

RESUMO

The main goal of this study was to evaluate the potential of collagen meshes derived from porcine dermis as scaffolds for repairing pelvic organ prolapses. Mechanical properties of collagen meshes with different cross-linking percentages before and after Adipose Derived Stromal Cells (ADSC) seeding were studied as well as the cell-scaffold interaction. Uniaxial tensile tests of the collagen meshes with three different cross-linking percentages (full-, partial-, and noncross-linked) were carried out along orthogonal directions. Their mechanical properties were studied with the same tests before and after seeding with human derived adipose stem cells (ADSC) after 1 and 7 days. Histological analyses were performed to determine adhesion and proliferation of ADSC. Significant differences in mechanical properties of the unseeded meshes were observed between each orthogonal direction independently of the cross-linking percentage. A better cell adhesion rate was observed in the cross-linked meshes. An increase in the mechanical properties after cell seeding was observed with a direct relation with the degree of cross-linking. All meshes analyzed showed a marked anisotropy that should be taken into account during the surgical procedure. The cross-linking treatment increased cell adhesion and the mechanical properties of the collagen meshes after seeding. These results suggest that the mechanical properties of this type of collagen mesh could be useful as scaffolds for repair of pelvic organ prolapse.


Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Colágeno/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Teste de Materiais , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Humanos , Indóis/metabolismo , Coloração e Rotulagem , Estresse Mecânico , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Sus scrofa
14.
PLoS One ; 4(11): e7809, 2009 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-19915669

RESUMO

BACKGROUND: The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles. METHODOLOGY/PRINCIPAL FINDINGS: to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes. CONCLUSIONS/SIGNIFICANCE: Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.


Assuntos
Epigênese Genética , Células-Tronco/citologia , Tecido Adiposo/metabolismo , Adulto , Diferenciação Celular , Metilação de DNA , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição Genética
15.
Transfusion ; 49(9): 1901-10, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19497059

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function. STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF). RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-gamma was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells. DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential.


Assuntos
Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Proliferação de Células , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Adulto Jovem
16.
Eur J Heart Fail ; 10(11): 1065-72, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18805052

RESUMO

BACKGROUND: The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms. METHODS AND RESULTS: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56(+)), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56(-)). Gene expression revealed up-regulation of pro-angiogenic (PGF), anti-apoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (p<0.05) and a decrease in apoptosis of cardiomyocytes (p<0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-beta, and IGF-1 1 month after transplant. CONCLUSIONS: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.


Assuntos
Transplante de Células/métodos , Mioblastos Esqueléticos/transplante , Infarto do Miocárdio/cirurgia , Comunicação Parácrina/fisiologia , Animais , Apoptose , Biópsia , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Glicosilfosfatidilinositóis , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neovascularização Fisiológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA/genética , Ratos , Ratos Nus , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Resultado do Tratamento , Troponina I/biossíntese , Troponina I/genética
17.
Br J Haematol ; 142(4): 571-82, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18537972

RESUMO

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Antígenos CD34/metabolismo , Linhagem Celular Tumoral/metabolismo , Sobrevivência Celular , Ciclina D , Ciclinas/metabolismo , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSC70/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Análise em Microsséries , Transdução de Sinais
18.
Leuk Res ; 32(5): 709-16, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17942153

RESUMO

The aim of our study was to determine the potential mechanism(s) implicated in Imatinib resistance in patients with Ph+ ALL. Resistance of Ph+ ALL cells to Imatinib-induced apoptosis was associated with lack of inhibition of Akt phosphorylation. Addition of the PI3K inhibitor LY294002 to Imatinib significantly increased apoptosis of Ph+ ALL cells. Interestingly, expression of PTEN was reduced in Ph+ ALL cells which was due to PTEN promoter hypermethylation. Treatment of Ph+ ALL cells with 5-Aza-2'-deoxycytidine was associated with an increased expression of PTEN and an increase in cell apoptosis. These results suggest that Imatinib resistance in patients with ALL may be dependent at least in part to PTEN down-regulation due to the abnormal promoter hypermethylation and support the potential role of de-methylating agents for the treatment of patients with Ph+ ALL.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Metilação de DNA , PTEN Fosfo-Hidrolase/genética , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , Benzamidas , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/fisiologia , Humanos , Mesilato de Imatinib , Fosfatidilinositol 3-Quinases/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia
19.
Blood ; 109(6): 2634-42, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17090652

RESUMO

Many stem cell types have been shown to differentiate into endothelial cells (ECs); however, their specification to arterial or venous endothelium remains unexplored. We tested whether a specific arterial or venous EC fate could be induced in human multipotent adult progenitor cells (hMAPCs) and AC133(+) cells (hAC133(+)). In vitro, in the presence of VEGF(165), hAC133(+) cells only adopted a venous and microvascular EC phenotype, while hMAPCs differentiated into both arterial and venous ECs, possibly because hMAPCs expressed significantly more sonic hedgehog (Shh) and its receptors as well as Notch 1 and 3 receptors and some of their ligands. Accordingly, blocking either of those pathways attenuated in vitro arterial EC differentiation from hMAPCs. Complementarily, stimulating these pathways by addition of Delta-like 4 (Dll-4), a Notch ligand, and Shh to VEGF(165) further boosted arterial differentiation in hMAPCs both in vitro and in an in vivo Matrigel model. These results represent the first demonstration of adult stem cells with the potential to be differentiated into different types of ECs in vitro and in vivo and provide a useful human model to study arteriovenous specification.


Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular , Neovascularização Fisiológica , Antígeno AC133 , Células-Tronco Adultas/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Diferenciação Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Nus , Microscopia Eletrônica , Fragmentos de Peptídeos/farmacologia , Peptídeos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologia
20.
Clin Transl Oncol ; 8(9): 647-63, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17005467

RESUMO

Most human cancers derive from a single cell targeted by genetic and epigenetic alterations that initiate malignant transformation. Progressively, these early cancer cells give rise to different generations of daughter cells that accumulate additional mutations, acting in concert to drive the full neoplastic phenotype. As we have currently deciphered many of the gene pathways disrupted in cancer, our knowledge about the nature of the normal cells susceptible to transformation upon mutation has remained more elusive. Adult stem cells are those that show long-term replicative potential, together with the capacities of self-renewal and multi-lineage differentiation. These stem cell properties are tightly regulated in normal development, yet their alteration may be a critical issue for tumorigenesis. This concept has arisen from the striking degree of similarity noted between somatic stem cells and cancer cells, including the fundamental abilities to self-renew and differentiate. Given these shared attributes, it has been proposed that cancers are caused by transforming mutations occurring in tissue-specific stem cells. This hypothesis has been functionally supported by the observation that among all cancer cells within a particular tumor, only a minute cell fraction has the exclusive potential to regenerate the entire tumor cell population; these cells with stem-like properties have been termed cancer stem cells. Cancer stem cells can originate from mutation in normal somatic stem cells that deregulate their physiological programs. Alternatively, mutations may target more committed progenitor cells or even mature cells, which become reprogrammed to acquire stem-like functions. In any case, mutated genes should promote expansion of stem/progenitor cells, thus increasing their predisposition to cancer development by expanding self-renewal and pluripotency over their normal tendency towards relative quiescency and proper differentiation.


Assuntos
Transformação Celular Neoplásica , Células-Tronco Neoplásicas , Animais , Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Humanos , Leucemia/genética , Linfoma/genética , MicroRNAs , Mutação , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/fisiologia , Oncogenes , Telomerase , Telômero
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