RESUMO
Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton's jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.
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Cell culture conditions influence several biological and biochemical features of stem cells (SCs), including the membrane lipid profile, thus limiting the use of SCs for cell therapy approaches. The present study aims to investigate whether the in vitro culture may alter the membrane fatty acid signature of human Amniotic Epithelial Cells (hAECs). The analysis of the membrane fatty acid composition of hAECs cultured in basal medium showed a loss in polyunsaturated fatty acids (PUFA), in particular in omega-6 (ω-6) content, compared to freshly isolated hAECs. The addition to the basal culture medium of a chemically defined and animal-free tailored lipid supplement, namely Refeed®, partially restored the membrane fatty acid signature of hAECs. Although the amelioration of the membrane composition did not prolong hAECs culture lifespan, Refeed® influenced cell morphology, counteracted the onset of senescence, and increased the migratory capacity as well as the ability of hAECs to inhibit Peripheral Blood Mononuclear Cell (PBMC) proliferation. This study provides new information on hAEC features during culture passages and demonstrates that the maintenance of the membrane fatty acid signature preserved higher cell quality during in vitro expansion, suggesting the use of lipid supplementation for SC expansion in cell-based therapies.
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BACKGROUND: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). METHODS: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. RESULTS: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. CONCLUSIONS: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.
Assuntos
Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Diferenciação Celular , Membrana Celular/química , Proliferação de Células , Suplementos Nutricionais , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Placenta/citologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Cultura Primária de CélulasRESUMO
Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.
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Infecções por Herpesviridae/virologia , Células-Tronco Mesenquimais/virologia , Placenta/virologia , Adulto , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Suscetibilidade a Doenças , Embrião de Mamíferos , Feminino , Infecções por Herpesviridae/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Células-Tronco Mesenquimais/patologia , Placenta/patologia , Gravidez , Células VeroRESUMO
Following experimental hind limb denervation in rats, this study demonstrates that oxidative stress occurs and advances an hypothesis about its origin. In fact: (i) ROS are formed; (ii) membrane lipids are oxidized; (iii) oxidized ion channels and pumps may lead to increased [Ca(2+)](i); all the above mentioned events increase with denervation time. In the denervated muscle, (iv) mRNA abundance of cytoprotective and anti-oxidant proteins (Hsp70, Hsp27, Sod1, Catalase, Gpx1, Gpx4, Gstm1), as well as (v) SOD1 enzymatic activity and HSP70i protein increase; (vi) an unbalance in mitochondrial OXPHOS enzymes occurs, presumably leading to excess mitochondrial ROS production; (vii) increased cPLA2alpha expression (mRNA) and activation (increased [Ca(2+)](i)) may lead to increased hydroperoxides release. Since anti-oxidant defences appear inadequate to counterbalance increased ROS production with increased denervation time, an anti-oxidant therapeutic strategy seems to be advisable in the many medical conditions where the nerve-muscle connection is impaired.
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Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Estresse Oxidativo , Animais , Cálcio/metabolismo , Feminino , Canais Iônicos/metabolismo , Bombas de Íon/metabolismo , Lipídeos de Membrana/metabolismo , Denervação Muscular , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND AIMS: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) are currently under evaluation in phase III clinical trials for inflammatory bowel disease and other intestinal disease manifestations. The therapeutic efficacy of these treatments may derive from a combination of the differentiation, trophic and immunomodulatory abilities of the transplanted cells. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specific functions and hold advantages for engraftment and contribution in the gastrointestinal environment. METHODS: Intestinal specimens were collected, and the mucosa and submucosa mechanically separated and enzymatically digested. Mesenchymal stromal populations were isolated, expanded and characterized under conditions commonly used for MSC. The differentiation potential, trophic effect and immunomodulatory ability were investigated. Results We successfully isolated and extensively expanded populations showing the typical MSC profile: CD29+, CD44+, CD73+, CD105+ and CD166+, and CD14(-), CD34(-) and CD45(-). Intestinal mucosal (IM) MSC were also CD117+, while submucosal cultures (ISM MSC) showed CD34+ subsets. The cells differentiated toward osteogenic, adipogenic and angiogenic commitments. Intestinal-derived MSC were able to induce differentiation and organization of intestinal epithelial cells (Caco-2) in three-dimensional collagen cultures. Immunomodulatory activity was evidenced in co-cultures with normal heterologous phytohemagglutinin-stimulated peripheral blood mononuclear cells. Conclusions Multipotent MSC can be isolated from intestinal mucosal and submucosal tissues. IM MSC and ISM MSC are able to perform trophic and immunomodulatory functions. These findings could open a pathway for novel approaches to intestinal disease treatment.
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Separação Celular/métodos , Imunomodulação , Doenças Inflamatórias Intestinais/terapia , Intestinos/citologia , Células-Tronco Mesenquimais/citologia , Transplante de Células-Tronco , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fito-Hemaglutininas/farmacologiaRESUMO
BACKGROUND: The interest in stem cell (SC) isolation from easily accessible clinical specimens is booming. The lack of homogeneity in pluri/multipotent SC preparation blurs standardization, which however is recommended for successful applications. Multipotent mesenchymal SCs (MSCs) in fact express a broad panel of surface antigens, which limit the possibility of sorting homogeneous preparations by using an immunotag-based method. METHODS: We present a tag-less, flow-assisted method to purify, distinguish, and sort pluri/multipotent SCs obtained from clinical specimens, based on differences in the biophysical properties that cells acquire when in suspension under fluidic conditions. A suspension of cells in a transport fluid is injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min), sorted cells are collected. RESULTS: We obtained baseline separation between MSCs and epithelial cells, which are important contaminants of isolated MSCs. The extent of separation is evaluated by flow cytometry through detection of a specific epithelial antigen. MSCs from various human sources also prove to have different, characteristic, highly-reproducible fractionation profiles. Finally, we evaluated the dissimilar differentiation potential among cell fractions obtained from sorting a single MSC source. After differentiation induction, a fraction displayed a differentiation yield close to 100%, whereas unfractionated cells contained only 40% of responding cells. CONCLUSIONS: The results demonstrate that the method presented is able to obtain selected and well-characterized living MSCs with an increased differentiation yield. Its reduced cost, full biocompatibility, and scale-up potential could make this method an effective procedure for stem cell selection.
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Células Epiteliais/citologia , Citometria de Fluxo/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Adipogenia/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Separação Celular/métodos , Células Cultivadas , Células Epiteliais/fisiologia , Humanos , Manejo de Espécimes , Coloração e RotulagemRESUMO
Growing interest in stem cell research has led to the development of a number of new methods for isolation. The lack of homogeneity in stem cell preparation blurs standardization, which however is recommended for successful applications. Among stem cells, mesenchymal stem cells (MSCs) are promising candidates for cell therapy applications. This paper presents a fractionation protocol based on a tag-less, flow-assisted method of purifying, distinguishing and sorting MSCs. The protocol entails a suspension of cells in a transport fluid being injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min) sorted cells are collected. The protocol has been applied to the improvement of MSC isolation, with a specific view to reducing cell manipulation operations, keeping instrumental simplicity and increasing analytical information for cell characterization. Applications such as MSC purification from epithelial contaminants, MSC characterization from various human sources and sorting of MSC subpopulations with high differentiation potential are described. The low cost, full biocompatibility and scale-up potential of the protocol presented could make the procedure attractive for stem cell selection.
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Separação Celular/métodos , Análise de Injeção de Fluxo/métodos , Células-Tronco/citologia , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , HumanosRESUMO
Thrombocytopenia with absent radii (TAR) syndrome is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. Its expression includes skeletal, hematologic, and cardiac system abnormalities. According to some authors, the association of disparate skeletal and hematologic abnormalities is related to simultaneous development of the heart, radii, and megakaryocytes at 6 to 8 weeks' gestation. Thrombocytopenia that generally presents at birth or during the neonatal period can also occur subsequently. Data as to the physiopathology of TAR syndrome are scanty because of the low frequency of the disease and frequent unavailability of samples for bone marrow. The few studies on colony formation suggest that thrombocytopenia could be due to a decreased response to thrombopoietin that affects both proliferation and differentiation. The genetic basis of this syndrome remains unclear because c-mpl gene mutations are not a likely cause of thrombocytopenia and they are also frequent in the normal population. This is also the case for the mutations to the multifunctional growth factor transforming growth factor (TGF)-beta2 gene as described in our laboratory. Finally, the deletion on chromosome 1q21.1 described by Klopocki and colleagues is not considered sufficient to determine the TAR syndrome phenotype. We have reported that bone marrow adherent stromal cells from patients with TAR syndrome do not express CD105 antigen (expressed in normal mesenchymal cells), part of the receptor complex for TGF-beta1 and TGF-beta3. Thus, the hypothesis that the clinical phenotype of TAR could derive from damage to a common osteo/chondrogenic and hemopoietic progenitor warrants further study.
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Anormalidades Múltiplas/metabolismo , Doenças Genéticas Inatas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Trombocitopenia/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Antígenos CD/genética , Antígenos CD/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/metabolismo , Endoglina , Regulação da Expressão Gênica/genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Síndrome , Trombocitopenia/genética , Trombocitopenia/patologia , Trombopoetina/genética , Trombopoetina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at approximately 60% VO2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1alpha mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.
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Vasos Coronários/fisiologia , Miocárdio/metabolismo , Neovascularização Fisiológica/fisiologia , Condicionamento Físico Animal , Animais , Biomarcadores/metabolismo , Vasos Coronários/ultraestrutura , Ventrículos do Coração/metabolismo , Ventrículos do Coração/ultraestrutura , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC). RESULTS: The recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells. CONCLUSION: The current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.
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Âmnio/citologia , Diferenciação Celular , Endotélio Vascular/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Adipócitos/citologia , Separação Celular , Células Cultivadas , Condrócitos/citologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Células Musculares/citologia , Osteoblastos/citologia , GravidezRESUMO
BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potential source of MSCs instead of bone marrow (BM). METHODS: Flow cytometric analysis showed that DP-MSCs and BM-MSCs were equally SH2, SH3, SH4, CD29 and CD 166 positive. The in vitro proliferative kinetics of MSCs were measured by 3H-thymidine incorporation uptake. The immunosuppressive function of MSCs was then tested by coculturing PHA-stimulated allogeneic T cells with or without MSCs for 3 days. RESULTS: BM-MSCs could be differentiated in vitro into osteogenic, chondrogenic and adipogenic lineages. DP-MSCs showed osteogenic and adipocytic differentiation, but did not differentiate into chondrocytes. Although DP-MSCs grow rapidly in vitro between day 3 and day 8 of culture and then decrease their proliferation by day 15, BM-MSCs have a stable and continuous proliferation over the same period of time. The addition of DP-MSCs or BM-MSCs resulted in 91 +/- 4% and 75 +/- 3% inhibition of T cell response, respectively, assessed by a 3H-thymidine assay. CONCLUSIONS: Dental pulp is an easily accessible and efficient source of MSCs, with different kinetics and differentiation potentialities from MSCs as isolated from the bone marrow. The rapid proliferative capacity together with the immunoregulatory characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation.