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To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κß was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.
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Vitronectina , Gelatina de Wharton , Humanos , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Hipoxia/metabolismo , Cordón Umbilical , Vitronectina/metabolismo , Gelatina de Wharton/metabolismo , Células MadreRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs. METHODS: WJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array. RESULTS: When cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G0/G1 phase with a corresponding reduction in the percentage of cells in G2/M phase. Blebbistatin-induced G0/G1 arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle. CONCLUSIONS: Our study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G0/G1 arrest and alteration in the expression levels of certain key cell cycle-related genes.
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Células Madre Mesenquimatosas/citología , Miosina Tipo II/metabolismo , Gelatina de Wharton/citología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Fase G1/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Miosina Tipo II/antagonistas & inhibidores , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Cordón Umbilical/citologíaRESUMEN
Mesenchymal stromal cells (MSCs) show promise in cell-based transplantations and regenerative medicine applications. MSCs from Wharton's jelly (WJ) of umbilical cord can be easily harvested and exhibit greater proliferative activity than bone marrow MSCs. It is important to develop a practical cryopreservation technique to effectively store umbilical cord for potential future applications. Successful cryopreservation would allow access to umbilical cord from the same donor for repeated WJ MSC-based transplantations. For therapeutic applications, one should be able to obtain clinically-relevant quality and quantity of MSCs from cryopreserved tissues. In this study, we optimised a serum-free formulation of 10% dimethyl sulfoxide (DMSO) and 0.2M sucrose for cryopreservation of umbilical cord tissue. Slow freezing and rapid thawing were adopted. MSCs harvested from WJ of cryopreserved umbilical cord could undergo robust expansion, differentiate to mesodermal lineages and express MSC-characteristic surface antigens. The cumulative cell yield, however, was less compared to corresponding fresh cord tissue.
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Criopreservación/métodos , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Gelatina de Wharton/citología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Crioprotectores/metabolismo , Dimetilsulfóxido/metabolismo , Femenino , Humanos , Recién Nacido , Masculino , Células Madre Mesenquimatosas/metabolismo , Sacarosa/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) are known to facilitate angiogenesis and promote neo-vascularization via secretion of trophic factors. Here, we explored the molecular mechanism adopted by ADAMTS13 in modulating the expression of some key angiogenic markers in human umbilical cord-derived MSCs under serum-deprivation stress. Wharton's jelly MSCs (WJ-MSCs) were isolated from the perivascular region of human umbilical cords by explant culture. ADAMTS13 was upregulated at both mRNA and protein levels in WJ-MSCs under serum-deprivation stress. Correspondingly, some key angiogenic markers were also seen to be upregulated. By screening signaling pathways, p38 and JNK pathways were identified as negative and positive regulators for expression of ADAMTS13, and the angiogenic markers, respectively. Our results also indicated the Notch pathway and p53 as other probable partners modulating the expression of ADAMTS13 and the angiogenic markers. Knockdown of ADAMTS13 using siRNA led to reversal in the expression of these angiogenic markers. Further, ADAMTS13 was shown to act via the EphrinB2/EphB4 axis followed by ERK signaling to control expression of the angiogenic markers. Interestingly, stronger expression levels were noted for ADAMTS13, VEGF and PDGF under a more stringent nutrient stress condition. Thus, we highlight a novel role of ADAMTS13 in WJ-MSCs under nutrient stress condition.
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Células Madre Mesenquimatosas , Gelatina de Wharton , Humanos , Efrinas/metabolismo , Cordón Umbilical , Transducción de Señal , Diferenciación Celular , Células Cultivadas , Proliferación Celular , Proteína ADAMTS13/metabolismoRESUMEN
Mesenchymal stem cells (MSCs), also referred to as "medicinal signaling cells," have gained prominence as candidates for cell-based therapy and in clinical trials owing to their regenerative and therapeutic properties. Here, we present a protocol for isolating MSCs from the decidua basalis layer of human placenta using an explant culture approach. We describe steps for collecting, disinfecting, and plating placental tissue. We then detail procedures for characterizing the isolated MSCs through flow cytometry and in vitro differentiation.
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Células Madre Mesenquimatosas , Placenta , Humanos , Embarazo , Femenino , Decidua , Citometría de Flujo , Diferenciación CelularRESUMEN
BACKGROUND AIMS: Because of their multilineage differentiation capacity, immunomodulatory role and homing ability, mesenchymal stromal cells (MSC) are emerging as a new therapeutic strategy for treating a variety of disorders. Although bone marrow (BM) is the best characterized source of MSC, Wharton's jelly (WJ) of the umbilical cord holds great promise as an alternative. As delivery direct to the site of injury is not always feasible, efficient homing of MSC to the site of injury is critical for inducing tissue repair and regeneration. MSC express a wide variety of growth factors, chemokines and receptors that are important for cell migration, homing and re-establishment of blood supply for recovery of damaged tissues. METHODS: Detailed chemokine and receptor gene expression profiles of WJ MSC were established, and subsequently compared with those of BM-derived MSC using a polymerase chain reaction (PCR) array. Secretion of growth factors was analyzed and evaluated using culture supernatant from WJ and BM MSC. RESULTS: Our results revealed a differential expression pattern of the chemokines and their receptors between WJ- and BM-derived MSC. Several Glutamic acid-Leucine-Arginine; ELR-positive CXC chemokine genes and secretion of growth factors, which promote angiogenesis, were found to be up-regulated in WJ MSC. CONCLUSIONS: To understand better the localization and mechanism of tissue repair by transplanted WJ MSC, we attempted chemokine and their receptor transcription profiling, followed by analysis of growth factors secreted by WJ MSC, and compared them against those of BM MSC. The data suggest that MSC from different sources can be explored for distinct therapeutic roles.
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Células de la Médula Ósea/patología , Quimiocinas CXC/genética , Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Receptores de Quimiocina/genética , Gelatina de Wharton/metabolismo , Movimiento Celular/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Trasplante de Células Madre Mesenquimatosas , Neovascularización Fisiológica/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Nicho de Células MadreRESUMEN
BACKGROUND: Due to their immunomodulatory and trophic support functions, mesenchymal stem cells (MSCs) are promising in the field of cell-based regenerative medicine. However, MSC survival post-transplantation is challenged by various microenvironment stress factors. Here, we investigated the role of vitronectin (VTN) in the survival strategy of MSCs under serum deprivation stress condition. METHODS: Proliferation kinetics and cell adhesion of MSCs under serum deprivation were determined from population doublings and cell-matrix de-adhesion studies, respectively. mRNA and protein expression levels of VTN were confirmed by qRT-PCR and Western blotting, respectively. Immunofluorescence technique revealed distribution of VTN under serum deprivation stress. siRNA and inhibitor-based studies were performed to confirm the role and regulation of VTN. Apoptosis and cell cycle status of MSCs were assessed using flow cytometric analysis. RESULTS: Subjecting MSCs to serum deprivation led to significant increase in cell spread area and cell-matrix adhesion. An upregulation of VTN expression was noted with an arrest in G0/G1 phase of cell cycle and no appreciable apoptotic change. Pro-survival PI3kinase pathway inhibition led to further increase in VTN expression with no apoptotic change. siRNA-mediated inhibition of VTN resulted in reversal in G0/G1 cell cycle arrest and a marked increase in apoptosis, suggesting a role of VTN in preventing serum deprivation-induced apoptotic cell death. In addition, p65 knockdown resulted in downregulation of VTN establishing an association between NF-κß pathway and VTN. CONCLUSIONS: VTN was identified as a survival factor in providing protection from serum deprivation-induced apoptosis in MSCs.
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Células Madre Mesenquimatosas , Apoptosis , Adhesión Celular , Ciclo Celular , Células Cultivadas , Vitronectina/genéticaRESUMEN
Mesenchymal stromal cells (MSCs) are clinically beneficial for regenerative treatment of chronic inflammation and autoimmune disorders. However, to attain maximum efficacy from the transplanted MSCs, evaluation of its interaction with the microenvironment, becomes critical. Fever being an important hallmark of inflammation, we investigated the effect of febrile temperature stress on adhesion and migration of umbilical cord-derived MSCs. 40 °C-exposure altered cellular morphology with significant cell flattening, delayed cell-matrix de-adhesion response and slower migration of MSCs, accompanied by suppressed directionality ratio and cell trajectory. Corresponding to the observed changes, mRNA expression of extracellular matrix genes like COLs and VTN were upregulated, while matrix metalloproteinase MMP-1, showed a significant downregulation. NF-κß pathway inhibition at 40 °C, led to reversal of gene expression pattern, cell spreading, de-adhesion dynamics and migration rate. Independent knockdown of p65 and p53 at 40 °C indicated inhibitory role of p65/p53/p21 axis in regulation of MMP-1 expression. P21 inhibits JNK activity, and JNK pathway inhibition at 40 °C resulted in further downregulation of MMP-1. Hence, our study provides the first evidence of cell migration getting adversely affected in MSCs under elevated temperature stress due to an inverse relationship between p65/p53/p21 and MMP1 with a possible involvement of the JNK pathway.
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Adhesión Celular , Movimiento Celular , Fiebre/metabolismo , Células Madre Mesenquimatosas/fisiología , FN-kappa B/metabolismo , Transducción de Señal , Estrés Fisiológico , Biomarcadores , Adhesión Celular/genética , Movimiento Celular/genética , Matriz Extracelular/metabolismo , Fiebre/etiología , Expresión Génica , Humanos , Modelos BiológicosRESUMEN
Mesenchymal stem cells (MSCs) are successful for their therapeutic application in immune and inflammatory contexts due to their anti-inflammatory, trophic, and immunomodulatory roles. However, though MSCs have the potential to provide regenerative treatment toward a wide range of devastating diseases, massive cell death of transplanted MSCs remains an obstacle to overcome. The relation between MSCs and inflammation is multifactorial and challenging to comprehend. Fever is a critical component of the inflamed microenvironment. Also, the choice of MSC source could be critical in determining the fate of transplanted cells under stress conditions. Here we investigated the thermosensitivity of Wharton's jelly MSCs (WJ-MSCs) to elevated temperature in the physiological fever range. We explored the effect of febrile range temperature on morphology, viability, proliferation kinetics, and cell cycle status of WJ-MSCs. WJ-MSCs adopted a flattened morphology at 40°C, and our data from proliferation kinetics study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that WJ-MSCs had reduced proliferation and viability at 40°C compared with control cultures. There was also a G0/G1 cell cycle arrest, which was further confirmed by messenger RNA (mRNA) levels of genes specific for different stages of cell cycle. On evaluating p53 status, we observed an increase in p53 protein expression and its nuclear localization in WJ-MSCs exposed to 40°C. Its downstream effector p21 too was upregulated. Moreover, this temperature-induced p53 induction was inhibited on exposure to 40°C in the presence of NF-κB pathway inhibitor, pyrrolidinedithiocarbamate (PDTC) or endonuclease-prepared small interfering RNA (esiRNA) targeting p65. Febrile temperature exposure did not affect the senescence status of WJ-MSCs. The MSC-specific surface antigen profile at 40°C was similar to control WJ-MSCs. Our findings suggest that under febrile temperature stress conditions, WJ-MSCs exhibit G0/G1 cell cycle arrest and reduction in viable cell count, while retaining their basic characteristics, with an underlying interplay of p53 and NF-κB pathway.
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Respuesta al Choque Térmico , Células Madre Mesenquimatosas/metabolismo , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Cordón Umbilical/citología , División Celular , Células Cultivadas , Senescencia Celular , Humanos , Células Madre Mesenquimatosas/citología , FN-kappa B/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mesenchymal stem cells (MSCs) are multipotent precursor cells which have been isolated from different vascularized tissue sources. Due to their paracrine function of secreting trophic and immunomodulatory molecules, MSCs are successfully used in cell-based transplantations and provide an alternative medical paradigm for treating a variety of devastating disorders. Umbilical cord is a medical waste with a large, readily available donor pool. Since umbilical cord is a fetal tissue, MSCs derived from it are considered more primitive with proliferative and differentiation advantages over adult MSCs. We define here a simple, efficient, and reproducible protocol to isolate MSCs from WJ of human umbilical cord using a nonenzymatic procedure. Under the optimized culture conditions, the WJ-MSCs undergo robust proliferation, can be expanded up to 15-20 passages and express the characteristic MSC surface antigens. They can be differentiated into mesodermal lineages in vitro.
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Células Madre/citología , Cordón Umbilical/citología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Gelatina de Wharton/citologíaRESUMEN
We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic beta-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet "progenitor" cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse beta-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from beta-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal "progenitors" might not represent the best possible source for generation of physiologically competent beta-cells for treatment of diabetes.
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Células Epiteliales/citología , Células Secretoras de Insulina/citología , Mesodermo/citología , Animales , Sitios de Ligazón Microbiológica/genética , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Células Cultivadas , Expresión Génica , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Secretoras de Insulina/metabolismo , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Transgénicos , RatasRESUMEN
Mesenchymal stem cells (MSCs) are currently considered as 'medicinal signaling cells' and a promising resource in regard to cell-based regenerative therapy. Umbilical cord is a human term perinatal tissue which is easily attainable, and a promising source of stem cells with no associated ethical concerns. MSCs have been isolated from different regions of the umbilical cord and Wharton's jelly (WJ) is the gelatinous matrix that surrounds and provides protection to the umbilical cord blood vessels. Being more primitive, MSCs from human umbilical cord exhibit greater proliferative capacity and immunosuppressive ability as compared to adult stem cells which gives them a therapeutic advantage. To meet the requirements for cell therapy, it is important to generate MSCs at a clinical scale by following steps which are not time consuming or labor intensive. Here we present a simple, efficient protocol for isolation of MSCs from WJ of human umbilical cord by explant culture method which is reproducible and also, cost effective.
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We report the synthesis and dynamical behavior of a carbon dot (CD) with near 100% photoluminescence quantum yield in water for a very large pH range (1-12). This CD exhibits a rotational correlational time of only â¼130 ps, signifying the whole CD is not exhibiting photoluminescence. Unlike most carbon-based nanoparticles (which act as a quencher of fluorescence), this CD could act as a donor, and the Förster model could account for the experimental observables for the resonance energy transfer (RET) experiment quite well. Based on two dynamical measurements, it could be shown that the fluorescing moiety is located inside the core of the CD. Importantly, for this CD, RET experiments could be performed with a very low concentration (500 nM) of the acceptor. This kind of electrostatics-driven RET at very low concentration is quite important in bioimaging. This ultrabright CD is nontoxic and useful for bioimaging in mesenchymal stem cells.
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The efficacy of mesenchymal stem cell (MSC) therapy is currently limited by low retention and poor survival of transplanted cells as demonstrated by clinical studies. This is mainly due to the harsh microenvironment created by oxygen and nutrient deprivation and inflammation at the injured sites. The choice of MSC source could be critical in determining fate and cellular function of MSCs under stress. Our objective here was to investigate the influence of ischemia-like stress on Wharton's jelly MSCs (WJ-MSCs) from human umbilical cord to assess their therapeutic relevance in ischemic diseases. We simulated conditions of ischemia in vitro by culturing WJ-MSCs in 2% oxygen in serum deprived and low glucose medium. Under these conditions, WJ-MSCs retained viable population of greater than 80%. They expressed the characteristic MSC surface antigens at levels comparable to the control WJ-MSCs and were negative for the expression of costimulatory molecules. An upregulation of many ECM and adhesion molecules and growth and angiogenic factors contributing to wound healing and regeneration was noted in the ischemic WJ-MSC population by a PCR array. Their migration ability, however, got impaired. Our findings provide evidence that WJ-MSCs might be therapeutically beneficial and potent in healing wounds under ischemic conditions.
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Transplantation of pancreatic islets can provide long-lasting insulin independence for diabetic patients, but the current islet supply is limited. Here we describe a new in vitro system that utilizes adult human pancreatic islet-enriched fractions to generate hormone-producing cells over 3-4 weeks of culture. By labeling proliferating cells with a retrovirus-expressing green fluorescent protein, we show that in this system hormone-producing cells are generated de novo. These hormone-producing cells aggregate to form islet-like cell clusters. The cell clusters, when tested in vitro, release insulin in response to glucose and other secretagogues. After transplantation into immunodeficient, nondiabetic mice, the islet-like cell clusters survive and release human insulin. We propose that this system will be useful as an experimental tool for investigating mechanisms for generating new islet cells from the postnatal pancreas, and for designing strategies to generate physiologically competent pancreatic islet cells ex vivo.
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Técnicas de Cultivo de Célula , Insulina/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Animales , Péptido C/metabolismo , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Tipo 1/cirugía , Glucosa/farmacología , Proteínas Fluorescentes Verdes , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/ultraestructura , Ratones , Ratones Endogámicos NOD , Ratones SCID , FenotipoRESUMEN
It is the promise of regeneration and therapeutic applications that has sparked an interest in mesenchymal stem cells (MSCs). Following infusion, MSCs migrate to sites of injury or inflammation by virtue of their homing property. To exert optimal clinical benefits, systemically delivered MSCs need to migrate efficiently and in adequate numbers to pathological areas in vivo. However, underlying molecular mechanisms responsible for MSC migration are still not well understood. The Wharton's jelly (WJ) of the umbilical cord is an attractive source of MSCs for stem cell therapy because of its abundant availability and painless collection. In this study, we attempted to identify the role of nonmuscle myosin II (NMII), if any, in the migration of WJ-derived MSCs (WJ-MSCs). Expression of NMII isoforms, NMIIA, and NMIIB was observed both at RNA and protein levels in WJ-MSCs. Inhibition of NMII or its regulator ROCK, by pharmacological inhibitors, resulted in significant reduction in the migration of WJ-MSCs as confirmed by the scratch migration assay and time-lapse microscopy. Next, trying to dissect the role of each NMII isoform in migration of WJ-MSCs, we found that siRNA-mediated downregulation of NMIIA, but not NMIIB expression, led to cells failing to retract their trailing edge and losing cell-cell cohesiveness, while exhibiting a nondirectional migratory pathway. Migration, moreover, is also dependent on optimal affinity adhesion, which would allow rapid attachment and release of cells and, hence, can be influenced by extracellular matrix (ECM) and adhesion molecules. We demonstrated that inhibition of NMII and more specifically NMIIA resulted in increased gene expression of ECM and adhesion molecules, which possibly led to stronger adhesions and, hence, decreased migration. Therefore, these data suggest that NMII acts as a regulator of cell migration and adhesion in WJ-MSCs.
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Diferenciación Celular/fisiología , Movimiento Celular , Células Madre Mesenquimatosas/citología , Miosina Tipo II/metabolismo , Cordón Umbilical/citología , Gelatina de Wharton/citología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Regeneración/fisiología , Trasplante de Células Madre/métodosRESUMEN
INTRODUCTION: Mesenchymal stromal/stem cells (MSCs) for clinical use have largely been isolated from the bone marrow, although isolation of these cells from many different adult and fetal tissues has been reported as well. One such source of MSCs is the Whartons Jelly (WJ) of the umbilical cord, as it provides an inexhaustible source of stem cells for potential therapeutic use. Isolation of MSCs from the umbilical cord also presents little, if any, ethical concerns, and the process of obtaining the cord tissue is relatively simple with appropriate consent from the donor. However, a great majority of studies rely on the use of bovine serum containing medium for isolation and expansion of these cells, and porcine derived trypsin for dissociating the cells during passages, which may pose potential risks for using these cells in clinical applications. It is therefore of high priority to develop a robust production process by optimizing culture variables to efficiently and consistently generate MSCs that retain desired regenerative and differentiation properties while minimizing risk of disease transmission. METHODS: We have established a complete xeno-free, serum-free culture condition for isolation, expansion and characterization of WJ-MSCs, to eliminate the use of animal components right from initiation of explant culture to clinical scale expansion and cryopreservation. Growth kinetics, in vitro differentiation capacities, immunosuppressive potential and immunophenotypic characterization of the cells expanded in serum-free media have been compared against those cultured under standard fetal bovine serum (FBS) containing medium. We have also compared the colony-forming frequency and genomic stability of the large scale expanded cells. Secretome analysis was performed to compare the angiogenic cytokines and functional angiogenic potency was proved by Matrigel assays. RESULTS: Results presented in this report identify one such serum-free, xeno-free medium for WJ expansion. Cells cultured in serum-free, xeno-free medium exhibit superior growth kinetics and functional angiogenesis, alongside other MSC characteristics. CONCLUSIONS: We report here that WJ-MSCs cultured and expanded in Mesencult XF, SF Medium retain all necessary characteristics attributed to MSC for potential therapeutic use.
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Técnicas de Cultivo de Célula , Células Madre Mesenquimatosas/citología , Gelatina de Wharton/citología , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Senescencia Celular , Medios de Cultivo , Medio de Cultivo Libre de Suero , Inestabilidad Genómica , Humanos , XenobióticosRESUMEN
Mesenchymal stem cells (MSCs) have become an attractive tool for tissue engineering and targets in clinical transplantation due to their regeneration potential and immuno-suppressive capacity. Although MSCs derived from bone marrow are the most widely used, their harvest requires an invasive procedure. The umbilical cord, which is discarded at birth, can provide an inexhaustible source of stem cells for therapy. The Wharton's jelly-derived MSCs (WJ-MSCs), from the umbilical cord, have been shown to have faster proliferation rates and greater expansion capability compared with adult MSCs. The standard isolation and in vitro culture protocol for WJ-MSCs utilizes fetal bovine serum (FBS) or calf serum as a nutrient supplement. However, FBS raises potential safety concerns such as transmission of viral/prion disease and may initiate xenogeneic immune reactions against bovine antigens. Therefore, for therapeutic applications, there is an urgent requirement to establish an alternative nutrient supplement which would favor cell proliferation, retain MSC characteristics, and prove safe in human subjects. In the present study, we isolated and expanded WJ-MSCs in 5% pooled, allogeneic human serum (HS) supplemented with 2 ng/mL of basic fibroblast growth factor. For cell dissociation, porcine trypsin was replaced with TrypLE, a recombinant enzyme, and a protease-free protocol was adapted for isolation of MSCs from WJ. We determined their growth kinetics, in vitro differentiation potential, surface marker expression, and colony-forming unit potential and compared them against standard WJ-MSC cultures expanded in 10% FBS. All these parameters matched quite well between the two MSC populations. To test whether there is any alteration in gene expression on switching from FBS to HS, we analyzed a panel of stem cell and early lineage markers using Taqman® low density array. No significant deviation in gene expression was observed between the two populations. Thus we established an efficient, complete xeno-free protocol for propagation of human WJ-MSCs.
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Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.
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Proliferación Celular , Células Madre Mesenquimatosas/citología , Oxígeno/metabolismo , Cordón Umbilical/citología , Biomarcadores , Diferenciación Celular , Hipoxia de la Célula , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Cariotipificación , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic fibroblast growth factor (bFGF) supplementation in the growth medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of 15 days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.