RESUMO
BACKGROUND: Inflamm-aging is associated with the rate of aging and is significantly related to diseases such as Alzheimer's disease, Parkinson's disease, atherosclerosis, heart disease, and age-related degenerative diseases such as type II diabetes and osteoporosis. This study aims to evaluate the safety and efficiency of autologous adipose tissue-derived mesenchymal stem cell (AD-MSC) transplantation in aging-related low-grade inflammation patients. METHODS: This study is a single-group, open-label, phase I clinical trial in which patients treated with 2 infusions (100 million cells i.v) of autologous AD-MSCs were initially evaluated in 12 inflamm-aging patients who concurrently had highly proinflammatory cytokines and 2 of the following 3 diseases: diabetes, dyslipidemia, and obesity. The treatment effects were evaluated based on plasma cytokines. RESULTS: During the study's follow-up period, no adverse effects were observed in AD-MSC injection patients. Compared to baseline (D-44), the inflammatory cytokines IL-1α, IL-1ß, IL-8, IL-6, and TNF-α were significantly reduced after 180 days (D180) of MSC infusion. IL-4/IL-10 at 90 days (D90) and IL-2/IL-10 at D180 increased, reversing the imbalance between proinflammatory and inflammatory ratios in the patients. CONCLUSION: AD-MSCs represent a potential intervention to prevent age-related inflammation in patients. TRIAL REGISTRATION: ClinicalTrials.gov number is NCT05827757, first registered on 13th Oct 2020.
Assuntos
Tecido Adiposo , Citocinas , Inflamação , Transplante de Células-Tronco Mesenquimais , Transplante Autólogo , Humanos , Feminino , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Pessoa de Meia-Idade , Citocinas/sangue , Inflamação/sangue , Resultado do Tratamento , Idoso , Envelhecimento , Mediadores da Inflamação/sangue , Fatores de Tempo , Fatores Etários , AdultoRESUMO
The transplantation of mesenchymal stem cell (MSC) sheets derived from human umbilical cords (hUCs) was investigated in this study as a potential application in treating myocardial infarction (MI). Two groups of hUC-MSC sheets were formed by populating LunaGelTM, which are 3D scaffolds of photo-crosslinkable gelatin-based hydrogel with two different cell densities. An MI model was created by ligating the left anterior descending coronary artery of healthy BALB/c mice. After two weeks, the cell sheets were applied directly to the MI area and the efficacy of the treatment was evaluated over the next two weeks by monitoring the mice's weight, evaluating the left ventricle ejection fraction, and assessing the histology of the heart tissue at the end of the experiment. Higher cell density showed significantly greater efficiency in MI mice treatment in terms of weight gain and the recovery of ejection fraction. The heart tissue of the groups receiving cell sheets showed human-CD44-positive staining and reduced fibrosis and apoptosis. In conclusion, the hUC-MSC sheets ameliorated heart MI injury in mice and the efficacy of the cell sheets improved as the number of cells increased.
RESUMO
Hepatic stellate cell (HSC) activation via the autophagy pathway is a critical factor in liver fibrogenesis. This study tests the hypothesis that chloroquine (CQ) treatment can prevent autophagy and HSC activation in vitro and in vivo in bile-duct-ligated (BDL) mice. Sham-operated and BDL mice were treated with either PBS or CQ in two 60 mg/kg doses the day (D) before and after surgery. On day 2 (2D), HSCs were isolated, and their biological activities were evaluated by measuring intracellular lipid content, α-sma/collagen, and expression of autophagy lc3, sqstm1/p62 markers. The treatment efficacy on liver function was evaluated with serum albumin, transaminases (AST/ALT), and hepatic histology. Primary HSCs were treated in vitro for 24 h with CQ at 0, 2.5, 5, 10, 30, and 50 µM. Autophagy and HSC activation were assessed after 2D of treatment. CQ treatment improved serum AST/ALT, albumin, and bile duct proliferation in 2D BDL mice. This is associated with a suppression of HSC activation, shown by higher HSC lipid content and collagen I staining, along with the blockage of HSC autophagy indicated by an increase in p62 level and reduction in lc3 staining. CQ 5 µM inhibited autophagy in primary HSCs in vitro by increasing p62 and lc3 accumulation, thereby suppressing their in vitro activation. The autophagy inhibitor CQ reduced HSC activation in vitro and in vivo. CQ improved liver function and reduced liver injury in BDL mice.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Camundongos , Animais , Cirrose Hepática/metabolismo , Células Estreladas do Fígado/metabolismo , Bile/metabolismo , Cloroquina/farmacologia , Ductos Biliares/metabolismo , Colágeno/metabolismo , Autofagia , LipídeosRESUMO
Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the main characteristics of HCC and ncRNAs are described. The involvement of ncRNAs in HCC is then presented over five sections: (a) miRNAs, (b) lncRNAs, (c) circRNAs, (d) ncRNAs and drug resistance and (e) ncRNAs and liver fibrosis. Overall, this work provides the reader with the most recent state-of-the-art approaches in this field, highlighting key trends and opportunities for more advanced and efficacious HCC treatments.
RESUMO
Scaffold-based culture is necessary for hepatic stellate cells (HSCs) because HSCs are promptly autoactivated under plastic conditions. Our research aims to investigate the potential and role of fibrin scaffold in reducing autoactivation, maintaining cell function, and extending the in vitro culture time of primary HSCs. HSCs were isolated from BALB/c mice and cultured on the surface of plastic, Matrigel, and fibrin gel. HSC's characteristics, including recovery, morphology, proliferation, lipid droplet (LD) storage, and activation were evaluated. Cell recovery was 86%, 80%, and 60% in fibrin, Matrigel, and plastic, respectively (P < 0.05). HSCs cultured on a plastic dish were autoactivated until day 7 with high proliferation, loss of cytoplasmic LD lipid droplets, and increased expression of activation markers, including alpha-smooth muscle actin (α-sma) and collagen type I. In contrast, these phenomena were reduced in Matrigel and fibrin-based cultures (P < 0.05). HSC culture in fibrin scaffold was associated with altered expression of cell adhesion molecules, including increased E-cadherin and inhibited N-cadherin. HSCs were more stellate-like in morphology in fibrin than in the Matrigel scaffold. Interestingly, fibrin-scaffold-embedded culture was able to maintain HSC quiescent state for up to 14 days in vitro. Fibrin gel could provide a potential scaffold for primary HSC culture while preserving cell function and extending primary HSC in vitro culture time.NEW & NOTEWORTHY Fibrin gel is appropriate for maintaining quiescence characteristics in primary culture of mouse hepatic stellate cells. Embedded culture of hepatic stellate cells in fibrin gel simulates in vivo cell morphology. Stiffness and adhesion molecules of fibrin gel play a crucial role in the hepatic stellate cell's primary culture.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Camundongos , Animais , Células Estreladas do Fígado/metabolismo , Células Cultivadas , Cirrose Hepática/metabolismo , Colágeno Tipo I/metabolismoRESUMO
Despite the progress made in the diagnoses and therapy of gastrointestinal cancers, these diseases are still plagued by a high mortality. Thus, novel therapeutic approaches are urgently required. In this regard, small interfering RNA (siRNA), double-stranded RNA molecules able to specifically target the mRNA of pathological genes, have the potential to be of therapeutic value. To be effective in the human body, siRNAs need to be protected against degradation. Additionally, they need to target the tumor, leaving the normal tissue untouched in an effort to preserve organ function. To accomplish these tasks, siRNAs have been formulated with smart delivery systems such has polymers and lipids. While siRNA protection is not particularly difficult to achieve, their targeting of tumor cells remains problematic. Here, after introducing the general features of gastrointestinal cancers, we describe siRNA characteristics together with representative delivery systems developed for gastrointestinal cancers. Afterward, we present a selection of research papers employing siRNAs against upper- and lower- gastrointestinal cancers. For the liver, we also consider papers using siRNAs to combat liver cirrhosis, a relevant risk factor for liver cancer development. Finally, we present a brief description of clinical trials employing siRNAs for gastrointestinal cancers.
RESUMO
BACKGROUND: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. METHODS: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. RESULTS: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. CONCLUSION: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.
RESUMO
BACKGROUND AND AIM: Hepatic stellate cells (HSCs) activation, a critical event in liver fibrosis, has been recently shown to be related to autophagy. Determine whether chloroquine (CQ) could affect (i) the activation of HSC in vivo and (ii) the hepatic damage in a mice acute liver injury model. METHODS: The acute liver injury was induced in BALB/c mice by carbon tetrachloride (CCl4 group); 24 h before and after CCl4 administration animals were treated by CQ (CCl4 + CQ group). As control, mice treated by olive oil were considered. After 48 h from CCl4 /olive oil administration, blood samples, liver tissues, and HSCs were harvested for analysis. RESULTS: In vivo, CQ attenuates CCl4 -induced acute liver damage as evidenced by (i) the reduction of liver enlargement, (ii) the reduction of liver swelling and necrosis also supported by a certain decrease of circulating transaminases level, and (iii) the reduction of liver fibrosis evaluated by collagen deposition and α-sma protein expression. In HSCs isolated from CQ treated group, we observed the inhibition of autophagy proved by the increase in p62 protein and the decrease of lc3 protein. In addition, CQ reduced the expression of the HSCs activation markers α-sma/collagen-I and down-regulated the expression of the proliferative marker ki67. CONCLUSION: The autophagy attenuation exerted by CQ together with the reduction of the expression of the proliferation marker in HSCs can lessen the acute liver damage potentially opening the way to novel therapeutic approaches for hepatic fibrosis.
Assuntos
Autofagia , Cloroquina , Células Estreladas do Fígado , Animais , Autofagia/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Cloroquina/farmacologia , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Hepatocellular carcinoma (HCC) is the sixth most common type of tumor and the second leading cause of tumor-related death worldwide. Liver cirrhosis is the most important predisposing factor for HCC. Available therapeutic approaches are not very effective, especially for advanced HCC, which is the most common form of the disease at diagnosis. New therapeutic strategies are therefore urgently needed. The use of animal models represents a relevant tool for preclinical screening of new molecules/strategies against HCC. However, several issues, including animal husbandry, limit the use of current models (rodent/pig). One animal model that has attracted the attention of the scientific community in the last 15 years is the zebrafish. This freshwater fish has several attractive features, such as short reproductive time, limited space and cost requirements for husbandry, body transparency and the fact that embryos do not show immune response to transplanted cells. To date, two different types of zebrafish models for HCC have been developed: the transgenic zebrafish and the zebrafish xenograft models. Since transgenic zebrafish models for HCC have been described elsewhere, in this review, we focus on the description of zebrafish xenograft models that have been used in the last five years to test new molecules/strategies against HCC.
RESUMO
Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC's undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 µM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.
Assuntos
Células Estreladas do Fígado/citologia , Animais , Células Cultivadas , Meios de Cultura , Iohexol/análise , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,ß-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Galactose , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Camundongos , Polímeros , RNA Interferente PequenoRESUMO
INTRODUCTION AND OBJECTIVES: Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. MATERIALS AND METHODS: For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. RESULTS: CD90 mRNA expression was higher in HCC than in SNT for 8-fold (pâ¯<â¯0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (pâ¯<â¯0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. CONCLUSIONS: We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Doxorrubicina/uso terapêutico , Neoplasias Hepáticas/patologia , Antígenos Thy-1/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-IdadeRESUMO
Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.
Assuntos
Gotículas Lipídicas/metabolismo , Perilipina-2/metabolismo , Proteólise , Ubiquitinação , Acetilação , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Perilipina-2/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Achaete-scute homolog 1 is a lineage oncogene of high-grade pulmonary neuroendocrine tumors. Due to the relatively few studies investigating the epigenetic regulation of achaete-scute homolog 1 expression, we wanted to address whether DNA methylation of the achaete-scute homolog 1 CpG island is associated with clinicopathological features in pulmonary neuroendocrine tumors and to investigate its effect on the expression of this gene. Here, We performed multiplex immunohistochemistry (PerkinElmer, Waltham, MA, USA) to check for achaete-scute homolog 1 and Notch homolog 1 expression in 139 pulmonary neuroendocrine tumor samples. Quantitative measurements of achaete-scute homolog 1 CpG island methylation were conducted using the MassARRAY EpiTYPER (Sequenom, San Diego, CA, USA). The correlation between immunohistochemistry data, methylation data, and clinicopathological information was analyzed. Achaete-scute homolog 1 methylation levels were increased in pulmonary neuroendocrine tumors compared to those in normal controls (0.107 vs 0.061, p < 0.001), and among the achaete-scute homolog 1 CpG island, only CpG_6 and CpG_7.8 showed higher methylation levels in pulmonary neuroendocrine tumors (0.208 and 0.135, respectively) compared to those in normal lung tissues (0.072 and 0.087, respectively; p < 0.001). Moreover, the methylation level of CpG_6.7.8 was higher in patients with stage I pulmonary neuroendocrine tumors than in patients with stage II/III pulmonary neuroendocrine tumors (0.19 ± 0.16 vs 0.14 ± 0.07, p = 0.012). The hypermethylation of CpG_6.7.8 showed an inverse correlation with achaete-scute homolog 1 protein expression (r = -0.408, p = 0.007, Spearman test). Finally, we found that CpG_6.7.8 of the achaete-scute homolog 1 CpG island is frequently hypermethylated in early-stage pulmonary neuroendocrine tumors, and this aberrant hypermethylation is negatively correlated with achaete-scute homolog 1 expression in this tumor spectrum.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Metilação de DNA/genética , Neoplasias Pulmonares/genética , Tumores Neuroendócrinos/genética , Receptor Notch1/genética , Idoso , Linhagem da Célula/genética , Ilhas de CpG/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Tumores Neuroendócrinos/patologiaRESUMO
Because of self-renewal, strong proliferation in vitro, abundant sources for isolation, and a high differentiation capacity, mesenchymal stem cells are suggested to be potentially therapeutic for liver fibrosis/cirrhosis. In this study, we evaluated the treatment effects of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) on mouse liver cirrhosis induced by carbon tetrachloride. Portal and tail vein transplantations were examined to evaluate the effects of different injection routes on the liver cirrhosis model at 21 days after transplantation. BM-MSCs transplantation reduced aspartate aminotransferase/alanine aminotransferase levels at 21 days after injection. Furthermore, BM-MSCs induced positive changes in serum bilirubin and albumin and downregulated expression of integrins (600- to 7000-fold), transforming growth factor, and procollagen-α1 compared with the control group. Interestingly, both injection routes ameliorated inflammation and liver cirrhosis scores. All mice in treatment groups had reduced inflammation scores and no cirrhosis. In conclusion, transplantation of BM-MSCs via tail or portal veins ameliorates liver cirrhosis in mice. Notably, there were no differences in treatment effects between tail and portal vein administrations. In consideration of safety, we suggest transfusion of bone marrow-derived mesenchymal stem cells via a peripheral vein as a potential method for liver fibrosis treatment.
RESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are an attractive source of stem cells for clinical applications. These cells exhibit a multilineage differentiation potential and strong capacity for immune modulation. Thus, MSCs are widely used in cell therapy, tissue engineering, and immunotherapy. Because of important advantages, umbilical cord blood-derived MSCs (UCB-MSCs) have attracted interest for some time. However, the applications of UCB-MSCs are limited by the small number of recoverable UCB-MSCs and fetal bovine serum (FBS)-dependent expansion methods. Hence, this study aimed to establish a xenogenic and allogeneic supplement-free expansion protocol. METHODS: UCB was collected to prepare activated platelet-rich plasma (aPRP) and mononuclear cells (MNCs). aPRP was applied as a supplement in Iscove modified Dulbecco medium (IMDM) together with antibiotics. MNCs were cultured in complete IMDM with four concentrations of aPRP (2, 5, 7, or 10%) or 10% FBS as the control. The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion, the percentage of successfully isolated cells in the primary culture, surface marker expression, and in vitro differentiation potential following expansion. RESULTS: The results showed that primary cultures with complete medium containing 10% aPRP exhibited the highest success, whereas expansion in complete medium containing 5% aPRP was suitable. UCB-MSCs isolated using this protocol maintained their immunophenotypes, multilineage differentiation potential, and did not form tumors when injected at a high dose into athymic nude mice. CONCLUSION: This technique provides a method to obtain UCB-MSCs compliant with good manufacturing practices for clinical application.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Separação Celular/métodos , Separação Celular/normas , Sangue Fetal/citologia , Fidelidade a Diretrizes/normas , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Bovinos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Nus , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Fatores de TempoRESUMO
INTRODUCTION: Adipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency. METHODS: ADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage. RESULTS: PRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs. CONCLUSION: Pretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.
Assuntos
Tecido Adiposo/metabolismo , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Animais , Cartilagem Articular , Diferenciação Celular , Humanos , Imunofenotipagem , Camundongos , Plasma Rico em Plaquetas , Regeneração , Transplante de Células-TroncoRESUMO
BACKGROUND: Breast cancer stem cells with a CD44(+)CD24(-) phenotype are the origin of breast tumors. Strong CD44 expression in this population indicates its important role in maintaining the stem cell phenotype. Previous studies show that CD44 down-regulation causes CD44(+)CD24(-) breast cancer stem cells to differentiate into non-stem cells that are sensitive to antitumor drugs and lose many characteristics of the original cells. In this study, we determined tumor suppression in non-obese severe combined immunodeficiency mice using CD44 shRNA therapy combined with doxorubicin treatment. METHODS: Tumor-bearing non-obese severe combined immunodeficiency mice were established by injection of CD44(+)CD24(-) cells. To track CD44(+)CD24(-) cells, green fluorescence protein was stably transduced using a lentiviral vector prior to injection into mice. The amount of CD44 shRNA lentiviral vector used for transduction was based on CD44 down-regulation by in vitro CD44 shRNA transduction. Mice were treated with direct injection of CD44 shRNA lentiviral vector into tumors followed by doxorubicin administration after 48 hours. The effect was evaluated by changes in the size and weight of tumors compared with that of the control. RESULTS: The combination of CD44 down-regulation and doxorubicin strongly suppressed tumor growth with significant differences in tumor sizes and weights compared with that of CD44 down-regulation or doxorubicin treatment alone. In the combination of CD44 down-regulation and doxorubicin group, the tumor weight was significantly decreased by 4.38-fold compared with that of the control group. CONCLUSION: These results support a new strategy for breast cancer treatment by combining gene therapy with chemotherapy.
RESUMO
BACKGROUND: Breast cancer stem cells (BCSCs) are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs). METHODS: We isolated a breast cancer cell population (CD44+CD24- cells) from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. RESULTS: Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. CONCLUSIONS: Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.