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1.
Life Sci ; 238: 116970, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639395

RESUMO

AIMS: The reciprocity between stem cells and biomaterials is an essential topic in bone tissue engineering. Bone marrow mesenchymal stromal cells (BMSCs) have attracted considerable attention in regenerative medicine owing to their ability to self-renew and differentiate into osteoblasts, and more importantly, their immunomodulatory effects on the immune response. Ideal biomaterials should be osteo-inductive, environmentally sustainable, and economical. Our previous study showed that hydrolyzed fish collagen (HFC) can meet each of the above requirements. However, it is still unclear whether BMSCs maintain their immunomodulatory properties after osteogenic differentiation induced by HFC. MAIN METHODS: Non-commercial sources of BMSCs were isolated from Sprague-Dawley (SD) rats. Osteogenically differentiated BMSCs induced by HFC and undifferentiated BMSCs were co-cultured with PBMC or NR 8383 macrophages, respectively. Cell proliferation of PBMC was examined using a BrdU uptake assay. In addition, the IL-6, TGF-ß1, IL-10, PGE2, and nitric oxide levels were determined. The expressions of TSG-6 (TNF-stimulated gene 6) and IDO (indoleamine 2, 3-dioxygenase) genes were analyzed using qRT-PCR. KEY FINDINGS: The results revealed that HFC-induced BMSCs suppressed the proliferation of PBMC. The expression levels of anti-inflammatory mediators including IL-6, TGF-ß1, and PGE2 significantly increased after 48 h of co-culture. Moreover, the nitric oxide production increased during osteogenesis induced by HFC, whereas the level of TSG-6 and IDO remained unchanged after osteogenic differentiation. HFC-BMSCs inhibited the inflammatory mediator production (IL-1ß, TNF-α) in LPS-stimulated macrophages. SIGNIFICANCE: Taken together, these findings suggest that the immunomodulation ability is still retained in osteogenically differentiated BMSCs induced by HFC.


Assuntos
Diferenciação Celular/imunologia , Proliferação de Células , Colágeno/farmacologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/imunologia , Osteogênese/imunologia , Engenharia Tecidual , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Peixes , Hidrólise , Leucócitos Mononucleares/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
2.
Mater Sci Eng C Mater Biol Appl ; 103: 109833, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349499

RESUMO

The different lineage-specific biological properties of articular cartilage and subchondral bone present a great challenge in the construction of bi-lineage scaffolds for simultaneous osteochondral regeneration. To overcome this challenge, strontium incorporated calcium silicate (Sr-CS) ceramic was prepared for bi-lineage formation of scaffolds in this study. The positive result of Sr-CS in the regeneration of osteochondral defects was first proven by its improved effect on the osteogenesis and chondrogenesis induction of mesenchymal stem cells (MSCs). After that, scaffold-mediated macrophage polarization between classically activated inflammatory macrophages (termed M1Ф) and alternatively activated inflammatory macrophages (termed M2Ф) was assayed to investigate whether the incorporation of Sr into calcium silicate could alter host-to-scaffold immune response. Furthermore, the interactions between Sr-CS pretreated macrophages and MSCs differentiation were performed to prove the enhancement effect of suppressed inflammatory response on osteogenesis and chondrogenesis. In vivo transplantation showed that the Sr-CS scaffolds distinctly improved the regeneration of cartilage and subchondral bone, as compared to the calcium silicate scaffolds. On the one hand, the mechanism attributes to enhancement of strontium on the osteogenic and chondrogenic differentiation of MSCs. On the other hand, the reason can partially be attributed to suppressed synovial inflammatory response, which has improved effects on enhancement of osteogenesis and chondrogenesis. These findings suggest that monophasic Sr-CS scaffolds with a bi-lineage conducive property and an inflammatory response regulatory property represents a viable strategy for simultaneous regeneration of osteochondral defects.


Assuntos
Condrogênese/efeitos dos fármacos , Fatores Imunológicos , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Osteogênese/efeitos dos fármacos , Estrôncio , Tecidos Suporte/química , Animais , Compostos de Cálcio/química , Compostos de Cálcio/farmacocinética , Compostos de Cálcio/farmacologia , Células Cultivadas , Condrogênese/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacocinética , Fatores Imunológicos/farmacologia , Osteogênese/imunologia , Coelhos , Silicatos/química , Silicatos/farmacocinética , Silicatos/farmacologia , Estrôncio/química , Estrôncio/farmacocinética , Estrôncio/farmacologia
3.
J Immunol Res ; 2019: 7059680, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31321244

RESUMO

Background: Adipose-derived mesenchymal stem cells (ADMSCs) can promote healing and inhibit inflammation/immune response in local tissues, while the detailed mechanism remains unknown. Results: ADMSCs and peritoneal macrophages were collected from C57BL/6 mice. The culture medium (CM) from ADMSCs (24 hours cultured) was collected. The CM was added to the Mφ culture system with lipopolysaccharide (LPS) or IL-4/IL-13 or blank. And those Mφ cultures without adding CM were used as controls. A series of classification markers and signaling pathways for Mφ polarization were detected by using flow cytometry, RT-PCR, and western blotting. Furthermore, the cell viability of all the groups was detected by CCK8 assay. After CM induction in different groups, M1-Mφ markers and M2a-Mφ were decreased; however, M2b/c-Mφ markers increased. STAT3/SOCS3 and STAT6/IRF4 were suppressed in all 3 CM-treated groups. Moreover, the cell viability of all 3 groups which were induced by CM significantly increased as compared to that of the control groups without adding CM. Conclusion: ADMSCs can induce nonactivated macrophage and M1-Mφ into M2b/c-Mφ. Downregulation of the STAT3 and STAT6 pathway may involve in this process. This data shows that the anti-inflammatory role of ADMSC in local tissues may be partly due to their effect on Mφ to M2b/c-Mφ.


Assuntos
Tecido Adiposo/citologia , Macrófagos Peritoneais/imunologia , Células-Tronco Mesenquimais/imunologia , Animais , Diferenciação Celular/imunologia , Sobrevivência Celular , Inflamação , Fatores Reguladores de Interferon/metabolismo , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
4.
Genes (Basel) ; 10(6)2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234442

RESUMO

Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and loss of function. Adipose tissue harbors mesenchymal stem/stromal cells (MSC), or medicinal signaling cells as suggested by Caplan (Caplan, 2017), used in autologous transplantation in many clinical settings. The aim of the study was to characterize a stromal vascular fraction from microfragmented lipoaspirate (SVF-MLA) applied for cartilage treatment in OA and compare it to that of autologous lipoaspirate (SVF-LA). Samples were first stained using a DuraClone SC prototype tube for the surface detection of CD31, CD34, CD45, CD73, CD90, CD105, CD146 and LIVE/DEAD Yellow Fixable Stain for dead cell detection, followed by DRAQ7 cell nuclear dye staining, and analyzed by flow cytometry. In SVF-LA and SVF-MLA samples, the following population phenotypes were identified within the CD45- fraction: CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells (SA-ASC). The immunophenotyping profile of SVF-MLA was dominated by a reduction of leukocytes and SA-ASC, and an increase in EP, evidencing a marked enrichment of this cell population in the course of adipose tissue microfragmentation. The role of EP in pericyte-primed MSC-mediated tissue healing, as well as the observed hormonal implication, is yet to be investigated.


Assuntos
Túnica Adventícia/imunologia , Cartilagem/metabolismo , Imunofenotipagem , Osteoartrite/tratamento farmacológico , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Túnica Adventícia/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Osteoartrite/imunologia , Osteoartrite/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/imunologia
5.
Med Sci Monit ; 25: 4457-4468, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31201771

RESUMO

BACKGROUND The treatment of inflammatory bowel disease (IBD) is still not satisfactory and novel technologies are clinically needed. This study aimed to examine the effect of mesenchymal stromal cells (MSCs) coated with the anti-vascular cell adhesion molecule 1 (VCAM 1) antibody on experimental colitis. MATERIAL AND METHODS The antibody was coated onto the MSCs isolated from male BALB/C mice to generate anti-VCAM 1 antibody-coated MSC (V-MSC). The Transwell assay was used to detect migration rate. 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to generate experimental colitis. MSCs were injected intravenously into experimental models. Weight changes, disease activity index, and histological changes were evaluated. The SRY gene were used for cell tracking. Expression of Ki67 and claudin 1 was used to measure local repair using immunohistochemistry. T helper (Th)1, Th2, Th17, and T regulatory cells were counted. RESULTS V-MSCs were successfully generated through coating MSCs with VCAM1 antibody. Analysis showed that the V-MSCs had similar surface types and differentiation as uncoated MSCs. Transwell assays showed that V-MSCs had higher migration rate than MSCs. After injection of V-MSCs, the expression of the SRY gene was enhanced in diseased colon and all indices (including weight changes, DAI score, histological changes, and the expressions of Ki67 and claudin 1) recovered rapidly. The ratio of proinflammatory Th1 and Th17 cells decreased, but the ratio of anti-inflammatory Th2 and Treg cells increased after the treatment. CONCLUSIONS V-MSCs enhance homing and modulating immune balance in the experimental colitis, suggesting that they are potentially useful for treating inflammatory bowel disease or other immune diseases.


Assuntos
Colite/patologia , Células-Tronco Mesenquimais/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Diferenciação Celular , Proliferação de Células , Colite/imunologia , Colo/patologia , Modelos Animais de Doenças , Imunomodulação/imunologia , Doenças Inflamatórias Intestinais/patologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia
6.
Int Immunopharmacol ; 74: 105663, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200338

RESUMO

Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSC-EVs) are taken more seriously as immunomodulatory and anti-inflammatory agents. We studied the therapeutic effects of MSC-EVs on allergic contact dermatitis (ACD), a typical T cell-mediated disorder. A contact hypersensitivity (CHS) mouse model for ACD was established and treated by intravenous MSC-EVs injection. We found that human umbilical cord MSC-EVs could significantly prevent the pathology of CHS, including reduced ear swelling and leukocyte infiltration. Injection of MSC-EVs significantly inhibited CD8+IFN-γ+ cytotoxic T (Tc1) cells and CD4+IFN-γ+ type 1 helper T (Th1) cells, and reduced the level of pro-inflammatory Tumor Necrosis Factor-alpha (TNF-α) and interferon gamma (IFN-γ), and induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) and the level of anti-inflammatory IL-10. In vitro, MSC-EVs also suppressed Tc1 and Th1 cells and induced Tregs and the related cytokines, further indicating the immune regulatory role of MSC-EVs. Interestingly, PKH26-labeled MSC-EVs were found to be directly internalized by CD3+ T cells, resulting in reduced signal transducer and activator of transcription 1 (STAT1) protein levels in vitro. In summary, MSC-EVs can prevent the onset of CHS by inhibiting Tc1 and Th1 immune responses and inducing the Tregs phenotype in vivo and in vitro. The mechanism by which MSC-EVs influence CD3+ T cells might partially involve targeting STAT1 in vitro. Therefore, MSC-EVs are ideal candidates for cell-free immunomodulatory therapy for T cell-mediated diseases such as ACD.


Assuntos
Dermatite de Contato/imunologia , Vesículas Extracelulares/imunologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/imunologia , Linfócitos T/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Humanos , Masculino , Camundongos Endogâmicos BALB C
7.
Cells ; 8(5)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052214

RESUMO

Inflammatory bowel disease (IBD) is caused by a dysregulated immune response against normal components of the intestinal microflora combined with defective functioning of anti-inflammatory pathways. Currently, all therapies approved for IBD manipulate the immune system by inhibiting pro-inflammatory mechanisms, such as tumor necrosis factor-α, gut-homing α4ß7 integrin, interleukin-12/interleukin-23, and Janus kinases. However, some IBD patients are non-responders to these drugs, which are also associated with serious side effects. Thus, it has been hypothesized that therapies aimed at restoring anti-inflammatory signals, by exploiting the tolerogenic potential of cytokines (interleukin-10, transforming growth factor-ß, granulocyte macrophage colony-stimulating factor), immune cells (regulatory T cells, tolerogenic dendritic cells), or mesenchymal stem cells, might offer promising results in terms of clinical efficacy with fewer side effects. In this review, we provide new insights into putative novel treatments aimed at restoring anti-inflammatory signaling pathways in IBD.


Assuntos
Anti-Inflamatórios/uso terapêutico , Citocinas/antagonistas & inibidores , Células Dendríticas/imunologia , Inflamação/terapia , Doenças Inflamatórias Intestinais/terapia , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores/imunologia , Humanos , Imunoterapia
8.
Cell Biol Int ; 43(8): 899-909, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31081266

RESUMO

Previous studies have shown that the ovarian failure in autoimmune-induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta-derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol-requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X-box binding protein 1 (XBP1), up-regulated 78 kDa glucose-regulated protein (GRP78) and caspase-12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis-induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation-mediating ovarian function recovery.


Assuntos
Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Placenta/citologia , Insuficiência Ovariana Primária , Animais , Caspase 12/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Feminino , Células da Granulosa/citologia , Proteínas de Choque Térmico/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ovário/metabolismo , Gravidez , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/terapia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo
9.
Immunobiology ; 224(4): 585-594, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31072631

RESUMO

Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunomodulação , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Tecido Adiposo/citologia , Apoptose , Biomarcadores , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Imunofenotipagem , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo
10.
PLoS One ; 14(5): e0216862, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086407

RESUMO

Polytrauma (PT) is a life-threatening disease and a major global burden of injury. Mesenchymal stromal cells (MSC) might be a therapeutic option for PT patients due to their anti-inflammatory and regenerative potential. We hypothesised that the inflammatory response of MSC is similar after exposure to selected trauma-relevant factors to sera from PT patients (PTS). Therefore, we investigated the effects of a mixture of defined factors, supposed to play a role on MSC in the early phase of PT. Additionally, in a translational approach we investigated the effect of serum from PT patients on MSC in vitro. MSC were incubated with a PT cocktail in physiological (PTCL) and supra-physiological (PTCH) concentrations or PTS. The effect on gene expression and protein secretion of MSC was analysed by RNA sequencing, ELISA and Multiplex assays of cell culture supernatant. Stimulation of MSC with PTCH, PTCL or IL1B led to significant up- or downregulation of 470, 183 and 469 genes compared to unstimulated MSC at 6 h. The intersection of differentially expressed genes in these groups was very high (92% overlap with regard to the PTCL group; treated for 6 h). Cytokine secretion profile of MSC revealed that IL1B mimics the effect of a more complex PT cocktail as well. However, there was only a minor proportion of overlapping differentially expressed genes between the MSC group stimulated with early times of PTS and the MSC group stimulated with PTCH, PTCL and IL1B. In conclusion, the effect of sera from PT patients on MSC activation cannot be simulated by the chosen trauma-relevant factors. Furthermore, we conclude that while IL1B might be useful to prime MSC prior to therapeutic application, it might not be as useful for the in vitro study of functional properties of MSC in the context of PT.


Assuntos
Inflamação/imunologia , Células-Tronco Mesenquimais/imunologia , Traumatismo Múltiplo/imunologia , Adulto , Células Cultivadas , Citocinas/sangue , Citocinas/imunologia , Feminino , Humanos , Inflamação/sangue , Inflamação/complicações , Inflamação/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Traumatismo Múltiplo/sangue , Traumatismo Múltiplo/complicações , Traumatismo Múltiplo/patologia , Adulto Jovem
11.
Cells ; 8(5)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096722

RESUMO

Multipotent mesenchymal stromal cells (MSCs) have emerged as potent therapeutic agents for multiple indications. However, recent evidence indicates that MSC function is compromised in the physiological post-injury milieu. In this study, bone marrow (BM)- and adipose-derived (AD)-MSCs were preconditioned in hypoxia with or without inflammatory mediators to potentiate their immunotherapeutic function in preparation for in vivo delivery. Human MSCs were cultured for 48 hours in either normoxia (21% O2) or hypoxia (2% O2) with or without the addition of Cytomix, thus creating 4 groups: 1) normoxia (21%); 2) Cytomix-normoxia (+21%); 3) hypoxia (2%); and 4) Cytomix-hypoxia (+2%). The 4 MSC groups were subjected to comprehensive evaluation of their characteristics and function. Preconditioning did not alter common MSC surface markers; nonetheless, Cytomix treatment triggered an increase in tissue factor (TF) expression. Moreover, the BM-MSCs and AD-MSCs from the +2% group were not able to differentiate to chondrocytes and osteoblasts, respectively. Following Cytomix preconditioning, the metabolism of MSCs was significantly increased while viability was decreased in AD-MSCs, but not in BM-MSCs. MSCs from both tissues showed a significant upregulation of key anti-inflammatory genes, increased secretion of IL-1 receptor antagonist (RA), and enhanced suppression of T-cell proliferation following the Cytomix treatment. Similarly, following a lipopolysaccharide challenge, the Cytomix-treated MSCs suppressed TNF-α secretion, while promoting the production of IL-10 and IL-1RA. These preconditioning approaches facilitate the production of MSCs with robust anti-inflammatory properties. AD-MSCs preconditioned with Cytomix under normoxia appear to be the most promising therapeutic candidates; however, safety concerns, such as thrombogenic disposition of cells due to TF expression, should be carefully considered prior to clinical translation.


Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Células da Medula Óssea/imunologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Células-Tronco Mesenquimais/imunologia , Tromboplastina/metabolismo , Células da Medula Óssea/citologia , Hipóxia Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Humanos , Mediadores da Inflamação/imunologia , Interleucina-10/metabolismo , Células-Tronco Mesenquimais/citologia , Fator de Necrose Tumoral alfa/metabolismo
12.
Cells ; 8(5)2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117301

RESUMO

BACKGROUND: The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards.


Assuntos
Técnicas de Cultura de Células/métodos , Imunomodulação/fisiologia , Células-Tronco Mesenquimais/imunologia , Cordão Umbilical/citologia , Geleia de Wharton/imunologia , Proliferação de Células , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Humanos , Cariótipo , Fenótipo , Medição de Risco
13.
Transfusion ; 59(S2): 1593-1600, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30980752

RESUMO

BACKGROUND: Cellular therapeutic agents may benefit trauma patients by modulating the immune response to injury, and by reducing inflammation and vascular leakage. Administration of allogeneic mesenchymal stromal cells (MSCs) shows some benefit in preclinical and clinical trials, but less testing has been performed with other cell types. Human primary fibroblasts (FBs) were compared to MSCs in assays designed to evaluate MSCs to determine if these assays actually evaluate properties unique to MSCs or whether related cell types perform similarly. STUDY DESIGN AND METHODS: MSC-related surface marker expression, tissue factor, and human leukocyte antigen-D related were evaluated by flow cytometry, and in vitro adipogenic and osteogenic differentiation potential were determined. Procoagulant activity was determined by thromboelastography. Two potency assays correlated with immunomodulation potential were utilized: the mixed lymphocyte reaction and indoleamine 2,3-dioxygenase enzyme activity assays. RESULTS: Human primary FBs performed similarly to MSCs in assays designed to evaluate MSC characteristics and potency. Although similar for MSC-positive cell surface marker expression, FBs did not show robust adipose differentiation and expressed some level of markers not expected on MSCs. CONCLUSIONS: Human primary FBs are very similar to human MSCs, at least in assays currently used to evaluate MSC potency. Preclinical and clinical testing are required to determine if FBs show similar activity to MSCs in vivo. If FBs show inferior activity in vivo, development of new MSC-specific potency assays will be necessary to evaluate properties relevant to their unique clinical benefits.


Assuntos
Diferenciação Celular , Fibroblastos/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Técnicas de Cultura de Células , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/imunologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia
14.
Int Immunopharmacol ; 72: 264-274, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31005036

RESUMO

Extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) have shown repairing effects in tissue damage. However, their efficacy and mechanism in the treatment of ulcerative colitis (UC), a type of chronic inflammatory bowel disease, are unclear. To investigate the effects and possible mechanism of EVs in UC treatment, we established an in vitro model using lipopolysaccharide (LPS)-treated macrophages and an in vivo dextran sulfate sodium (DSS)-induced mouse model to mimic UC. In vitro, EVs promoted the proliferation and suppressed inflammatory response in LPS-induced macrophages, as demonstrated by the up-regulation of pro-inflammatory factors (TNF-α, IL-6, and IL-12) and down-regulation of the anti-inflammatory factor IL-10. In the in vivo model, EV administration ameliorated the symptoms of UC by reducing weight loss, disease activity index, and colon mucosa damage and severity while increasing colon length. This was additionally accompanied by the increase in IL-10 and TGF-ß levels and the decline in VEGF-A, IFN-γ, IL-12, TNF-α, CCL-24, and CCL-17 levels. In terms of the mechanism, EVs promoted M2-like macrophage polarization, characterized by the increase in the M2 marker CD163. Furthermore, the positive effect of EVs on UC repair seemed to be related to the JAK1/STAT1/STAT6 signaling pathway. Collectively, BMSC-derived EVs exerted positive therapeutic effects against DSS-induced UC, which could be due to a negative inflammatory response.


Assuntos
Colite Ulcerativa/imunologia , Vesículas Extracelulares/imunologia , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Citocinas/imunologia , Sulfato de Dextrana , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Fenótipo
15.
Int J Exp Pathol ; 100(2): 102-113, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017330

RESUMO

A major translational barrier to the use of stem cell (SC)-based therapy in patients with myocardial infarction (MI) is the lack of a clear understanding of the mechanism(s) underlying the cardioprotective effect of SCs. Numerous paracrine factors from SCs may account for reduction in infarct size, but myocardial salvage associated with transdifferentiation of SCs into vascular cells as well as cardiomyocyte-like cells may be involved too. In this study, bone marrow-derived rat mesenchymal SC (MSCs) were microencapsulated in alginate preventing viable cell release while supporting their secretory phenotype. The hypothesis on the key role of paracrine factors from MSCs in their cardioprotective activity was tested by comparison of the effect of encapsulated vs free MSCs in the rat model of MI. Intramyocardial administration of both free and encapsulated MSCs after MI caused reduction in scar size (12.1 ± 6.83 and 14.7 ± 4.26%, respectively, vs 21.7 ± 6.88% in controls, P = 0.015 and P = 0.03 respectively). Scar size was not different in animals treated with free and encapsulated MSC (P = 0.637). These data provide evidence that MSC-derived growth factors and cytokines are crucial for cardioprotection elicited by MSC. Administration of either free or encapsulated MSCs was not arrhythmogenic in non-infarcted rats. The consistency of our data with the results of other studies on the major role of MSC secretome components in cardiac protection further support the theory that the use of live, though encapsulated, cells for MI therapy may be replaced with heart-targeted-sustained delivery of growth factors/cytokines.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Infarto do Miocárdio/terapia , Alginatos , Animais , Arritmias Cardíacas/etiologia , Células Cultivadas , Cicatriz/patologia , Citoproteção/fisiologia , Composição de Medicamentos , Ecocardiografia , Imunofenotipagem , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/imunologia , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Comunicação Parácrina/fisiologia , Ratos Wistar , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/fisiologia
16.
Rheumatol Int ; 39(5): 819-826, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30944956

RESUMO

We aimed to assess the immunoregulatory effects of secretory factors produced by adipose tissue-derived MSC (AT-MSC) on Th17 and Treg subsets from patients with rheumatoid arthritis (RA). 17 patients with active disease matching the ACR/EULAR 2010 criteria for RA were included. Patients' peripheral blood mononuclear cells (PBMC) were cultured in AT-MSC-conditioned medium (AT-MSCcm) and in control medium. The cytokine production of AT-MSC and PBMC was quantified by ELISA. Th17 and Treg were determined by flow cytometry. AT-MSCcm contained: IL-6, IL-17, IL-21, CCL2, CCL5, IL-8, sVEGF-A and PGE2. Cultivation of patients' PBMC with AT-MSCcm increased TGF-ß1 (8318 pg/ml; IQR 6327-11,686) vs control medium [6227 pg/ml (IQR 1681-10,148, p = 0.013)]. PBMC cultivated with AT-MSCcm downregulated TNF-α, IL-17A, and IL-21 compared to control PBMC: 5 pg/ml IQR (1.75-11.65) vs 1 pg/ml (IQR 0.7-1.9), p = 0.001; 4.2 pg/ml (IQR 3.1-6.1) vs 2.3 pg/ml (IQR.75-5.42), p = 0.017; 66.9 pg/ml (IQR 40.6-107.2) vs 53 pg/ml (IQR 22-73), p = 0.022. Th17 decreased under the influence of AT-MSCcm: 10.13 ± 3.88% vs 8.98 ± 3.58%, p = 0.02. CD4+FoxP3+, CD4+CD25-FoxP3+, and CD4+CD25+FoxP3+ was 11.35 ± 4.1%; 7.13 ± 3.12% and 4.22 ± 2% in control PBMC. Accordingly, CD4+FoxP3+, CD4+CD25-FoxP3+, and CD4+CD25+FoxP3+ significantly increased in PBMC cultured with AT-MSCcm: 15.6 ± 6.1%, p = 0.001; 9.56 ± 5.4%, p = 0.004 and 6.04 ± 3.6%, p = 0.001. All these effects could define MSC-based approaches as adequate avenues for further treatment development in RA.


Assuntos
Artrite Reumatoide/imunologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Tecido Adiposo/citologia , Adulto , Quimiocina CCL2/imunologia , Quimiocina CCL5/imunologia , Meios de Cultivo Condicionados , Dinoprostona/imunologia , Feminino , Humanos , Interleucina-17/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Interleucinas/imunologia , Masculino , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular/imunologia
17.
Cell Prolif ; 52(3): e12595, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953394

RESUMO

OBJECTIVES: Mesenchymal stem cells (MSCs) could regulate the function of various immune cells. It remains unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of induction of anti-inflammatory purinergic signalling. MATERIAL AND METHODS: We obtained human bone marrow mesenchymal stem cells (BMMSCs) and dental pulp stem cells (DPSCs). NK cells were isolated from peripheral blood of healthy volunteers. Activated NK cells were cultured with MSCs. Proliferation assay, apoptosis analysis, activating or inhibitory receptor expression and degranulation assay were used to explore NK cells' function. High-performance liquid chromatography was used to investigate the purinergic signalling in activated NK cells. RESULTS: Both DPSCs and BMMSCs could impair proliferation and promote apoptosis of activated NK cells. Also, activated NK cells could cause DPSCs to lyse. Furthermore, the expression of activating NK cells' receptors was decreased, but inhibitory receptors of NK cells were elevated following co-cultivation. NK cells acquired CD73 expression, while MSCs could release ATP into the extracellular space where nucleotides were converted into adenosine (ADO) following co-culture system. Under the existence of exogenous 2-chloroadenosine (CADO), the cytotoxic capacity of NK cells was remarkably depressed in a concentration-dependent manner. CONCLUSIONS: DPSCs and BMMSCs could depress NK cells' function by hydrolysing ATP to ADO using CD39 and CD73 enzymatic activity. Our data suggested that DPSCs might represent a new strategy for treating immune-related diseases by regulating previously unrecognized functions in innate immune responses.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/imunologia , 2-Cloroadenosina/farmacologia , 5'-Nucleotidase/metabolismo , Apoptose , Proliferação de Células , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas Ligadas por GPI/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células K562 , Células Matadoras Naturais/citologia , Ativação Linfocitária , Purinas/metabolismo , Receptores de Células Matadoras Naturais/efeitos dos fármacos , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais
18.
Transpl Immunol ; 55: 101205, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30946889

RESUMO

Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.


Assuntos
Doenças da Medula Óssea , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Tecido Adiposo , Aloenxertos , Animais , Doenças da Medula Óssea/etiologia , Doenças da Medula Óssea/imunologia , Doenças da Medula Óssea/patologia , Doenças da Medula Óssea/terapia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Células-Tronco Mesenquimais/patologia , Camundongos
19.
Res Vet Sci ; 124: 212-222, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30925336

RESUMO

Little information is currently available on therapeutic features of bovine mesenchymal stem cells (MSCs), despite the development of large animal experimental models including cattle may open alternative strategies for investigating MSC physiology and eventual applications for regenerative therapy. The aim of the present study was to compare in vitro immunomodulatory and immunogenic potentials of bovine fetal MSCs (bfMSCs) derived from bovine fetal bone marrow (BM-MSCs) and adipose tissue (AT-MSCs). Immunomodulatory analyses in bfMSCs were performed by determination of the effect of interferon-γ (IFNγ) on mRNA levels of indoleamine 2, 3-dioxygenase (IDO), transforming growth factor ß1 (TGFß1), prostaglandin E receptor 2 (PTGER2), interleukin-6 and -10 (IL-6 and IL-10), and IDO enzymatic activity. The effect of conditioned medium from IFNγ-stimulated bfMSCs on the proliferation of alloantigen-activated peripheral blood lymphocytes (PBLs) was assessed. Immunogenicity of bfMSCs was determined by quantification of mRNA levels of major histocompatibility complex I and II (MHC-I and -II), CD80 and CD86, and the proportion of cells positive for MHC-I and -II by flowcytometry (FACS) analyses. IFNγ treatment increased IL-6, PTGER2 and IDO gene expression and activity in bfMSCs but did not affect suppressive effect on proliferation of PBLs. Lower proportion of AT-MSCs expressed MHC-I and MHC-II in comparison to BM-MSCs. In conclusion, BM-MSCs and AT-MSCs upregulated expression of immunomodulatory genes in a similar way after IFNγ stimuli. BM-MSCs and AT-MSCs in basal condition and treated with IFNγ displayed similar in vitro immunomodulatory ability. Lower expression of MHC-I and MHC-II suggest that AT-MSCs might be less immunogenic compared to BM-MSCs.


Assuntos
Tecido Adiposo/metabolismo , Células da Medula Óssea/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Animais , Medula Óssea/metabolismo , Bovinos , Feto
20.
PLoS One ; 14(3): e0212642, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870461

RESUMO

Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans.


Assuntos
Mobilização de Células-Tronco Hematopoéticas , Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Proliferação de Células , Separação Celular , Cavalos , Linfócitos/citologia , Linfócitos/imunologia , Células-Tronco Mesenquimais/citologia , Transplante Autólogo
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