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AIMS: Allogeneic stem cell therapy is more logistically suitable compared with autologous cell therapy for large-scale patient treatment. We aim to investigate the clinical safety and efficacy profile of the allogeneic adipose tissue derived mesenchymal stromal cell product (CSCC_ASC) as an add-on therapy in patients with chronic non-ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) < 40%. METHODS AND RESULTS: This is a single-centre investigator-initiated randomized phase I/II study with direct intra-myocardial injections of 100 million allogeneic CSCC_ASC. A total of 30 HFrEF patients with New York Heart Association (NYHA) class ≥II despite optimal anticongestive heart failure medication and plasma NT-proBNP > 300 pg/mL (>35 pmol/L) were included and randomized 2:1 to CSCC_ASC or standard care. The primary endpoint left ventricular end systolic volume (LVESV) and other echo related parameters were analysed by an investigator blinded for treatment allocation. No difference in serious adverse events was observed between groups. LVESV decreased significantly from baseline to 6 months follow-up in the ASC group (153.7 ± 53.2 mL and 128.7 ± 45.6 mL, P < 0.001) and remained unchanged in the standard care group (180.4 ± 39.4 mL and 186.7 ± 48.9 mL, P = 0.652). There was a significant difference between the groups in LVESV change (31.3 ± 11.0 mL, P = 0.009). The difference from baseline to follow-up between the two groups in left ventricular end diastolic volume (LVEDV) was 18.7 ± 12.4 mL, P = 0.146 and in left ventricular ejection fraction (LVEF) -7.8 ± 2.1%, P = 0.001. Considering the baseline values of LVESV, LVEDV and LVEF as covariates, the difference between groups for change from baseline to follow-up resulted in a P-value of 0.056, 0.076, and 0.738, respectively. NYHA class and self-reported health did also improve significantly in the ASC group compared with the standard care group (0.7 ± 0.2, P = 0.001 and -12.8 ± 5.3, P = 0.025; respectively). There was no difference in NT-proBNP (-371 ± 455 pmol/L, P = 0.422) or in 6 min walk test (12 ± 31 m, P = 0.695) between groups. CONCLUSIONS: Intramyocardial injections of allogeneic CSCC_ASC in patients with chronic non-ischaemic HFrEF was safe and improved LVESV, LVEF, NYHA class, and self-reported health compared with standard care group.
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Doxorubicin (DOX) is a common and highly effective chemotherapeutic. However, its use is limited by cardiotoxic effects and the lack of methods to detect these at early time points. In the present study, we evaluated if [64Cu]Cu-NODAGA-E[(cRGDyK)]2 positron emission tomography-computed tomography ([64Cu]Cu-RGD PET/CT) could detect cardiotoxicity in a rat model of DOX-induced heart failure. Male Lewis rats were divided into two groups and treated with either a cumulative dose of 15 mg/kg of DOX or left untreated. Cardiac anatomy and function were assessed using magnetic resonance imaging at baseline and in week 8. [64Cu]Cu-RGD PET/CT scans were performed in week 4. DOX treatment led to a decline in pump function as well as an increase in cardiac and thymic uptake of [64Cu]Cu-RGD. In addition, DOX altered cardiac gene expression, led to infiltration of immune cells, reduced endothelial content, and increased interstitial fibrosis. Furthermore, concentrations of inflammatory plasma proteins were increased in the DOX group. In conclusion, DOX treatment resulted in the development of cardiotoxicity and heart failure, which could be detected using [64Cu]Cu-RGD PET/CT at early time points. [64Cu]Cu-RGD uptake in the myocardial septum and thymus predicted a low left ventricular ejection fraction in week 8.
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Radioisótopos de Cobre , Modelos Animais de Doenças , Doxorrubicina , Insuficiência Cardíaca , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ratos Endogâmicos Lew , Animais , Doxorrubicina/efeitos adversos , Doxorrubicina/toxicidade , Ratos , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/diagnóstico por imagem , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos Heterocíclicos com 1 Anel , Cardiotoxicidade/etiologia , Acetatos/química , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons/métodos , Coração/efeitos dos fármacos , Coração/diagnóstico por imagem , Compostos RadiofarmacêuticosRESUMO
BACKGROUND: Adipose-derived mesenchymal stem/stromal cells (ASCs) are proposed as a new xerostomia treatment. The study evaluated the long-term safety and effectiveness of allogeneic ASCs in radiation-induced xerostomia among patients with previous oropharyngeal cancer. METHODS: This study constitutes 3-year follow-up on the original 10 patients who received allogeneic ASCs injections to the submandibular and parotid glands as part of the MESRIX-II trial. The MESRIX-II trial included the preliminary 4-month follow-up. The primary endpoint was long-term safety. Secondary endpoints were effectiveness evaluated by changes in salivary flow rate and patient-reported outcomes (PROs). Immune response was evaluated by assessing the development of donor-specific antibodies (DSA). FINDINGS: All 10 MESRIX-II patients completed the long-term follow-up (ie, no missing data). During the long-term follow-up, 2 patients encountered a significant adverse event, which was determined to be unrelated to the treatment. No DSAs were detectable at 3 years. The stimulated salivary flow rate increased significantly from an average of 0.66 mL/minute at baseline to 0.86 mL/minute at follow-up, corresponding to an increase of 0.20 [95% CI 0.08 to 0.30] mL/minute, or approximately 30%. Among the PROs, sticky saliva symptoms were reduced, with a -20.0 [95% CI -37.3 to -2.7] units. INTERPRETATION: In conclusion, this study is the first to present long-term follow-up outcomes of allogeneic ASC treatment as a therapeutic option for radiation-induced xerostomia. The study found that ASC treatment appears safe, and there were no indications of adverse immune responses at the 3-year follow-up. Further studies are warranted to evaluate the findings in larger settings.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Xerostomia , Humanos , Xerostomia/etiologia , Xerostomia/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Células-Tronco Mesenquimais/citologia , Seguimentos , Transplante Homólogo/métodos , Resultado do TratamentoRESUMO
The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs' secretome which act in concert with VEGF to improve angiogenesis.
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Tecido Adiposo , Técnicas de Cocultura , Neovascularização Fisiológica , Comunicação Parácrina , Células Estromais , Humanos , Células Estromais/metabolismo , Células Estromais/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/citologia , AngiogêneseRESUMO
PURPOSE: No effective treatment exists for radiation-induced xerostomia. The objective of this study was to compare the effect of adipose-derived mesenchymal stem/stromal cell (ASC) injection, relative to placebo, on salivary gland function in patients with radiation-induced xerostomia. PATIENT AND METHODS: In this single-centre, double-blind, placebo-controlled trial, patients with hyposalivation were randomised to receive ultrasound-guided injections of allogeneic ASCs or placebo into the submandibular glands. Patients were followed for 4 months. We evaluated unstimulated whole salivary flow rate (UWS), stimulated salivary flow rate, and patient-reported outcomes. Adverse events were recorded and immune response determined in blood samples. RESULTS: We enrolled 120 patients. ASC treatment resulted in a statistically significant UWS increase of 0.04 [95% confidence interval (CI), 0.02-0.06] mL/min (38%) compared with pretreatment baseline whereas placebo treatment did not cause a significant increase [0.01 (95% CI, -0.01 to 0.04) mL/min (21%)]. Both the ASC and placebo treatment yielded notable symptom reductions, with dry mouth decreasing by 13.6 and 7.7 units, sticky saliva decreased by 14.8 and 9.3 units, swallowing difficulties decreased by 7.9 and 8.0 units, and the summary score of the Xerostomia Questionnaire decreased 5.9 and 5.1 units for the ASC and placebo arms, respectively. We found no statistically significant group difference between the ASC and placebo arms for any of the outcomes. CONCLUSIONS: We could not confirm superiority of the ASC relative to placebo. ASC therapy significantly improved UWS in previous patients with head and neck cancer, whereas placebo resulted in an insignificant increase.
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Neoplasias de Cabeça e Pescoço , Transplante de Células-Tronco Mesenquimais , Xerostomia , Humanos , Xerostomia/etiologia , Xerostomia/terapia , Masculino , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/complicações , Transplante de Células-Tronco Mesenquimais/métodos , Pessoa de Meia-Idade , Idoso , Adulto , Células-Tronco Mesenquimais/citologia , Lesões por Radiação/terapia , Lesões por Radiação/etiologia , Método Duplo-Cego , Resultado do Tratamento , Glândulas Salivares/efeitos da radiação , Radioterapia/efeitos adversosRESUMO
PURPOSE: This double-blinded randomized clinical trial aimed to evaluate the efficacy of injecting allogeneic adipose-derived mesenchymal stem cells (ASCs) into the lacrimal gland (LG) for the treatment of dry eye disease (DED) secondary to Sjögren's syndrome (SS). METHODS: Fifty-four participants with severe DED secondary to SS were included and allocated to either ASCs (n = 20), vehicle (n = 20), or a non-randomized observation group (n = 14). The intervention groups received a single injection of either ASCs or an active comparator (vehicle, Cryostor® CS10) into the LG in one eye, while the observation group received lubricating eye drops only. The primary outcome measure was changes in Ocular Surface Disease Index (OSDI) score and secondary outcome measures were non-invasive tear break-up time, tear meniscus height, Schirmer's test, and Oxford score within a 12-month follow-up. RESULTS: A significant reduction in OSDI score was observed in the ASCs and vehicle groups compared to the observation group. In addition, the ASCs group demonstrated a significant increase in non-invasive tear break-up time compared to the vehicle group at the 4-week follow-up and to the observation group at the 12-month follow-up. A significant improvement in ocular surface staining, tear osmolarity, and Schirmer test score from baseline was also observed in the ASCs group; however, these changes were not significant compared to the other groups. CONCLUSION: Improvement of subjective and objective signs and symptoms of DED was observed in both intervention groups following injection into the LG compared to the observation group. Future studies should investigate the mode-of-action of both injection treatments.
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Síndromes do Olho Seco , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Síndrome de Sjogren , Humanos , Síndrome de Sjogren/complicações , Síndrome de Sjogren/terapia , Síndrome de Sjogren/diagnóstico , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/terapia , Síndromes do Olho Seco/diagnóstico , Lágrimas/metabolismoRESUMO
BACKGROUND: A predominant side effect of radiotherapy for head and neck cancer is salivary gland hypofunction and xerostomia leading to debilitating oral disorders and impaired quality of life (QoL). Intraglandular mesenchymal stem cell therapy has shown promising results as a treatment for xerostomia. METHODS: This is a randomised, double-blinded, placebo-controlled, parallel-group, prospective, single-centre trial investigating the safety, tolerability, and effectiveness of allogeneic stem cells as a treatment for radiation-induced hyposalivation and xerostomia for previous head and neck cancer patients. We will include a total of 120 patients who previously have been treated with radiotherapy for a head and neck cancer in Denmark. Participants will be randomly assigned using block randomisation to one of two parallel groups in a 1:1 ratio to receive ultrasound-guided injection of allogeneic adipose-derived mesenchymal stem cell (ASC) (n = 60) or placebo (n = 60) into the submandibular glands. Placebo will consist of CryoStor10 (BiolifeSolutions), the freeze media for ASCs containing 10% dimethyl sulfoxide (DMSO). The primary endpoint is change in unstimulated whole saliva flow rate. The secondary endpoints are change in stimulated whole saliva flow rate, QoL, and composition of saliva. Further secondary endpoints are safety and immune response (human leukocyte antigen (HLA) response) to the stem cells will be assessed. Patients are evaluated at baseline (before treatment), after 4 months, and after 12 months. All study personnel, except study personnel thawing and preparing the treatment for injection, and participants will be blinded to group assignment. Unblinded study personnel will not participate in the outcome assessment. DISCUSSION: The trials will investigate the efficacy and safety of ASC injection to the submandibular gland as a potential new treatment for post-radiation xerostomia. We hope the results will pave the way for a clinically relevant treatment to ameliorate patients with xerostomia, a severely hampering condition. TRIAL REGISTRATION: The study is approved by the Danish Data Protection Agency (protocol number P-2020-1164), the National Ethics Committee protocol number: (Protocol number: 1802872), and the Danish Medical Agency (2018-000348-24). The protocol was registered at the ClinicalTrials.gov database (NCT04776538).
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Neoplasias de Cabeça e Pescoço , Células-Tronco Mesenquimais , Xerostomia , Humanos , Qualidade de Vida , Estudos Prospectivos , Xerostomia/etiologia , Xerostomia/terapia , Neoplasias de Cabeça e Pescoço/radioterapia , Ensaios Clínicos Controlados Aleatórios como Assunto , Ensaios Clínicos Fase II como AssuntoRESUMO
Small extracellular vesicles (EVs) and their cargo are an important component of cell-to-cell communication in cardiac disease. Allogeneic adipose derived stem cells (ADSCs) are thought to be a potential approach for cardiac regenerative therapy in ischemic heart disease. The SCIENCE study investigated the effect of ADSCs administered via intramyocardial injection on cardiac function in patients with ischemic heart disease. The aim of this substudy, based on samples from 15 patients, was to explore small EV miRNA dynamics after treatment with ADSCs compared to a placebo. Small EVs were isolated at several timepoints after the percutaneous intramyocardial application of ADSCs. No significant effect of ADSC treatment on small EV concentration was detected. After 12 months, the expression of miR-126 decreased significantly in ADSC patients, but not in the placebo-treated group. However, all cardiac miRNAs correlated with plasma cardiac biomarkers. In line with the overall negative results of the SCIENCE study, with the exception of one miR, we did not detect any significant regulation of small EV miRNAs in this patient collective.
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Vesículas Extracelulares , Insuficiência Cardíaca , MicroRNAs , Isquemia Miocárdica , Humanos , MicroRNAs/genética , Tecido Adiposo , Vesículas Extracelulares/genética , Células-Tronco , Isquemia Miocárdica/genética , Isquemia Miocárdica/terapiaRESUMO
An increasing number of patients are living with chronic ischemic cardiomyopathy (ICM) and/or heart failure. Treatment options and prognostic tools are lacking for many of these patients. Our aim was to investigate the prognostic value of imaging angiogenesis and macrophage activation via positron emission tomography (PET) in terms of functional improvement after cell therapy. Myocardial infarction was induced in rats. Animals were scanned with [18F]FDG PET and echocardiography after four weeks and randomized to allogeneic adipose tissue-derived stromal cells (ASCs, n = 18) or saline (n = 9). Angiogenesis and macrophage activation were assessed before and after treatment by [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE. There was no overall effect of the treatment. Rats that improved left ventricular ejection fraction (LVEF) had higher uptake of both [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE at follow-up (p = 0.006 and p = 0.008, respectively). The uptake of the two tracers correlated with each other (r = 0.683, p = 0.003 pre-treatment and r = 0.666, p = 0.004 post-treatment). SUVmax at follow-up could predict improvement in LVEF (p = 0.016 for [68Ga]Ga-RGD and p = 0.045 for [64Cu]Cu-DOTATATE). High uptake of [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE PET after injection of ASCs or saline preceded improvement in LVEF. The use of these tracers could improve the monitoring of heart failure patients in treatment.
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AIMS: The aim of the SCIENCE trial was to investigate whether a single treatment with direct intramyocardial injections of adipose tissue-derived mesenchymal stromal cells (CSCC_ASCs) was safe and improved cardiac function in patients with chronic ischaemic heart failure with reduced ejection fraction (HFrEF). METHODS AND RESULTS: The study was a European multicentre, double-blind, placebo-controlled phase II trial using allogeneic CSCC_ASCs from healthy donors or placebo (2:1 randomization). Main inclusion criteria were New York Heart Association (NYHA) class II-III, left ventricular ejection fraction (LVEF) <45%, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels >300 pg/ml. CSCC_ASCs or placebo (isotonic saline) were injected directly into viable myocardium. The primary endpoint was change in left ventricular end-systolic volume (LVESV) at 6-month follow-up measured by echocardiography. A total of 133 symptomatic HFrEF patients were included. The treatment was safe without any drug-related severe adverse events or difference in cardiac-related adverse events during a 3-year follow-up period. There were no significant differences between groups during follow-up in LVESV (0.3 ± 5.0 ml, p = 0.945), nor in secondary endpoints of left ventricular end-diastolic volume (-2.0 ± 6.0 ml, p = 0.736) and LVEF (-1.6 ± 1.0%, p = 0.119). The NYHA class improved slightly within the first year in both groups without any difference between groups. There were no changes in 6-min walk test, NT-proBNP, C-reactive protein or quality of life the first year in any groups. CONCLUSION: The SCIENCE trial demonstrated safety of intramyocardial allogeneic CSCC_ASC therapy in patients with chronic HFrEF. However, it was not possible to improve the pre-defined endpoints and induce restoration of cardiac function or clinical symptoms.
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Insuficiência Cardíaca , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Humanos , Doença Crônica , Qualidade de Vida , Volume Sistólico , Resultado do Tratamento , Função Ventricular Esquerda , Método Duplo-CegoRESUMO
AIMS: Patients suffering from chronic ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) have reduced quality-of-life, repetitive hospital admissions, and reduced life expectancy. Allogeneic cell therapy is currently investigated as a potential treatment option after initially encouraging results from clinical autologous and allogeneic trials in patients with HFrEF. We aimed to investigate the allogeneic Cardiology Stem Cell Centre Adipose tissue derived mesenchymal Stromal Cell product (CSCC_ASC) as an add-on therapy in patients with chronic HFrEF. METHODS AND RESULTS: This is a Danish multi-centre double-blinded placebo-controlled phase II study with direct intra-myocardial injections of allogeneic CSCC_ASC. A total of 81 HFrEF patients were included and randomized 2:1 to CSCC_ASC or placebo injections. The inclusion criteria were reduced left ventricular ejection fraction (LVEF ≤ 45%), New York Heart Association (NYHA) class II-III despite optimal anti-congestive heart failure medication and no further revascularization options. Injections of 0.3 mL CSCC_ASC (total cell dose 100 × 106 ASCs) (n = 54) or isotonic saline (n = 27) were performed into the viable myocardium in the border zone of infarcted tissue using the NOGA Myostar® catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA). The primary endpoint, left ventricular end systolic volume (LVESV), was evaluated at 6-month follow-up. The safety was measured during a 3-years follow-up period. RESULTS: Mean age was 67.0 ± 9.0 years and 66.6 ± 8.1 years in the ASC and placebo groups, respectively. LVESV was unchanged from baseline to 6-month follow-up in the ASC (125.7 ± 68.8 mL and 126.3 ± 72.5 mL, P = 0.827) and placebo (134.6 ± 45.8 mL and 135.3 ± 49.6 mL, P = 0.855) group without any differences between the groups (0.0 mL (95% CI -9.1 to 9.0 mL, P = 0.992). Neither were there significant changes in left ventricular end diastolic volume or LVEF within the two groups or between groups -5.7 mL (95% CI -16.7 to 5.3 mL, P = 0.306) and -1.7% (95% CI -4.4. to 1.0, P = 0.212), respectively). NYHA classification and 6-min walk test did not alter significantly in the two groups (P > 0.05). The quality-of-life, total symptom, and overall summary score improved significantly only in the ASC group but not between groups. There were 24 serious adverse events (SAEs) in the ASC group and 11 SAEs in the placebo group without any significant differences between the two groups at 1-year follow-up. Kaplan-Meier plot using log-rank test of combined cardiac events showed an overall mean time to event of 30 ± 2 months in the ASC group and 29 ± 2 months in the placebo group without any differences between the groups during the 3 years follow-up period (P = 0.994). CONCLUSIONS: Intramyocardial CSCC_ASC injections in patients with chronic HFrEF were safe but did not improve myocardial function or structure, nor clinical symptoms.
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Insuficiência Cardíaca , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Isquemia Miocárdica , Humanos , Pessoa de Meia-Idade , Idoso , Insuficiência Cardíaca/terapia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/terapia , Volume Sistólico , Função Ventricular Esquerda , Transplante de Células-Tronco Mesenquimais/métodos , DinamarcaRESUMO
The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 via TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.
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Leucócitos Mononucleares , Células-Tronco Mesenquimais , Células Cultivadas , Células-Tronco Mesenquimais/metabolismo , Imunomodulação , Células Estromais , MitógenosRESUMO
BACKGROUND: Hyposalivation and xerostomia (dry mouth), are the leading site-effects to treatment of head and neck cancer. Currently, there are no effective therapies to alleviate radiation-induced hyposalivation. Adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) have shown potential for restoring salivary gland function. However, the mode of action is unknown. The purpose of the present study was therefore to characterize the effect of AT-MSC therapy on the salivary proteome in previously irradiated head and neck cancer patients. METHODS: Whole saliva was collected from patients with radiation-induced salivary gland hypofunction (n = 8) at baseline, and 120 days after AT-MSC treatment, and from healthy controls (n = 10). The salivary proteome was characterized with mass spectrometry based proteomics, and data was compared within the AT-MSC group (baseline versus day 120) and between AT-MSC group and healthy controls. Significance levels between groups were determined by using double-sided t-test, and visualized by means of principal component analysis, volcano plots and cluster analysis. RESULTS: Here we show that 140 human proteins are significantly differentially expressed in saliva from patients with radiation-induced hypofunction versus healthy controls. AT-MSC treatment induce a significant impact on the salivary proteome, as 99 proteins are differentially expressed at baseline vs. 120 days after treatment. However, AT-MSC treatment does not restore healthy conditions, as 212 proteins are significantly differentially expressed in saliva 120 days after AT-MSCs treatment, as compared to healthy controls. CONCLUSION: The results indicate an increase in proteins related to tissue regeneration in AT-MSCs treated patients. Our study demonstrates the impact of AT-MSCs on the salivary proteome, thereby providing insight into the potential mode of action of this novel treatment approach.
Currently, there are no effective treatments to ease dry mouth, which is a leading long-term side effect of radiation treatment for head and neck cancer. However, treatment with stem cells has shown potential for restoring function of the salivary glands, which are damaged due to radiation. We compared proteins in saliva of previously radiation-treated patients with healthy non-irradiated persons and found differences in the levels of 140 proteins. After stem cell treatment of irradiated patients, we found changes in the salivary content of proteins related to tissue regeneration. Our study demonstrates the impact of stem cell treatment on proteins in saliva, thereby providing insight into the potential mode of action of this treatment approach for patients with radiation-induced dry mouth. Consequently, this could potentially help to improve treatment of dry mouth in the future.
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As the interest in cell-based therapies continue to increase, so does the need for assays detailing potency and providing platforms for identifying mechanisms of action. For most clinical implications of mesenchymal stromal cells, the immunomodulatory effect is crucial. While the suppressive potential on lymphocyte proliferation is well-described in literature, reproducible and standardized assays to document and quantify it varies from research group to research group and between methodologies. The aim of the present study was to utilize flowcytometry to quantify proliferation and identify measurements to increase the assay sensitivity to treatment with adipose tissue-derived stromal cells (ASC). Lymphocyte proliferation was induced by the unspecific mitogen phytohemagglutinin or by alloreactivity towards an irradiated donor in a mixed lymphocyte reaction. Addition of ASC did not change the composition of T cells, B cells, NK cells, NKT cell types considerably; likewise, no increases in proliferation were observed upon inclusion of ASC, demonstrating that ASC does not evoke an additive response. On the contrary, the suppressive effect of ASC was documented. By applying different gating strategies and curve fitting, the sensitivity was increased, and dose-response relationships established. Flow cytometric evaluation allows for more detailed identification of the lymphocytes affected by ASC and constitute a significant asset in future unraveling of modes and mechanisms of action, as well as quantification of potency.
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Tecido Adiposo , Mitógenos , Proliferação de Células , Células Cultivadas , Fito-Hemaglutininas/farmacologia , Células Estromais/metabolismoRESUMO
BACKGROUND: Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. METHODS: An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-ß stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell-cell contacts. The cultures were screened using RT2 PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. RESULTS: TGF-ß stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. CONCLUSIONS: The co-culture model provides effective stimulation of NHDF monocultures by TGF-ß for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs' capabilities and underlying mechanisms related to ECM formation and remodeling.
Assuntos
Fibronectinas , Metaloproteinase 2 da Matriz , Células Cultivadas , Técnicas de Cocultura , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos , Fibronectinas/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The emerging field of advanced therapy medicinal products (ATMP) holds promise of treating a variety of diseases. Adipose-derived stromal cells (ASCs) are currently being marketed or tested as cell-based therapies in numerous clinical trials. To ensure safety and efficacy of treatments, high-quality products must be manufactured. A good manufacturing practice (GMP) compliant and consistent manufacturing process including validated quality control methods is critical. Product design and formulation are equally important to ensure clinical feasibility. Here, we present a GMP-compliant, xeno-free, and semiautomated manufacturing process and quality controls, used for large-scale production of a cryopreserved off-the-shelf ASC product and tested in several phase I and II allogeneic clinical applications.
RESUMO
Individual patient data (IPD)-based meta-analysis (ACCRUE, meta-analysis of cell-based cardiac studies, NCT01098591) revealed an insufficient effect of intracoronary cell-based therapy in acute myocardial infarction. Patients with ischemic heart failure (iHF) have been treated with reparative cells using percutaneous endocardial, surgical, transvenous or intracoronary cell delivery methods, with variable effects in small randomized or cohort studies. The objective of this meta-analysis was to investigate the safety and efficacy of percutaneous transendocardial cell therapy in patients with iHF. Two investigators extracted the data. Individual patient data (IPD) (n = 8 studies) and publication-based (n = 10 studies) aggregate data were combined for the meta-analysis, including patients (n = 1715) with chronic iHF. The data are reported in accordance with PRISMA guidelines. The primary safety and efficacy endpoints were all-cause mortality and changes in global ejection fraction. The secondary safety and efficacy endpoints were major adverse events, hospitalization and changes in end-diastolic and end-systolic volumes. Post hoc analyses were performed using the IPD of eight studies to find predictive factors for treatment safety and efficacy. Cell therapy was significantly (p < 0.001) in favor of survival, major adverse events and hospitalization during follow-up. A forest plot analysis showed that cell therapy presents a significant benefit of increasing ejection fraction with a mean change of 2.51% (95% CI: 0.48; 4.54) between groups and of significantly decreasing end-systolic volume. The analysis of IPD data showed an improvement in the NYHA and CCS classes. Cell therapy significantly decreased the end-systolic volume in male patients; in patients with diabetes mellitus, hypertension or hyperlipidemia; and in those with previous myocardial infarction and baseline ejection fraction ≤ 45%. The catheter-based transendocardial delivery of regenerative cells proved to be safe and effective for improving mortality and cardiac performance. The greatest benefit was observed in male patients with significant atherosclerotic co-morbidities.
RESUMO
No effective therapy exists for the most common long-term side effect of radiation therapy for head and neck cancer (HNC)-xerostomia. The objective was to evaluate safety and provide proof of concept for efficacy of allogeneic adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) injected into the major salivary glands of irradiated patients. This open-label, first-in-human, phase 1b, and single-center trial was conducted with repeated measurements days 0, 1, 5, and 30 and 4 months. Eligible patients with objective and subjective signs of radiation-induced salivary gland damage after treatment of oropharyngeal squamous cell carcinoma stages I-II (UICC 8) were enrolled. Twenty-five million cryopreserved AT-MSCs were injected into each submandibular and 50 million AT-MSCs into each parotid gland. Data were collected on adverse events, unstimulated and stimulated whole saliva (UWS and SWS) flow rates and saliva composition, patient-reported outcomes (EORTC QLQ-H&N35 and Xerostomia Questionnaire [XQ]), blood samples and salivary gland scintigraphy. Data were analyzed using repeated measures linear mixed models. Ten patients (7 men, 3 women, 59.5 years [range: 45-70]) were treated in 4 glands. No treatment-related serious adverse events occurred. During 4 months, UWS flow rate increased from 0.13 mL/minute at baseline to 0.18 mL/minute with a change of 0.06 (P = .0009) mL/minute. SWS flow rate increased from 0.66 mL/minute at baseline to 0.75 mL/minute with a change of 0.09 (P = .017) mL/minute. XQ summary score decreased by 22.6 units (P = .0004), EORTC QLQ-H&N35 dry mouth domains decreased by 26.7 (P = .0013), sticky saliva 23.3 (P = .0015), and swallowing 10.0 (P = .0016). Our trial suggests treatment of the major salivary glands with allogenic AT-MSCs is safe, warranting confirmation in larger trials.
Assuntos
Neoplasias de Cabeça e Pescoço , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Lesões por Radiação , Xerostomia , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Masculino , Lesões por Radiação/etiologia , Lesões por Radiação/terapia , Xerostomia/etiologia , Xerostomia/terapiaRESUMO
AIMS: The long-term outcomes of the intracoronary delivery of autologous bone marrow-derived cells (BMCs) after acute myocardial infarction are not well established. Following the promising 1 year results of the REGENERATE-AMI trial (despite it not achieving its primary endpoint), this paper presents the analysis of the 5 year clinical outcomes of these acute myocardial infarction patients who were treated with an early intracoronary autologous BMC infusion or placebo. METHODS AND RESULTS: A 5 year follow-up of major adverse cardiac events (defined as the composite of all-cause death, recurrent myocardial infarction, and all coronary revascularization) and of rehospitalization for heart failure was completed in 85 patients (BMC n = 46 and placebo n = 39). The incidence of major adverse cardiac events was similar between the BMC-treated patients and the placebo group (26.1% vs. 18.0%, P = 0.41). There were no cases of cardiac death in either group, but an increase in non-cardiac death was seen in the BMC group (6.5% vs. 0%, P = 0.11). The rates of recurrent myocardial infarction and repeat revascularization were similar between the two groups. There were no cases of rehospitalization for heart failure in either group. CONCLUSION: This 5 year follow-up analysis of the REGENERATE-AMI trial did not show an improvement in clinical outcomes for patients treated with cell therapy. This contrasts with the 1 year results which showed improvements in the surrogate outcome measures of ejection fraction and myocardial salvage index.
Assuntos
Transplante de Medula Óssea , Infarto do Miocárdio , Transplante de Medula Óssea/métodos , Seguimentos , Humanos , Infarto do Miocárdio/terapia , Transplante Autólogo , Resultado do TratamentoRESUMO
Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.