Search

To evaluate the safety and feasibility of injecting allogeneic adipose-derived mesenchymal stem cells (ASCs) into the lacrimal gland (LG) as a treatment of aqueous deficient dry eye disease (ADDE).In this open-label, 5-visit clinical trial (baseline, treatment and weeks 1, 4 and 16) seven subjects with ADDE received one transconjunctival injection of allogeneic ASCs into the LG in one eye. The ASC product contained 22 million ASCs/ml and the injected volume was maximally 50% of the LG volume as determined on magnetic resonance imaging (MRI). Treatment related adverse events (AEs) were assessed at each visit (primary endpoint). Ocular Surface Disease Index (OSDI), tear osmolarity, tear film breakup time (TBUT), corneal staining (Oxford grade) and Schirmer's I test were assessed at each timepoint.No AEs related to the study treatment were observed. Mean follow-up time was 126 days after treatment. The mean OSDI score decreased from 58.9 ± 20.6 at baseline to 34.1 ± 21.6 (p 0.002). In the study eye mean tear osmolarity decreased from 312.9 ± 10.4 to 291.6 ± 10.9 mosm/l (p 0.002), mean TBUT increased from 3.7 ± 1.5 to 7.1 ± 1.9 s (p 0.002), mean Schirmer's I test increased from 4.6 ± 0.7 to 8.1 ± 3.1 mm/5 min (p 0.03), while mean Oxford grade showed a trend towards a decrease from 2.4 ± 0.7 to 1.3 ± 1 (p 0.10).Our trial suggests that injection of allogeneic ASCs into the LG is a safe and feasible treatment of severe ADDE. A randomized placebo-controlled trial aimed at elucidating the therapeutic effect of allogeneic ASCs in a larger patient cohort from our research group is currently underway.

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.

AIMS The study assessed 4-year outcomes of intramyocardial injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in patients with ischaemic heart failure. METHODS AND RESULTS The MSC-HF trial was a randomized, double-blind, placebo-controlled trial. Patients were randomized 2:1 to intramyocardial injections of MSCs or placebo. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured by magnetic resonance imaging or computed tomography. Sixty patients aged 30-80 years with ischaemic heart failure, New York Heart Association class II-III, left ventricular ejection fraction (LVEF)

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-β 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-β 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-β 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.

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.

Background An increasing number of patients living with chronic ischemic cardiomyopathy (ICM) are potential candidates for cell therapy, including adipose tissue-derived mesenchymal stromal cells (ASC). However, proper assessment of clinical effects in relation to cellular effector mechanisms, such as angiogenesis, is currently lacking. Purpose To investigate the prognostic value of assessing angiogenesis non-invasively using novel PET imaging in terms of functional outcome after cell therapy in a homologous animal model of ICM. Methods Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Four weeks after infarction, the rats were scanned with [18F]-FDG and echocardiography, and based on left ventricular ejection fraction (LVEF) and infarct size randomized to allogeneic ASC treatment (n=14) or saline (n=9). Animals were treated using echo-guided trans-thoracic intramyocardial injections. Follow-up echocardiography was performed four weeks after treatment. Angiogenesis was assessed non-invasively using the [68Ga]-NODAGA-E[(cRGDyK)]2 (RGD) PET-tracer before treatment and two weeks after the treatment (Figure). The output was RGD-uptake by maximum standardized uptake value (SUVmax). Results RGD-uptake in the infarct area significantly decreased in the saline group (p=0.04), while this decline was not significant in the ASC group (p=0.58). There was no effect on LVEF of the cell therapy in this study (p=0.70). When the rats were grouped by RGD-uptake post treatment, the high RGD-uptake group (n=8) improved LVEF compared to the rats with medium or low RGD-uptake (n=15, p=0.04) irrespective of the treatment. This could indicate that non-invasive detection of a high degree of myocardial angiogenesis measured using RGD is predictive of improvement in cardiac pump function in ICM. Conclusions Following injection of ASC or saline, a high RGD-uptake precluded improvement in pump function. This is the first evidence of how uptake of the novel RGD tracer relates to changes in pump function in ICM and the results could affect the design of future clinical trials using regenerative therapy for ICM patients. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Aase and Ejnar Danielsens FondDoctor Sofus Carl Emil Friis and wife Olga Doris Friis' Scholarship Study design, RGD-uptake, and results

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.

Background Stem cell therapy is investigated as a treatment option for patients with ischemic heart disease. In this study, long-term safety and efficacy of autologous intra-myocardial injections of adipose-derived stromal cells (ASCs) was studied in patients with refractory angina. Methods Sixty patients with coronary artery stenosis and preserved left ventricular ejection fraction were 2:1 randomised to intramyocardial injections of ASCs or saline and followed for 3 years. Results For patients in the ASC group, the bicycle exercise time and the exercise performance in watt were un-changed (383 ± 30 s to 370 ± 44 s, P = 0.052 and 81 ± 6 to 78 ± 10, P = 0.123, respectively), but the performance in METs was reduced significantly (4.2 ± 0.3 to 4.0 ± 0.4, P = 0.027) during the follow-up period. However, in the same period, there was in the placebo group a significant decline in bicycle exercise time (437 ± 53 s to 383 ± 58 s, P = 0.001), the exercise performance measured in watt (87 ± 12 W to 80 ± 12 W, P = 0.019) and in METs (4.5 ± 0.4 to 4.1 ± 0.4, P = 0.002). Moreover, angina measured as CCS class was significantly reduced in the ASC group but not in the placebo group (2.5 ± 0.9 to 1.8 ± 1.2, P = 0.002 and 2.5 ± 0.8 to 2.1 ± 1.3, P = 0.186, respectively). However, no significant change was observed between the two groups. Conclusions Patients receiving ASCs had improved cardiac symptoms and unchanged exercise capacity, in opposition to deterioration in the placebo group. Trial registration ClinicalTrials.gov Identifier: NCT01449032. Registered 7 October 2011—Retrospectively registered, https://www.clinicaltrials.gov/ct2/show/NCT01449032?term=jens+kastrup&rank=7

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.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.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.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.

Non-ischemic dilated cardiomyopathy (NIDCM) constitutes one of the most common causes to non-ischemic heart failure. Despite treatment, the disease often progresses, causing severe morbidity and mortality, making novel treatment strategies necessary. Due to the regenerative actions of mesenchymal stem cells (MSCs), they have been proposed as a treatment for NIDCM. This systematic review aims to evaluate efficacy and mode of action (MoA) of MSC-based therapies in NIDCM. A systematic literature search was conducted in Medline (Pubmed) and Embase. A total of 27 studies were included (3 clinical trials and 24 preclinical studies). MSCs from different tissues and routes of delivery were reported, with bone marrow-derived MSCs and direct intramyocardial injections being the most frequent. All included clinical trials and 22 preclinical trials reported an improvement in cardiac function following MSC treatment. Furthermore, preclinical studies demonstrated alterations in tissue structure, gene, and protein expression patterns, primarily related to fibrosis and angiogenesis. Consequently, MSC treatment can improve cardiac function in NIDCM patients. The MoA underlying this effect involves anti-fibrosis, angiogenesis, immunomodulation, and anti-apoptosis, though these processes seem to be interdependent. These encouraging results calls for larger confirmatory clinical studies, as well as preclinical studies utilizing unbiased investigation of the potential MoA.

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10

We aimed to evaluate the effect of intramyocardial injections of autologous VEGF-A165-stimulated adipose-derived stromal cells (ASCs) in patients with refractory angina. MyStromalCell trial is a randomized double-blind placebo-controlled study including sixty patients with CCS/NYHA class II-III, left ventricular ejection fraction > 40%, and at least one significant coronary artery stenosis. Patients were treated with ASC or placebo in a 2 : 1 ratio. ASCs from the abdomen were culture expanded and stimulated with VEGF-A165. At 6 months follow-up, bicycle exercise tolerance increased significantly in time duration 22 s (95%CI −164 to 208 s) (P=0.034), in watt 4 (95%CI −33 to 41, 0.048), and in METs 0.2 (95%CI −1.4 to 1.8) (P=0.048) in the ASC group while there was a nonsignificant increase in the placebo group in time duration 9 s (95%CI −203 to 221 s) (P=0.053), in watt 7 (95%CI −40 to 54) (P=0.41), and in METs 0.1 (95%CI −1.7 to 1.9) (P=0.757). The difference between the groups was not significant (P=0.680, P=0.608, and P=0.720 for time duration, watt, and METs, resp.). Intramyocardial delivered VEGF-A165-stimulated ASC treatment was safe but did not improve exercise capacity compared to placebo. However, exercise capacity increased in the ASC but not in the placebo group. This trial is registered with ClinicalTrials.gov NCT01449032.

Background. While regenerative stem cell therapy for ischemic heart disease has moved into phase 3 studies, little is still known about retention and migration of cell posttransplantation. In human studies, the ability to track transplanted cells has been limited to labeling with radioisotopes and tracking using nuclear imaging. This method is limited by low resolution and short half-lives of available radioisotopes. Longitudinal tracking using magnetic resonance imaging (MRI) of myocardial injected cells labeled with iron oxide nanoparticles has shown promising results in numerous preclinical studies but has yet to be evaluated in human studies. We aimed to evaluate MRI tracking of mesenchymal stromal cells (MSCs) labeled with ultrasmall paramagnetic iron oxide (USPIO) nanoparticles after intramyocardial transplantation in patients with ischemic heart disease (IHD). Methods. Five no-option patients with chronic symptomatic IHD underwent NOGA-guided intramyocardial transplantation of USPIO-labeled MSCs. Serial MRI scans were performed to track labeled cells both visually and using semiautomated T2∗ relaxation time analysis. For safety, we followed symptoms, quality of life, and myocardial function for 6 months. Results. USPIO-labeled MSCs were tracked for up to 14 days after transplantation at injection sites both visually and using semiautomated regional T2∗ relaxation time analysis. Labeling of MSCs did not impair long-term safety of treatment. Conclusion. This was a first-in-man clinical experience aimed at evaluating the utility of MRI tracking of USPIO-labeled bone marrow-derived autologous MSCs after intramyocardial injection in patients with chronic IHD. The treatment was safe, and cells were detectable at injection sites up to 14 days after transplantation. Further studies are needed to clarify if MSCs migrate out of the injection area into other areas of the myocardium or if injected cells are washed out into the peripheral circulation. The trial is registered with ClinicalTrials.gov NCT03651791.

Background Improvements in medical and interventional therapies have transformed ischemic heart disease into a chronic illness for lot of patients. The disease is in progress and by time patients suffer from cardiac symptoms, reduced work capacity and decline in quality of life. Stem cell therapy is investigated as a treatment option for these patients. Purpose In this study, long-term safety and efficacy of autologous intra-myocardial injections of adipose-derived stromal cells (ASCs) were studied in patients with refractory angina. Methods Sixty patients were double-blinded 2:1 randomised to ASC or saline injections and followed for three years. The patients had significant angina due to ≥1 coronary artery stenosis but preserved left ventricular ejection fraction. ASCs were obtained from abdomen, ex vivo culture expanded and VEGF-A165 stimulated before delivery into the ischemic myocardium. Results The cardiac symptoms, CCS and NYHA classification, were significantly reduced in the ASC group during the three years follow-up period (2.5±0.9 to 1.8±1.2, P=0.002 and 2.4±0.6 to 2.2±0.8, P=0.007, respectively). However, no significant change was observed in CCS or NYHA in the placebo group during the follow-up period (2.5±0.8 to 2.1±1.3, P=0.186 and 2.7±0.6 to 2.4±0.8, P=0.314, respectively). Moreover, the number of weekly angina attacks reported was significantly reduced in the ASC group (P=0.017), but not in the placebo group (P=0.425). For patients in the ASC group, the bicycle exercise time (383±30s to 370±44s, P=0.052) and the exercise performance in watt were un-changed (81±6 to 78±10, P=0.123), but the performance in METs was reduced significantly (4.2±0.3 to 4.0±0.4, P=0.027) during the follow-up period. At the same time in the placebo group, there was a significant decline in bicycle exercise time (437±53s to 383±58s, P=0.001), the exercise performance measured in watt (87±12 watt to 80±12 watt, P=0.019) and in METs (4.5±0.4 to 4.1±0.4, P=0.002). In both groups, significant improved quality-of-life, angina stability, angina frequency and physical limitation score was observed but not for overall satisfaction score. Conclusion Patients receiving ASCs had improved cardiac symptoms during the three years follow-up period, which was not the case for patients in the placebo group. Moreover, patients receiving ASCs had unchanged exercise capacity, in opposition to deterioration in the placebo group. Acknowledgement/Funding Arvid Nilssons Foundation; Rigshospitalets Research Foundation; Aase and Ejnar Danielsens Foundation

Investigational cell-based therapeutics are rapidly heading towards pivotal clinical trials. The premise is that the scientific rationale is well defined, and that product quality reflects exactly this. In vitro potency assays are necessary tools for evaluating cell products, and with potency assays comes high demands for standardization and reproducibility of the methods involved. For demonstrating principles of cell therapeutics for allogeneic use or with claimed immunosuppressive efficacies, assays involving peripheral blood mononuclear cells (PBMC) are critical. Establishment of a cryopreserved bank of PBMC favors standardization, as it allows repeated use of a single donor and simultaneous testing of several donors. The first step to fulfil such potential is to ensure optimum conditions for preservation of PBMC function, and secondly to design assays which heightens the reproducibility. Emphasis should be put on application of the assay. The objective of the present study was to establish a methodological foundation for cell therapeutics to be tested, and several aspects were factored in, including cell concentrations and partial changes of medium. PBMC were isolated and cryopreserved in six formulations of cryoprotective medium consisting of fetal bovine serum (90%, 60%, and 30%) in combination with dimethyl sulfoxide (10% or 5%). The proliferative capacity of the cryopreserved cells was assayed by labeling with carboxyfluorescein succinimidyl ester and stimulation by phytohemagglutinin or in mixed lymphocyte reactions, analyzed by flow cytometry. To counter an eventual lag phase post thaw, the assays were designed to include two durations and to explore the possibility of reducing cell numbers, two cell concentrations. Qualitative and quantitative aspects of the staining were affected by formulation as well as design, stressing the importance of basic optimization for assay development. We conclude that the established methods allow for optimized preservation of function and will serve as a platform for further development of robust functional assays.

Background. Cell therapy for heart disease has been proven safe and efficacious, despite poor cell retention in the injected area. Improving cell retention is hypothesized to increase the treatment effect. In the present study, human adipose-derived stromal cells (ASCs) were delivered in an in situ forming alginate hydrogel following acute myocardial infarction (AMI) in rats. Methods. ASCs were transduced with luciferase and tested for ASC phenotype. AMI was inducted in nude rats, with subsequent injection of saline (controls), 1 × 106 ASCs in saline or 1 × 106 ASCs in 1% (w/v) alginate hydrogel. ASCs were tracked by bioluminescence and functional measurements were assessed by magnetic resonance imaging (MRI) and 82rubidium positron emission tomography (PET). Results. ASCs in both saline and alginate hydrogel significantly increased the ejection fraction (7.2% and 7.8% at 14 days and 7.2% and 8.0% at 28 days, resp.). After 28 days, there was a tendency for decreased infarct area and increased perfusion, compared to controls. No significant differences were observed between ASCs in saline or alginate hydrogel, in terms of retention and functional salvage. Conclusion. ASCs improved the myocardial function after AMI, but administration in the alginate hydrogel did not further improve retention of the cells or myocardial function.

Background aims. Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to inject the cells in an in situ cross-linked alginate hydrogel. Methods. ASCs from abdominal human tissue were embedded in alginate hydrogel and alginate hydrogel modified with Arg-Gly-Asp motifs (RGD-alginate) and cultured for 1 week. Cell viability, phenotype, immunogenicity and paracrine activity were determined by confocal microscopy, dendritic cell co-culture, flow cytometry, reverse transcriptase quantitative polymerase chain reaction, Luminex multiplex, and lymphocyte proliferation experiments. Results. ASCs performed equally well in alginate and RGD-alginate. After 1 week of alginate culture, cell viability was >93%. Mesenchymal markers CD90 and CD29 were reduced compared with International Society for Cellular Therapy criteria. Cells sedimented from the alginates during cultivation regained the typical level of these markers, and trilineage differentiation was performed by standard protocols. Hepatocyte growth factor mRNA was increased in ASCs cultivated in alginates compared with monolayer controls. Alginates and alginates containing ASCs did not induce dendritic cell maturation. ASCs in alginate responded like controls to interferon-gamma stimulation (licensing), and alginate culture increased the ability of ASCs to inhibit lymphocyte proliferation. Discussion. ASCs remain viable in alginates; they transiently change phenotype in alginate hydrogel but regain the phenotype of monolayer controls upon release. Cells maintain their paracrine potential while in alginates; the combination of ASCs and alginate is non-immunogenic and, in fact, immunosuppressive.

Aims Regenerative treatment with mesenchymal stromal cells (MSCs) has been promising in patients with ischaemic heart failure but needs confirmation in larger randomized trials. We aimed to study effects of intra-myocardial autologous bone marrow-derived MSC treatment in patients with severe ischaemic heart failure. Methods and results The MSC-HF trial is a randomized, double-blind, placebo-controlled trial. Patients were randomized 2 : 1 to intra-myocardial injections of MSC or placebo, respectively. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured by magnetic resonance imaging or computed tomography at 6 months follow-up. Sixty patients aged 30–80 years with severe ischaemic heart failure, New York Heart Association (NYHA) classes II–III, left ventricular ejection fraction (LVEF)

Background. Ischemic heart failure (IHF) has a poor prognosis in spite of optimal therapy. We have established a new allogeneic Cardiology Stem Cell Centre adipose-derived stromal cell (CSCC_ASC) product from healthy donors. It is produced without animal products, in closed bioreactor systems and cryopreserved as an off-the-shelf product ready to use. Study Design. A multicentre, double-blind, placebo-controlled phase II study with direct intramyocardial injections of allogeneic CSCC_ASC in patients with chronic IHF. A total of 81 patients will be randomised at 2 : 1 to CSCC_ASC or placebo. There is no HLA tissue type matching needed between the patients and the donors. Methods. The treatment will be delivered by direct injections into the myocardium. The primary endpoint is change in the left ventricle endsystolic volume at 6-month follow-up. Secondary endpoints are safety and changes in left ventricle ejection fraction, myocardial mass, stroke volume, and cardiac output. Other secondary endpoints are change in clinical symptoms, 6-minute walking test, and the quality of life after 6 and 12 months. Conclusion. The aim of the present study is to demonstrate safety and the regenerative efficacy of the allogeneic CSCC_ASC product from healthy donors in a double-blind, placebo-controlled, multicentre study in patients with IHF.

Number of stromal cells injected in patients with ischaemic heart disease (IHD) may be of importance for the treatment efficacy, which in turn may be influenced by various patient-related factors. In this study, we investigate whether patient-related factors influence the number of autologous stromal cells reached after in vitro culture expansion for clinical therapy.Culture expansion data from 111 patients with IHD treated with autologous stromal cells in three clinical trials were used. We correlated the final cell count after two passages of cultivation with different patient factors.There was a significant relation between body mass index (BMI) and the number of adipose derived stromal cells (ASCs) reached after culture expansion and for all patients included into the three studies (r = 0.375, p = .019 and r = 0.200, p = .036, respectively). Moreover, there was a significantly higher number of ASCs reached in patients with hypertension compared to those without hypertension and for all patients overall (68.8 ± 39.6 × 10Patient related factors such as BMI, hypertension and gender may influence the number of MSCs reached after in vitro culture expansion.

To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell tracking for years, but knowledge regarding possible cytotoxic ultrastructural changes subsequent to iron-oxide particle labeling is limited. Hence, the purpose of this study was to label MSCs with dextran-coated ultrasmall super-paramagnetic iron-oxide (USPIO) particles conjugated with the transduction sequence of trans-activator of transcription (TAT) (IODEX-TAT) and evaluate the effect of labeling on ultrastructure, viability, phenotype and proliferative capacity of the cells.MSCs were labeled with 5 and 10 μg IODEX-TAT/10(5) cells for 2, 6 and 21 hours. IODEX-TAT uptake and cellular ultrastructure were determined by electron microscopy. Cell viability was determined by propidium iodide staining and cell proliferation capacity by 5-bromo-2-deoxyuridine (BrdU) incorporation. Maintenance of stem cell surface markers was determined by flow cytometry. Results. IODEX-TAT labeling for 2, 6 and 21 h did not influence cellular ultrastructure or viability. Moreover, neither stem cell surface markers nor cell proliferation capacity was affected by labeling with IODEX-TAT.Our results demonstrate that labeling of MSCs for 21 h with a clinically relevant dose of 10 μg IODEX-TAT/10(5) cells is feasible and does not affect MSC ultrastructure, viability, phenotype or proliferation capacity.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6–8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up ( p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 ( p < 0.001), angina attacks per week from 13.8 to 3.2 ( p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week ( p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life ( p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Although, treatment of ischemic heart disease (IHD) has improved considerably within the last decades, it is still the main cause of death worldwide. Despite maximum treatment, many IHD patients suffer from refractory angina and heart failure, which severely limits their daily lives. Moreover, IHD is very costly for the health care system. Therefore, new treatment options and strategies are being researched intensely. Stem cell therapy to improve myocardial perfusion and stimulate growth of new cardiomyocytes could be a new way to go. Nevertheless, the results from clinical studies have varied considerably, probably due to the use of many different cell lines obtained from different tissues and the different patient populations. The present review will focus on treatment with the mesenchymal stromal cell from bone marrow and adipose tissue in animal and patients with acute and chronic IHD (CIHD).

Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.

Background Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation. Methods Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded into either T75 flasks or a Quantum system that had been coated with cryoprecipitate. The cultivation of ASCs from SVF was performed in 3 ways: flask to flask; flask to Quantum system; and Quantum system to Quantum system. In all cases, quality controls were conducted for sterility, mycoplasmas, and endotoxins, in addition to the assessment of cell counts, viability, immunophenotype, and differentiation potential. Results The viability of ASCs passage 0 (P0) and P1 was above 96%, regardless of cultivation in flasks or Quantum system. Expression of surface markers and differentiation potential was consistent with ISCT/IFATS standards for the ASC phenotype. Sterility, mycoplasma, and endotoxin tests were consistently negative. An average of 8.0 × 107 SVF cells loaded into a Quantum system yielded 8.96 × 107 ASCs P0, while 4.5 × 106 SVF cells seeded per T75 flask yielded an average of 2.37 × 106 ASCs—less than the number of SVF cells seeded. ASCs P1 expanded in the Quantum system demonstrated a population doubling (PD) around 2.2 regardless of whether P0 was previously cultured in flasks or Quantum, while ASCs P1 in flasks only reached a PD of 1.0. Conclusion: Manufacturing of ASCs in a Quantum system enhances ASC expansion rate and yield significantly relative to manual processing in T-flasks, while maintaining the purity and quality essential to safe and robust cell production. Notably, the use of the Quantum system entails significantly reduced working hours and thereby costs. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1080-9) contains supplementary material, which is available to authorized users.

Background Stem cell therapy is an emerging treatment modality in cardiovascular disease. The best cell type and delivery method in different cardiovascular diseases remain to be determined. Study Design The MSC-HF trial is a phase 2, single-center, double-blind, randomized, placebo-controlled trial of intramyocardial delivery of autologous bone-marrow derived mesenchymal stromal cells (MSCs) in patients with chronic ischemic heart failure. A total of 60 patients will be randomized in a 2:1 pattern to receive intramyocardial injections of either MSCs or placebo. Patients will be followed up for 12 months. Methods Bone marrow will be obtained by aspiration from the iliac crest. Mesenchymal stromal cells will be isolated, and culture will be expanded for 6 to 8 weeks. A total of 12 to 15 MSC or placebo injections will be placed in an ischemic viable region of the myocardium using the electromechanical NOGA-XP system (Biologics Delivery Systems Group, Johnson & Johnson, Irwindale, CA). Endpoints The primary endpoint is change in left ventricle end-systolic volume, measured by magnetic resonance imaging (MRI) or computed tomography (CT) at 6-month follow-up. Secondary endpoints are left ventricle ejection fraction, ventricular volumes, wall thickness, and systolic wall thickening measured by MRI or CT in addition to measurement of myocardial scar tissue by MRI. Other secondary endpoints are safety of treatment, clinical symptoms and functional capacity, weekly angina attacks, use of short-term nitroglycerine, and quality of life. Conclusion A randomized, double-blind, placebo-controlled, clinical trial of intramyocardial delivery of MSCs in patients with ischemic heart failure has been set up to confirm the positive findings in open-labeled clinical trials.

BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion. METHODS: We investigate the influence of cell density on homogeneity and differentiation during culture expansion of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS). MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q-PCR. RESULTS: The morphological observation and the phenotypic analyses showed that MSCs after 3 weeks cultivation constituted a phenotypically homogenous MSC cell population, which at low levels expressed genes for different cell lineages, confirming their multilineage plasticity, without actual differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which persists independent of cell density can be used for clinical therapies.

Introduction Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEGF stimulation on gene expression patterns of ASCs. Methods Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Microarray gene expressions were obtained using the Affymetrix HT HG-U133+ GeneChip®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms. Transcription of selected genes of interest was confirmed by quantitative PCR. Results Compared to ASCs in complete medium, 190 and 108 genes were significantly altered by serum deprivation and serum deprivation combined with VEGF, respectively. No significant differences in gene expression patterns between serum-deprived ASCs and serum-deprived ASCs combined with VEGF stimulation were found. Genes most prominently and significantly upregulated by both conditions were growth factors (IGF1, BMP6, PDGFD, FGF9), adhesion molecule CLSTN2, extracellular matrix-related proteins such as matricellular proteins SMOC2, SPON1 and ADAMTS12, and inhibitors of proliferation (JAG1). The most significantly downregulated genes included matrix metalloproteinases (MMP3, MMP1), and proliferation markers (CDKN3) and GREM2 (a BMP6 antagonist). Conclusion The decisive factor for the observed change in ASC gene expression proves to be serum starvation rather than VEGF stimulation. Changes in expression of growth factors, matricellular proteins and matrix metalloproteinases in concert, diverge from direct pro-angiogenic paracrine mechanisms as a primary consequence of the used protocol. In vitro serum starvation (with or without VEGF present) appears to favour cardioprotection, extracellular matrix remodelling and blood vessel maturation relevant for the late maturation phase in infarct healing.

The clinical consequence of the increasing incidence of coronary artery disease is a growing problem worldwide and ischemic heart disease remains the most common cause of death and a major cause of hospital admissions in industrialised countries. Early treatment with stabilizing drugs and mechanical revascularization by percutaneous coronary intervention or coronary by-pass surgery has reduced the mortality significantly. However, there is still a group of patients, which cannot be treated satisfactorily with these interventions. Treatment with genes encoding vascular growth factors or stem cells with the potential to regenerate the damaged myocardium is a relatively new approach. The results from early clinical studies on gene and stem cell therapy for cardiac regeneration in patients with acute or chronic ischemic heart disease have been inconsistent. Some of the discrepancy could be due to differences in study designs or patient selection. This review will present the conducted clinical trials and try to clarify the influence of patient selection, chosen cell type, cell source, delivery methods and mechanisms of action on the differences in results.

In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas.

BACKGROUND:Some studies indicate that a large part of the beneficial effect of physical activity on mortality is confined to a threshold effect of participation.METHODS:Self-reported physical activity was investigated in relation to all-cause mortality in the Danish Diet, Cancer and Health cohort, including 29,129 women and 26,576 men aged 50-64 years at baseline 1993-1997. Using Cox proportional hazards models we investigated the associations between mortality rate and leisure time physical activity by exploring 1) participation (yes/no) in each type of activity; 2) a simple dose-response relationship with hours spent on each activity, supplemented with indicators of participation in each activity; and 3) inflexion or nonmonotonic dose-response relationships using linear splines.RESULTS:A total of 2696 women and 4044 men died through March 2010. We found lower mortality with participation in sports (for women, mortality rate ratio = 0.75, 95% confidence interval = 0.69-0.81; for men, 0.78, 0.73-0.84), cycling (for women, 0.77, 0.71-0.84; for men, 0.90, 0.84-0.96), or gardening (for women, 0.84, 0.78-0.91; for men, 0.73, 0.68-0.79) and in men participating in do-it-yourself activity (0.77, 0.71-0.84). A weak adverse dose response was seen for walking and gardening, but the association was small (1-2% increase in mortality per additional hour). We found no signs of inflexion or nonmonotonic effects of additional hours spent on each activity.CONCLUSION:Mortality was lower with participation in specific leisure time physical activities, but not with more time spent on those activities. This could suggest that avoiding a sedative lifestyle is more important than a high volume of activity. Nonparticipation in these types of physical activity may be considered as risk factors. Some studies indicate that a large part of the beneficial effect of physical activity on mortality is confined to a threshold effect of participation.