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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.

Abstract 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).

Abstract 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.

Lynggaard et al. profile the salivary proteome and metaproteome in patients with head and neck cancer who have received radiation therapy and an intraglandular mesenchymal stem cell (MSC) treatment for radiation-induced xerostomia and in healthy controls. MSC therapy impacts the composition of the salivary proteome in the longer-term.

Abstract 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.

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.

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.

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.

Abstract 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

Allogeneic cell-based therapies using adipose tissue-derived stromal cells (ASCs) offer an off-the-shelf alternative to autologous therapy. An underlying assumption is that ASC can modulate the immune response of the recipient. However, in vitro models are required to explore and identify cell interactions and mechanisms of action, to ensure sufficient and sustained effects, and to document these. In this study, we shed light on the effect of ASC manufactured for clinical use on monocyte-derived dendritic cells and an inflammatory microenvironment. ASCs were isolated from healthy voluntary donors, expanded using a human platelet lysate in bioreactors, and cryopreserved as per clinical use. Monocyte-derived dendritic cells were generated by isolation of monocytes and differentiation with GM-CSF and IL-4. Dendritic cells were cocultured with different ratios of ASC and matured with LPS and IFN-γ. Dexamethasone was included as an immunosuppressive control. Dendritic cells were analyzed by flow cytometry for CD11c, CD40, CD80, CD83, CD86, PD-L1, and HLA-DR, and supernatants were analyzed for FGF2, HGF, IL-10, IL-12p70, LIF, MIF, PDGF, PlGF, and IDO. Reduced expression of maturation markers was observed on ASC-treated dendritic cells, while high levels of PD-L1 were maintained. Interestingly, the expression of CD83 was elevated. Escalating ratios of ASC did not affect the concentration of IL-10 considerably, whereas the presence of IL-12 was reduced in a dose-dependent manner. Besides offsetting the IL-12/IL-10 balance, the concentrations of IDO and MIF were elevated in cocultures. Concentrations of FGF2, HGF, LIF, and PIGF were high in ASC cocultures, whereas PDGF was depleted. In a robust coculture model, the addition of ASC to dendritic cells inhibited the dendritic maturation substantially, while inducing a less inflammatory and more tolerogenic milieu. Despite the exposure to dendritic cells and inflammatory stimuli, ASC resulted in supernatants with trophic factors relevant for regeneration. Thus, ASC can perform immunomodulation while providing a regenerative environment.

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.

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.

Abstract 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.

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.

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. 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.