Danish phase II trial using adipose tissue derived mesenchymal stromal cells for patients with ischaemic heart failure.

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.

Effect of allogeneic adipose tissue-derived mesenchymal stromal cell treatment in chronic ischaemic heart failure with reduced ejection fraction - the SCIENCE trial.

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.

GMP Compliant Production of a Cryopreserved Adipose-Derived Stromal Cell Product for Feasible and Allogeneic Clinical Use.

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.

Optimizing an immunomodulatory potency assay for Mesenchymal Stromal Cell.

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.

Adipose-derived stromal cells increase the formation of collagens through paracrine and juxtacrine mechanisms in a fibroblast co-culture model utilizing macromolecular crowding.

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.