Mesenchymal Stromal Cell-Based Products: Challenges and Clinical Therapeutic Options.

Mesenchymal stromal cell (MSC)-based advanced therapy medicinal products (ATMPs) are being tried in a vast range of clinical applications. These cells can be isolated from different donor tissues by using several methods, or they can even be derived from induced pluripotent stem cells or embryonic stem cells. However, ATMP heterogeneity may impact product identity and potency, and, consequently, clinical trial outcomes. In this review, we discuss these topics and the need to establish minimal criteria regarding the manufacturing of MSCs so that these innovative therapeutics may be better positioned to contribute to the advancement of regenerative medicine.

Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors.

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants.

Chromosomal aberrations after induced pluripotent stem cells reprogramming.

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton's jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton's jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.

Therapy with Cardiomyocytes Derived from Pluripotent Cells in Chronic Chagasic Cardiomyopathy.

Chagas disease discovered more than a century ago remains an incurable disease. The objective of this work was to investigate the therapeutic potential of cardiomyocytes derived from mouse embryonic stem cells (CM-mESC) in a model of chronic Chagasic cardiomyopathy (CCC). Mouse embryonic stem cells (mESC) were characterized, transduced with luciferase, and submitted to cardiac differentiation. CM-mESC were labeled with superparamagnetic iron oxide particles. To induce CCC, mice were infected with Brazil strain trypomastigotes. At 150 days post-infection (dpi), infected animals were treated with CM-mESC or PBS. Cells were detected by magnetic resonance imaging (MRI) and bioluminescence. Cardiac function was evaluated by MRI and electrocardiogram at 150 and 196 dpi. CCC mice showed significant differences in MRI and ECG parameters compared to non-infected mice. However, no differences were observed in contractile and electrical parameters between cell and PBS injected groups, 45 days after cell transplantation. Cells were detected 24 h after transplantation by MRI. CM-mESC bioluminescence tracking demonstrated over 90% decrease in signal 8 days after treatment. Nevertheless, the Infected + CM-mESC group showed a significant reduction in the percentage of collagen fibers when compared to the Infected + PBS group. In conclusion, CM-mESC therapy was not effective in reversing cardiac functional changes induced by Chagas disease despite some improvement in myocardial fibrosis.

Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy.

BACKGROUND: Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS: The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS: mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS: CM-mESC transplantation improves cardiac function in mice with DIC.

Cardiosphere-derived cells do not improve cardiac function in rats with cardiac failure.

BACKGROUND: Heart failure represents an important public health issue due to its high costs and growing incidence worldwide. Evidence showing the regenerative potential of postmitotic heart tissue has suggested the existence of endogenous cardiac stem cells in adult hearts. Cardiosphere-derived cells (CDC) constitute a candidate pool of such cardiac stem cells. Previous studies using acute myocardial infarction (MI) models in rodents demonstrated an improvement in cardiac function after cell therapy with CDC. We evaluated the therapeutic potential of CDC 60 days after MI in a rat model. METHODS: CDC were obtained from human discarded myocardial tissue and rat hearts by enzymatic digestion with collagenase II. At 10-15 days after isolation, small, round, phase-bright cells (PBCs) appeared on top of the adherent fibroblast-like cells. The PBCs were collected and placed on a nonadherent plate for 2 days, where they formed cardiospheres which were then transferred to adherent plates, giving rise to CDC. These CDC were characterized by flow cytometry. Wistar rats were submitted to MI through permanent occlusion of the anterior descending coronary artery. After 60 days, they were immunosuppressed with cyclosporine A during 10 days. On the third day, infarcted animals were treated with 5 × 105 human CDC (hCDC) or placebo through intramyocardial injection guided by echocardiogram. Another group of animals was treated with rat CDC (rCDC) without immunosuppression. hCDC and rCDC were stably transduced with a viral construct expressing luciferase under control of a constitutive promoter. CDC were then used in a bioluminescence assay. Functional parameters were evaluated by echocardiogram 90 and 120 days after MI and by Langendorff at 120 days. RESULTS: CDC had a predominantly mesenchymal phenotype. Cell tracking by bioluminescence demonstrated over 85% decrease in signal at 5-7 days after cell therapy. Cardiac function evaluation by echocardiography showed no differences in ejection fraction, end-diastolic volume, or end-systolic volume between groups receiving human cells, rat cells, or placebo. Hemodynamic analyses and infarct area quantification confirmed that there was no improvement in cardiac remodeling after cell therapy with CDC. CONCLUSION: Our study challenges the effectiveness of CDC in post-ischemic heart failure.

Generation of human iPS cell line ihFib3.2 from dermal fibroblasts.

The human ihFib3.2 iPS cell line was generated from dermal fibroblasts obtained from a healthy donor. Lentiviral particles were produced with the polycistronic hSTEMCCA vector with Oct4, Sox2, cMyc and Klf4 as reprogramming factors.