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Cardiovascular diseases represent the world's leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.

There is a constant need for improving embryo culture conditions in assisted reproduction. One possibility is to use mesenchymal stem/stromal cells derived from menstrual blood (mbMSCs), with an endometrial origin. In this study, we sought to analyze the expansion of mouse embryos in a direct coculture model with mbMSCs. Our results showed that after five passages, mbMSCs presented a spindle-shaped morphology, with surface markers that were comparable with the normal mesenchymal cell phenotype. mbMSCs could differentiate into adipogenic and osteogenic lineages and secrete angiopoetin-2 and hepatocyte growth factor. The coculture experiments employed 103 two-cell-stage embryos that were randomly divided into two groups: control (

Background The acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is one of the more frequent and severe processes, which, with very high mortality, affects renal structures and function. The first hours of lesion are critical for organ recovery, but the mechanisms of lesions to support early beneficial therapeutic actions are not well understood. AKI is characterized by intense production of anion superoxide (O2•-) and oxidative damage. In this study we investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administrated at the moment of reperfusion, suppresses the exacerbated mitochondrial O2•- formation in the first 24 h after I/R. Methods Rats were submitted to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received subcapsularly EVs (I/R+EVs group) or saline (I/R group). The 3rd group was of the false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and immediately used. EVs were characterized by cytometry (CD9, TSG101, CD63, CD81) and transmission electron microscopy. The probe Amplex Red™ was used to measure mitochondrial O2•- formation and MitoTracker® Orange to evaluate the mitochondrial electrical potential (Δψ). In vitro studies were carried out by using human renal cells (HK-2) co-cultured or not with EVs under hypoxia conditions. Results Administration of EVs to I/R rats restored O2•- formation to SHAM levels in all mitochondrial functional conditions: non-phosphorylating, phosphorylating, with ATP synthesis blocked and uncoupled. Proton leak was not modified. During this period, AKI remained active, as demonstrated by qRT-PCR analysis of the lesion biomarkers Kidney Injury Molecule-1 and Neutrophil Gelatinase-Associated Lipocaline. Tubular catalase and superoxide dismutase mRNA remained low. Heme oxygenase-1 (HO-1) was intensely upregulated. The co-cultures of HK-2 cells with EVs revealed decrease of apoptosis. Conclusions These findings provide evidence that the mechanisms in which rely the early recovery by EVs of the renal structure and function after I/R are those related with the normalization of the mitochondrial redox environment and open new vistas for the acellular treatment of AKI.

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.

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.

The COVID-19 pandemic had a profound impact on the operation of Brazilian hospital units, even those dedicated to non-infectious diseases. This study aims to describe the Covid-19 epidemic curve from a cardiovascular specialized nosocomial unit. All symptomatic employees were submitted to RT-qPCR. A total of 613 tests were performed on 548 employees between March 23, 2020, and June 4, 2020; with 45.7% positivity from the samples, representing 11.9% of the total employees. The epidemic curve showed a profound drop after first week of May. The data showed a high contamination rate despite widespread availability of personal protective equipment and employees’ training.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.

The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.

Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.

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. Electronic supplementary material The online version of this article (10.1186/s13287-018-0788-2) contains supplementary material, which is available to authorized users.

Adult peripheral nerves in vertebrates can regrow their axons and re-establish function after crush lesion. However, when there is extensive loss of a nerve segment, due to an accident or compressive damage caused by tumors, regeneration is strongly impaired. In order to overcome this problem, bioengineering strategies have been employed, using biomaterials formed by key cell types combined with biodegradable polymers. Many of these strategies are successful, and regenerated nerve tissue can be observed 12 weeks after the implantation. Mesenchymal stem cells (MSCs) are one of the key cell types and the main stem-cell population experimentally employed for cell therapy and tissue engineering of peripheral nerves. The ability of these cells to release a range of different small molecules, such as neurotrophins, growth factors and interleukins, has been widely described and is a feasible explanation for the improvement of nerve regeneration. Moreover, the multipotent capacity of MSCs has been very often challenged with demonstrations of pluripotency, which includes differentiation into any neural cell type. In this study, we generated a biomaterial formed by EGFP-MSCs, constitutively covering microstructured filaments made of poly-ε-caprolactone. This biomaterial was implanted in the sciatic nerve of adult rats, replacing a 12-mm segment, inside a silicon tube. Our results showed that six weeks after implantation, the MSCs had differentiated into connective-tissue cells, but not into neural crest-derived cells such as Schwann cells. Together, present findings demonstrated that MSCs can contribute to nerve-tissue regeneration, producing trophic factors and differentiating into fibroblasts, endothelial and smooth-muscle cells, which compose the connective tissue.

Background PRKAG2 cardiomyopathy is an autosomal dominant syndrome due to mutation in PRKAG2 gene, which encodes the γ2 subunit of the adenosine monophosphate (AMP)-activated protein kinase (AMPK). PRKAG2 syndrome leads to intracellular cardiomyocyte glycogen storage dysfunctions and clinical conditions that overlap hypertrophic cardiomyopathy, hampering its correct diagnosis. Our group identified a patient with a novel heterozygous missense mutation in PRKAG2 gene (c.1203C>A, p.His401Gln), with a malignant disease phenotype characterized by severe cardiac hypertrophy and cases of sudden death in young relatives. In this regard, induced pluripotent stem cells (iPSC) preserve the donor genome and can be differentiated into specialized cell types, being particularly interesting to study diseases that affect tissues which are difficult to access. Thus, this work aims to establish an in vitro study model of PRKAG2 cardiomyopathy through generation of human iPSC and its subsequent differentiation into patient-specific cardiomyocytes. Methods iPSC from patient with PRKAG2 mutation and from a health relative were generated from erythroblasts using CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) and their karyotype was analyzed by qPCR using hPSC Genetic Analysis Kit (Stemcell Technologies). The iPSC strains obtained were differentiated into cardiomyocytes by inducing mesoderma formation with glycogen synthase kinase inhibitor 3-β, CHIR99021 (5 µM), on day 0 and cardiogenic mesoderma formation with the Wnt inhibitor, Wnt-C59 (2 µM), on day 2. On day 30 of culture, cells were stained with periodic acid-Schiff (PAS) to assess cytosolic glycogen storage. Results Both iPSC strains have embryonic-like phenotype and normal karyotype. Moreover, preliminary differentiation studies suggest glycogen accumulation in cardiomyocytes derived from the iPSC strain with PRKAG2 mutation. Evaluation of pluripotency factors expression and the ability to differentiate in the 3 embryonic germinative layers are ongoing in iPSC strains. Conclusion At the end of the characterization of the generated iPSC and of iPSC-derived cardiomyocytes, is expected that this approach will make possible the establishment of an in vitro study platform to reveal molecular mechanisms triggered by the c.1203C>A mutation that may contribute to the development of new therapies and prognostic evaluation of patients with PRKAG2 syndrome.

Background Chagas disease is an infectious disease caused by the protozoan Trypanosoma cruzi and can lead to cardiac dysfunction characterized by arrhythmias and heart failure. The presence of circulating autoantibodies capable of recognizing G-protein coupled receptors (GPCR) and activating β-adrenergic and muscarinic receptors has been proposed as an interactive and relevant pathophysiological factor in the development of the disease. However, the interaction of the sera from chronic chagasic patients with human cardiomyocytes has never been tested. This study investigated if chronic chagasic sera previously shown to activate β-adrenergic and muscarinic receptors in rodent and rabbit hearts could also activate these receptors in human cardiomyocytes. Methods An induced pluripotent stem cell (iPS) line derived from a healthy donor, previously generated, and characterized in our laboratory, was differentiated into cardiomyocytes (CM-iPS) and their electrophysiology was characterized by Multielectrode Array (MEA) system. We evaluated the effects of an anti-β-adrenergic (Aβ82) and an anti-muscarinic serum (M32) on spontaneous beat rate (BR), field potential amplitude (FPA), and field potential duration (FPD). To minimize the effect of BR on FPD, FPD was corrected (FPDc) using an adaptation of the Bazett equation (FPDc = FPD/√BP), where BP is the interval between spontaneous beats. During recordings, CM-iPS were continuously perfused with Tyrode solution containing or not chagasic sera from patients diluted at 1:100 (v:v). Results Sera Aβ-82 induced an increase in BR (16,90±4,22 bpm to 26,60±5,62 bpm, n=5, p=0.0177), FPD (164,03±6,18 ms to 208,73±9,44 ms, n=5, p Conclusion We conclude that antibodies present in chronic chagasic patients‘ sera are capable of modulating GCPR activity in the human heart interacting with β-adrenergic and muscarinic receptors present in human cardiomyocytes.

Background/Objective One of the main cells in the liver is the hepatocyte, which regulates the metabolism of various nutrients and xenobiotics, mediates endocrine function and excretion of toxic substances. Hepatocytes function in vitro have been studied for many years using primary hepatocytes and immortalized cells. Nevertheless, these cells show phenotypic instability or loss of adult liver functions and altered expression of important transcription factors. As an alternative to obtain a stable genetic background with preserved function and in large quantities, hepatocytes are being generated through differentiation of induced pluripotent stem cells (iPSC). However, one of the main problems to be overcome is the phenotype maturation. In this context, the objective of this work was to compare two different hepatocyte differentiation protocols using spheroids derived from iPSC. Methods Immunofluorescence for OCT3/4 and Nanog was performed to characterized pluripotent phenotype of iPSC and hepatocyte differentiation was induced for 28 days (D28). Two plating strategies were adopted to form the definitive endoderm: iPSC-monolayers (M) and iPSC-spheroids (S). To induce definitive endoderm, activin A and CHIR99021 were added to both plating conditions for 5 days. Prior hepatic maturation, iPSC-monolayers were also aggregated to form spheroids (MS). Subsequently, HGF and SB431542 were added to MS and S medium for 23 days. Spheroids were evaluated by real time PCR and immunofluorescence for hepatocyte-specific markers at D28. Results At protein level, the iPSC showed a pluripotent character through the nuclear presence of OCT3/4 and NANOG. At the end of the differentiation protocol, S differentiation strategy exhibited contractile cells. Albumin and cardiac troponin T were detected into the same spheroid by immunofluorescence. The MS strategy showed no beating cells. Also, CYP3A4 and albumin were detected by immunofluorescence and detectable mRNA levels were expressed for HNF4α, transferrin and alpha-1 antitrypsin. Conclusions Hepatocyte differentiation using spheroids requires a monolayer endoderm formation step in order to avoid the presence contractile cells at the end of protocol. Moreover, spheroid cells exhibited a great mature hepatocyte phenotype and can be used as a powerful tool for cell therapy, liver bioengineering, studies of drug efficacy and toxicity assays.

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-β1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.

This is a pilot clinical study primarily designed to assess the feasibility and safety of X-ray-guided percutaneous intraspinal injection of allogeneic canine adipose tissue-derived mesenchymal stem cells in dogs with chronic spinal cord injury. Six dogs with chronic paraplegia (≥six months) were intraparenchymally injected with allogeneic cells in the site of lesion. Cells were obtained from subcutaneous adipose tissue of a healthy dog, cultured to passage 3, labeled with 99mTechnetium, and transplanted into the lesion by percutaneous X-ray-guided injection. Digital X-ray efficiently guided cell injection as 99mTechnetium-labeled cells remained in the injection site for at least 24 hours after transplantation. No adverse effects or complications (infection, neuropathic pain, or worsening of neurological function) were observed during the 16-week follow-up period after transplantation. Three animals improved locomotion as assessed by the Olby scale. One animal walked without support, but no changes in deep pain perception were observed. We conclude that X-ray-guided percutaneous intraspinal transplantation of allogeneic cells in dogs with chronic spinal cord injury is feasible and safe. The efficacy of the treatment will be assessed in a new study involving a larger number of animals.

Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Omics approaches have significantly impacted knowledge about molecular signaling pathways driving cell function. Induced pluripotent stem cells (iPSC) have revolutionized the field of biological sciences and proteomics and, in particular, has been instrumental in identifying key elements operating during the maintenance of the pluripotent state and the differentiation process to the diverse cell types that form organisms. This review covers the evolution of conceptual and methodological strategies in proteomics; briefly describes the generation of iPSC from a historical perspective, the state-of-the-art of iPSC-based proteomics; and compares data on the proteome and transcriptome of iPSC to that of embryonic stem cells (ESC). Finally, proteomics of healthy and diseased cells and organoids differentiated from iPSC are analyzed.

Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Background Anthracyclines (AC) are chemotherapeutic drugs used for treatment of various types of cancer. However, up to 26% of patients develop anthracycline-induced cardiotoxicity (AIC), ranging from mild arrhythmias to severe ventricular dysfunction. This study investigates the ability of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) to recapitulate in vitro the susceptibility that certain patients have to develop AIC. Methods Patients treated with cumulative dose ≥200 mg/m2 of AC were recruited, as well as age- and gender-matched healthy volunteers (HV). iPSCs were derived from erythroblasts, evaluated for pluripotency and genomic stability, and differentiated into CM. At day 30, CMs were treated for 72h with 0.01-100µM Doxorubicin (DOX) alone or in presence of 1-100µM cardioprotectant Dexrazoxane (DRZ), 1-100µM of its metabolite ADR925 or 1mM N-acetylcysteine (NAC). Treated CMs were evaluated for viability and sarcomeric organization. Next-Generation Sequencing (NGS) was performed for cardiac targets in iPSC (5 AC-sensitive, 5 AC-resistant). Results iPSCs presented normal karyotype (G-banding assay) and pluripotency (RT-PCR: Oct4, Sox2, Nanog, Klf4; flow cytometry: OCT4, SOX2, NANOG; spontaneous differentiation into three germ layers RT-PCR: Tubulinβ3, Bmp4, Afp). CMs expressed cardiac Troponin T (cTnT) (AC-S: 82.45±14.09%; HV: 90.45±4.99%; n=6). Increasing doses of DOX led to higher mortality rates (AC-S: 0.1µM–15.09±6.33%; 1µM–46.62±13.27%; 10µM–64.46±21.99%; 100µM–96.1±5.55%; n=10; HV: 0.1µM–3.65±16.37%; 1µM–27.35±25.56%; 10µM–56.2±25.82%; 100µM–89.75±11.63%; n=6). Cotreatment with cardioprotectants showed no significant difference in cell viability (IC50 in µM AC-S: DOX=3.18±2.95, n=10; DOX+100µM DRZ=2.84±2.24, n=5, p=0.82; DOX+100µM ADR925=5.56±2.91, n=4, p=0.19; DOX+1mM NAC=2.45±2.51, n=6, p=0.62; HV: DOX=10.23±10.07, n=6; DOX+100µM DRZ=1.39, n=1; DOX+100µM ADR925=6.56±7.02, n=3, p=0.59; DOX+1mM NAC=3.64±4.72, n=4, p=0.26). cTnT staining revealed sarcomeric disorganization for DOX≥10µM. NGS sequencing revealed 49 nonsynonymous variants, none of which associated with AIC. Only one of those variants, located at desmoplakin gene, was present in all 4 AC-S patients and absent in AC-R ones. Conclusion iPSC from AC-S and AC-R patients were successfully generated and differentiated into CM. Viability tests recapitulated the patient's clinical cardiotoxicity and confirmed iPSC-CM as a platform for drug screening.

Background Human induced pluripotent stem (iPS) cell-derived cardiomyocytes (hiPSC‐CMs) technology offers an unprecedented tool to study cardiac diseases. Long QT syndrome type 1 (LQT1) is an inherited cardiac channelopathy caused by mutations in KCNQ1, which encodes the main subunit of the potassium channel Kv 7.1, leading to delayed repolarizations and imposing potentially fatal arrhythmias on patients. Therefore, the in vitro patient-specific modeling of LQT1 is crucial to test the pathogenicity of the KCNQ1- I588T mutation. Objective The purpose of this study was to test a cellular model of LQT1 based on a KCNQ1-I588T mutation using hiPSC‐CMs. Methods Peripheral blood was collected from a proband (a 15-year-old LQT1 symptomatic patient carrying KCNQ1-I588T mutation), a carrier donor (non-symptomatic familial carrying KCNQ1 mutation), and an unrelated control donor. The erythroblast enriched fraction was reprogrammed into iPS using Sendai virus vector. iPS were characterized for pluripotency markers by RT-PCR and flow cytometry, in vitro spontaneous differentiation, and karyotype stability. iPS were differentiated into cardiomyocytes and electrophysiological studies were performed. Results The proband was clinically diagnosed with LQT1 with resting corrected QT interval equal to 516 ms. The KCNQ1 mutation (c.1763T>C; p.I588T) was identified by NGS in the proband DNA sample. By Sanger, the same mutation was identified in the familial carrier while it was absent in the control subject. Erythroblasts were successfully reprogrammed into iPS. All iPS expressed the 13 different pluripotent markers evaluated, differentiated into three germ layers and presented a normal chromosomic profile. The differentiation protocol generated cells positive for cardiac troponin T. As expected, proband hiPSC‐CMs exhibited prolongation of action potential duration (APD90 = 312 ± 79 ms) when compared to carrier donor and control donor hiPS-CMs (APD90 = 177 ± 68 and 271 ± 71 ms, respectively). Since the mutation is the same in both the proband and the carrier, a protective/damaging molecular regulation must be involved. We speculate that a polymorphism identified only in the proband (SCN5A c.1673A>G; p.H558R) may be involved in this mechanism. Conclusions We identified an LQT1 causative variant that successfully recapitulated LQT1 proband phenotype by in vitro modeling. We also propose the existence of a second variant with a damaging effect on LQT1 symptom development.

Background Anthracyclines are highly effective chemotherapeutic drugs commonly used to treat various types of tumors, however, they induce cardiotoxicity in about 30% of patients. So far, it is uncertain what induces individuals to develop anthracycline-induced cardiotoxicity (AIC), but it is well known that the damage is proportional to the cumulative dose used. In this context, the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) represents an alternative for in vitro disease modeling. However, most differentiation protocols use the ‘B27’ supplement, a compound rich in antioxidant molecules. It's likely that the usage of this supplement results in a cardioprotective effect on iPSC-CMs derived from AIC patients, possibly altering their ability to mimic the sensitive phenotype. This work aimed to establish a cardiac differentiation protocol for patient-specific iPSCs using a chemically defined medium (CDM) without antioxidant molecules. Methods A patient who was treated with anthracycline (240 mg/m2 doxorubicin, 300 mg/m2 epirubicin) and developed heart failure (ejection fraction = 33%) 7 years after the end of treatment was recruited, as well as a healthy unrelated donor. Patient-specific iPSCs underwent cardiac differentiation using two distinct protocols that modulate the Wnt canonical pathway: P1 - using B27 (RPMI 1640, B27 serum free supplement 1x or B27 minus insulin 1x) and P2 - CDM (RPMI 1640, 213 μg/mL L-ascorbic acid 2-phosphate, 500 μg/mL bovine serum albumin). After thirty days of culture (D30), the efficiency of differentiation was evaluated and compared between P1 and P2 through the expression of cardiac troponin T (cTnT) via flow cytometry. Results iPSCs derived from the sensitive patient (SP) and the healthy donor (HD) were successfully differentiated into CMs using both protocols (starting cell density SP: P1 and P2 = 4.2 × 105 cells/cm2; HD: P1 = 4.7 × 105 cells/cm2, P2 = 4.4 × 105 cells/cm2; CHIR99021 concentration SP: P1=9μM, P2=6μM; HD: P1=9μM, P2=7μM). CMs presented spontaneous contractions in vitro from D8. Both protocols attained high differentiation efficiencies, with no significant difference between them (cTnT SP: P1 = 82.45±14.09%, n=6; P2 = 81.13±12.99%, n=7, p=0.8635; HD: P1 = 90.45±4.99%, n=6; P2 = 91.80±3.68%, n=2). Conclusion A protocol for differentiating SP- and HD-iPSCs into CMs without using antioxidants has been successfully established, thus enabling its use in future drug screening experiments.

Background Human hepatocytes, within liver in vivo milieu, are capable of proliferation, but such characteristic is not reproducible in vitro when they were cultured over extended periods. In this scenario, induced pluripotent stem cells (iPS) arise as a promising source of hepatocytes, however, the differentiation outcome is still a challenge. In this study hepatocytes (iHep) differentiated from human iPS (hiPS) cells were characterized to evaluate the hepatic phenotype. Methods The hiPS cells differentiation protocol was carried out using RPMI medium supplemented from day 1 (D1) until D2 with B27- insulin (1%), Activin-A (100 ng/mL) and CHIR99021 (3 uM), from D3 to D5 the CHIR was removed. From D5 to D10, B27 (1%) and HGF (20 ng/mL) were added, and at D11 the HGF was replaced by SB431542 (1 uM) until the end of protocol, at D28. To evaluate differentiation's efficacy the expression and presence of hepatic markers were performed. Albumin (ALB), alfa-fetoprotein (AFP), hepatocyte nuclear factor4 (HNF4), CYP2D6, ASGR1 and Transferrin by qPCR, ALB and HNF4 by immunofluorescence, and ALB secretion by ELISA. Data were analyzed by Student's t test for paired variables and differences were considered significant at p Results The iHep cells expressed all hepatic genes evaluated, by qPCR, similar to HepG2. iHep also presented hepatic proteins (ALB and HNF4), detected by immunofluorescence. ELISA assay showed that iHep secreted ALB (96 ng/mL). As expected, HepG2 secreted ALB (263 ng/mL) and no ALB detection were observed in hiPS. The transcriptome analysis showed that iHep cells expressed hepatocyte-specific transcripts which were not found in hiPS. Conclusion The differentiation protocol from hiPS to iHep showed efficacy in generating hepatocyte-like cells and transcriptome analysis showed high genes similarity between iHep cells compared to human hepatocytes.

Background JLNS is a rare channelopathy caused by mutations in the KCNQ1 and KCNE1 genes that encodes the α and β subunits of the IKS current channel. Patients with JLNS have congenital bilateral deafness and prolongation of the QT interval on electrocardiogram (ECG), which can lead to arrhythmias, syncope, and a high risk of sudden death. Induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) can be a useful tool to recapitulate the disease phenotype in vitro enabling the comprehension of its molecular and cellular mechanisms. Methods The DNA of a proband with clinical symptoms of JVLNS was analyzed by next-generation sequencing. Sanger was done on samples from family members. iPSC were generated from the proband (Pac6), a cousin (Pac53) suspect of JLNS, the grandfather (Pac25), his sister (Pac24), and his mother (Pac8). iPSC were evaluated for euploidy and characterized for pluripotency by PCR, flow cytometry, and immunohistochemistry. iPSC were differentiated into CMs and its efficiency was assessed by flow cytometry. Electrophysiology was evaluated by intracellular microelectrode (PA) and microelectrode array (MEA). Results A homozygous variant c.1394-2A>G was identified in the KCNQ1 gene of Pac6 and Pac53. Both have congenital bilateral deafness and a QTc interval of 670 and 660ms respectively. Pac25 and Pac24 have no mutation and Pac8 have the variant in heterozygosis (Long QT syndrome type 1 - LQTS1). iPSC showed pluripotency markers in PCR and Flow Cytometry (OCT3/4, SOX2, NANOG, etc) and the capacity to differentiate in 3 embryonic layers (shown by immunohistochemistry and PCR). iPSC karyotype was normal, except for Pac53, in which there‘s a translocation between chromosomes 14 and 22. The iPSC-CM had differentiation efficiency above 70%. Surprisingly, electrophysiology functional analysis of action potential duration at 90% of repolarization (APD90) showed no significant differences between Pac6 (APD90 = 255,67 ± 39,19) and Pac53 (APD90 = 315,45 ± 74,71) compared to Pac24 (APD90 = 262,22 ± 62,81) and Pac25 (APD90 = 215,43 ± 73,83). Similar results were observed when analyzed by MEA. Conclusion This work is unique to modeling a non-described mutation in the KCNQ1 gene present in a family with two JLNS and one asymptomatic LQTS1. The complete understanding of why the CM-iPSC failed to mimetics in vitro disease demands further investigation challenging the cells with pro-arrhythmic drugs and iKS inhibitors.

Background/Objective Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a single point mutation in the lamin A gene that leads to the production of a truncated protein, Progerin, and accelerated aging, which culminates in the patient's death mainly due to cardiovascular complications. Since the pathogenesis of HGPS shares some mechanisms with normal aging, it gives an opportunity to understand the cellular and molecular mechanisms of this natural phenomenon. Therefore, the aim of the present study was to generate cardiomyocytes from induced pluripotent stem cells of HGPS patients (HGPS-CM) in order to provide a powerful in vitro platform for the study of age-related cardiac disease. Methods Induced pluripotent stem cell line was obtained from the Progeria Research Foundation Cell and Tissue Bank (HGPS-iPSC). HGPS-iPSC pluripotency was characterized by RT-PCR. G-banding patterns were evaluated so as to ensure that these cells have a normal karyotype. For cardiomyocyte differentiation, 2,1 × 105/cm2 and 4,2 × 105/cm2 HGPS-iPSC were cultured for 30 days in RPMI 1640 medium supplemented with recombinant human albumin and L-ascorbic acid 2-phosphate. Forty-eight hours later (day 0), 5, 6, or 7 µM CHIR99021 was added to induce mesoderm formation. On day 2, 2 µM Wnt-C59 was used to specify in cardiogenic mesoderm. Progerin and SERCA2A expression in HGPS-iPSC and HGPS-CM were analyzed by RT-PCR. Cardiac differentiation efficiency was analyzed by the percentage of positive cells for cardiac troponin T using flow cytometry. Moreover, immunostaining for the same protein was performed to evaluate the contractile structure. Results HGPS-iPSC expressed OCT3/4, NANOG, SOX2, KLF4, confirming the pluripotent state and exhibited normal karyotype after fifty passages. After 10 days of differentiation, it was possible to visualize contractile cells only in 5 µM CHIR99021 condition, for both cell concentrations. On the thirtieth day of differentiation, progerin and SERCA2A mRNA were only detected in HGPS-CM and approximately seventy percent were positive to cardiac troponin T. Moreover, the presence of troponin T revealed that HGPS-iPSC derived cardiomyocytes showed a striated pattern, resembling sarcomere structures. Conclusions In this work, we established an efficient protocol to obtain cardiomyocytes using iPSC from HGPS patients, which can be used as an important tool for the study of novel mechanisms underlying cardiac aging.

Several therapies are being developed to increase blood circulation in ischemic tissues. Despite bone marrow-derived mesenchymal stromal cells (bmMSC) are still the most studied, an interesting and less invasive MSC source is the menstrual blood, which has shown great angiogenic capabilities. Therefore, the aim of this study was to evaluate the angiogenic properties of menstrual blood-derived mesenchymal stromal cells (mbMSC) in vitro and in vivo and compared to bmMSC. MSC`s intrinsic angiogenic capacity was assessed by sprouting and migration assays. mbMSC presented higher invasion and longer sprouts in 3D culture. Additionally, both MSC-spheroids showed cells expressing CD31. mbMSC and bmMSC were able to migrate after scratch wound in vitro, nonetheless, only mbMSC demonstrated ability to engraft in the chick embryo, migrating to perivascular, perineural, and chondrogenic regions. In order to study the paracrine effects, mbMSC and bmMSC conditioned mediums were capable of stimulating HUVEC&rsquo, s tube-like formation and migration. Both cells expressed VEGF-A and FGF2. Meanwhile, PDGF-B was expressed exclusively in mbMSC. Our results indicated that mbMSC and bmMSC presented a promising angiogenic potential. However, mbMSC seems to have additional advantages since it can be obtained by non-invasive procedure and expresses PDGF-B, an important molecule for vascular formation and remodeling.

Background: Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by accelerated aging and high incidence of cardiovascular complications and metabolic alterations. However, the underlying mechanisms of this syndrome are not fully understood. Methods and Results: This study modelled HGPS using cardiomyocytes generated from induced pluripotent stem cells (iPSc) derived from a patient affected by the syndrome, characterizing the molecular and biophysical alterations found in cardiomyocytes obtained from three distinct differentiation protocols run in parallel with a control iPSc. Electrophysiology recordings were performed with intracellular microelectrodes and the Progeria cardiomyocytes fired action potentials similar to those from control iPSc-derived cardiomyocytes. High resolution respirometry performed on Oroboros system in isolated cardiomyocytes showed that Progeria cardiomyocytes have a tendency to decrease the oxygen consumption under basal condition and after FCCP, when compared to control cardiomyocytes (P=0.06). Furthermore, HGPS cardiomyocytes produced less ROS under basal and after 100uM Hydrogen peroxide treatment. However, mitotracker analysis shows no difference on mitochondrial content. Electron tomography and 3D reconstruction analysis suggest structural abnormalities in the mitochondria of Progeria cardiomyocytes. Proteomics analysis, performed with Liquid Chromatography tandem mass spectrometry (LC-MS/MS) in a high-resolution system (Q-Exactive Plus, Thermo Fischer), shows differential protein expression between Progeria (183 unique proteins) and Control (225 unique proteins) IPSc - derived cardiomyocytes. Conclusions: Our work demonstrate that iPSc- derived cardiomyocytes from Progeria patients have significant alterations in cellular respiration and protein expression.

Background: Diabetes mellitus is a severe chronic disease leading to systemic complications, including cardiovascular dysfunction. Previous cell therapy studies have obtained promising results with the use bone marrow mesenchymal stromal cells derived from healthy animals (MSCc) in diabetes animal models. However, the ability of MSC derived from diabetic rats to improve functional cardiac parameters is still unknown. Objectives: To investigate whether bone-marrow-derived MSC from diabetic rats (MSCd) would contribute to recover metabolic and cardiac electrical properties in other diabetic rats. Methods: Diabetes was induced in Wistar rats with streptozotocin. MSCs were characterized by flow cytometry, morphological analysis, and immunohistochemistry. Cardiac electrical function was analyzed using recordings of ventricular action potential. Differences between variables were considered significant when p < 0.05. Results: In vitro properties of MSCc and MSCd were evaluated. Both cell types presented similar morphology, growth kinetics, and mesenchymal profile, and could differentiate into adipogenic and osteogenic lineages. However, in an assay for fibroblast colony-forming units (CFU-F), MSCd formed more colonies than MSCc when cultured in expansion medium with or without hydrocortisone (1 µM). In order to compare the therapeutic potential of the cells, the animals were divided into four experimental groups: nondiabetic (CTRL), diabetic (DM), diabetic treated with MSCc (DM + MSCc), and diabetic treated with MSCd (DM + MSCd). The treated groups received a single injection of MSC 4 weeks after the development of diabetes. MSCc and MSCd controlled hyperglycemia and body weight loss and improved cardiac electrical remodeling in diabetic rats. Conclusions: MSCd and MSCc have similar in vitro properties and therapeutic potential in a rat model of diabetes induced with streptozotocin. Resumo Fundamentos: O diabetes mellitus é uma doença crônica grave que leva a complicações sistêmicas, como a disfunção cardiovascular. Estudos anteriores de terapia celular obtiveram resultados promissores com utilização de células estromais mesenquimais (CEM) derivadas de medula óssea de animais saudáveis (CEMc) em modelos de animais diabéticos. No entanto, a capacidade das CEM derivadas de ratos diabéticos em melhorar parâmetros cardíacos funcionais é ainda desconhecida. Objetivos: Investigar se CEM derivadas de medula óssea de ratos diabéticos (CEMd) poderiam contribuir para a recuperação metabólica e de propriedades elétricas cardíacas em outros ratos também com diabetes. Métodos: O diabetes foi induzido em ratos Wistar com estreptozotocina. As CEM foram caracterizadas por citometria de fluxo, análise morfológica e imunohistoquímica. A função elétrica cardíaca foi analisada através de registro do potencial de ação ventricular. As diferenças entre as variáveis foram consideradas significativas quando p < 0,05. Resultados: As propriedades in vitro das CEMc e CEMd foram avaliadas. Ambos os tipos celulares apresentaram morfologia, cinética de crescimento e perfil mesenquimal semelhante, e puderam ser diferenciadas em linhagens adipogênica e osteogênica. No entanto, em ensaios para unidades formadoras de colônias de fibroblastos (UFC-F), as CEMd formaram mais colônias em comparação às CEMc quando cultivadas em meio com ou sem hidrocortisona (1 µM). Para comparar o potencial terapêutico das células, os animais foram divididos em quatro grupos experimentais: não diabéticos (CTRL), diabéticos (DM), diabéticos tratados com CEMc (DM + CEMc) e diabéticos tratados com CEMd (DM + CEMd). Os grupos tratados receberam uma única injeção de CEM 4 semanas após o estabelecimento do diabetes. Ambas CEMc e CEMd controlaram a hiperglicemia e a perda de peso corporal e melhoraram o remodelamento elétrico cardíaco em ratos com diabetes. Conclusão: As CEMd e CEMc possuem propriedades in vitro e potencial terapêutico semelhante em um modelo de rato com diabetes induzido por estreptozotocina. (Arq Bras Cardiol. 2017; 109(6):579-589)

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.

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood-derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood-derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H(2)O(2), which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.

Aims: To assess the biodistribution of bone marrow mononuclear cells (BMMNC) delivered by different routes in patients with subacute middle cerebral artery ischemic stroke. Patients & methods: This was a nonrandomized, open-label Phase I clinical trial. After bone marrow harvesting, BMMNCs were labeled with technetium-99m and intra-arterially or intravenously delivered together with the unlabeled cells. Scintigraphies were carried out at 2 and 24 h after cell transplantation. Clinical follow-up was continued for 6 months. Results: Twelve patients were included, between 19 and 89 days after stroke, and received 1-5 × 10 8 BMMNCs. The intra-arterial group had greater radioactive counts in the liver and spleen and lower counts in the lungs at 2 and 24 h, while in the brain they were low and similar for both routes. Conclusion: BMMNC labeling with technetium-99m allowed imaging for up to 24 h after intra-arterial or intravenous injection in stroke patients.