Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.

Valproic acid downregulates heme oxygenase-1 independently of Nrf2 by increasing ubiquitination and proteasomal degradation.

AIMS: Heme oxygenase-1 (HO-1; HMOX1 in human, Hmox1 in mice) is an antioxidative enzyme affecting wide range of sub-cellular processes. It was shown to modulate tumor growth or vascular-related diseases, thus being putative molecular target for tailored therapies. Therefore it is of importance to elucidate novel compounds regulating HO-1 activity/expression and to delineate mechanisms of their action. In the present study we aimed to understand mode of action of valproic acid (VA), an antiepileptic drug, on HO-1 expression. RESULTS: We demonstrated that HO-1 expression is decreased by VA at protein but not mRNA level in human alveolar rhabdomyosarcoma cell line CW9019. Nrf2 transcription factor, the activator of HO-1 expression through ARE sequence, was excluded as a mediator of HO-1 decrease, as VA downregulated Bach1, a Nrf2 repressor, concomitantly upregulating ARE activation. Also miRNA-dependent inhibition was excluded as a mechanism of HMOX1 regulation. However, co-immunoprecipitation assay showed a higher level of ubiquitinated HO-1 after VA treatment. Accordingly, MG132, an inhibitor of proteasomal degradation, reversed the effect of VA on HO-1 suggesting that decrease in HO-1 expression by VA is through protein stability. The inhibitory effect of VA on HO-1 was also observed in murine cells including embryonic fibroblasts isolated from Nrf2-deficient mice, what confirms Nrf2-independent effect of the compound. Importantly, VA decreased also HO-1 expression and activity in murine skeletal muscles in vivo. CONCLUSION: Our data indicate that VA downregulates HO-1 by acting through ubiquitin-proteasomal pathway leading to decrease in protein level.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, in Contrast to Adipose Tissue-Derived Stromal Cells, Efficiently Improve Heart Function in Murine Model of Myocardial Infarction.

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration.

Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long-term culture.

AIMS: Mesenchymal stromal cells isolated from different tissues are claimed to demonstrate similar therapeutic potential and are often incorrectly named mesenchymal stem cells. However, through comparison of such cells is lacking. This study aimed to compare the transcriptome of mesenchymal cells of the same phenotype isolated from the heart muscle and epicardial fat of the same patient, before and after culture. METHODS AND RESULTS: Cells were isolated from biopsies of the right ventricle and epicardial fat collected from five patients (three men and two women, mean age 59.4 ± 2.6) who underwent heart transplantation due to ischaemic cardiomyopathy. In both tissues, immunophenotyping revealed three distinct populations: (i)CD31- CD45- CD90+ CD34+ CD146- , (ii) CD31- CD45- CD90+ CD34- CD146+ , and (iii) CD31- CD45- CD90- CD34- CD146+ , of which only the first one could be grown after sorting. Material for RNA-seq was collected from these cells before culture (250 cells) and at passage 6 (5000 cells). Transcriptomic analysis revealed that cells of the same phenotype (CD31- CD45- CD90+ CD34+ CD146- ) upon isolation preferentially clustered according to the tissue of origin, not to the patient from whom they were isolated. Genes up-regulated in the right ventricle-derived cells were related to muscle physiology while down-regulated genes included those encoding proteins with transmembrane signalling receptor activity. After six passages, heart-derived and fat-derived cells did not acquire similar transcriptome. Cells isolated from the right ventricle in comparison with their epicardial fat-derived counterparts demonstrated higher level of transcripts related, among others, to RNA processing and muscle development. The down-regulated genes were involved in the nucleosome assembly, DNA packaging and replication, and interleukin-7-mediated signalling pathway. Cells from epicardial fat demonstrated higher heterogeneity both before and after culture. Cell culture significantly changed gene expression profile within both tissues. CONCLUSIONS: This study is an essential indication that mesenchymal cells isolated from different tissues do not demonstrate similar properties. Phenotypic identification and ease of isolation cannot be considered as a criterion in any therapeutic utilization of such cells.

Critical View on Mesenchymal Stromal Cells in Regenerative Medicine.

SIGNIFICANCE: The belief in the potency of stem cells has resulted in the medical applications of numerous cell types for organ repair, often with the low adherence to methodological stringency. Such uncritical enthusiasm is mainly presented in the approaches employing so-called mesenchymal stem cells (MSC), for the treatment of numerous, unrelated conditions. However, it should be stressed that such broad clinical applications of MSC are mostly based on the belief that MSC can efficiently differentiate into multiple cell types, not only osteoblasts, chondrocytes and adipose cells. Recent Advances: Studies employing lineage tracing established more promising markers to characterize MSC identity and localization in vivo and confirmed the differences between MSC isolated from various organs. Furthermore, preclinical and clinical experiments proved that transdifferentiation of MSC is unlikely to contribute to repair of numerous tissues, including the heart. Therefore, the salvage hypotheses, like MSC fusion with cells in target organs or the paracrine mechanisms, were proposed to justify the widespread application of MSC and to explain transient, if any, effects. CRITICAL ISSUES: The lack of standardization concerning the cells markers, their origin and particularly the absence of stringent functional characterization of MSC, leads to propagation of the worrying hype despite the lack of convincing therapeutic efficiency of MSC. FUTURE DIRECTIONS: The adherence to rigorous methodological rules is necessary to prevent the application of procedures which can be dangerous for patients and scientific research on the medical application of stem cells. Antioxid. Redox Signal. 00, 000-000.

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

AIMS: The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. METHODS AND RESULTS: MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. CONCLUSION: In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.