Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

Background Cardiac-abundant microRNA-378a (miR-378a) is associated with postnatal repression of insulin–like growth factor 1 receptor (IGF-1R) controlling physiological hypertrophy and survival pathways. IGF-1/IGF-1R axis has been proposed as a therapeutic candidate against the pathophysiological progress of diabetic cardiomyopathy (DCM). We ask whether hyperglycemia-driven changes in miR-378a expression could mediate DCM progression. Methods Diabetes mellitus was induced by streptozotocin (STZ) (55 mg/kg i.p. for 5 days) in male C57BL/6 wild type (miR-378a+/+) and miR-378a knockout (miR-378a−/−) mice. As a parallel human model, we harnessed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM miR378a+/+ vs. hiPSC-CM miR378a−/−) subjected to high glucose (HG) treatment. Results We reported miR-378a upregulation in cardiac diabetic milieu arising upon STZ administration to wild-type mice and in HG-treated hiPSC-CMs. Pro-hypertrophic IGF-1R/ERK1/2 pathway and hypertrophic marker expression were activated in miR-378a deficiency and upon STZ/HG treatment of miR-378a+/+ specimens in vivo and in vitro suggesting miR-378a-independent hyperglycemia-promoted hypertrophy. A synergistic upregulation of IGF-1R signaling in diabetic conditions was detected in miR-378a−/− hiPSC-CMs, but not in miR-378a−/− hearts that showed attenuation of this pathway, pointing to the involvement of compensatory mechanisms in the absence of miR-378a. Although STZ administration did not cause pro-inflammatory or pro-fibrotic effects that were detected in miR-378a−/− mice, the compromised diabetic heart function observed in vivo by high-resolution ultrasound imaging upon STZ treatment was not affected by miR-378a presence. Conclusions Overall, data underline the role of miR-378a in maintaining basal cardiac structural integrity while pointing to miR-378a-independent hyperglycemia-driven cardiac hypertrophy and associated dysfunction

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

Generation and characterization of human induced pluripotent stem cells and hiPSC-derived cardiomyocytes lacking microRNA-378a

Kardiomiocyty uzyskane przez różnicowanie indukowanych pluripotencjalnych komórek macierzystych (hiPSC-CM) znalazły zastosowanie w eksperymentalnej medycynie regeneracyjnej, testowaniu leków oraz modelowaniu różnych chorób. Jednak ze względu na ich fenotyp przypominający kardiomiocyty płodowe nie mogą być modelem oddającym warunki kardiomiocytów występujących w dorosłym sercu, co znacząco ogranicza ich potencjał. Jak dotąd nie poznano ścieżek rozwojowych kierujących procesem dojrzewania kardiomiocytów zarówno w warunkach in vitro jak i in vivo. Jednak, coraz więcej prac wskazuje, że jednym z czynników niezbędnych w procesie dojrzewania hiPSC-CM, jest wprowadzenie kwasów tłuszczowych do pożywki hodowlanej. Teza ta ma odzwierciedlenie w procesach zachodzących przed oraz po narodzinach. Wraz z większą dostępnością tlenu oraz kwasów tłuszczowych dochodzi do zmiany metabolizmu kardiomiocytów z glikolizy na β-oksydację kwasów tłuszczowych. Towarzyszą temu liczne zmiany strukturalne i funkcjonalne kardiomiocytów. Jednym z mikroRNA (miRNA), którego ekspresja znacząco wzrasta po narodzinach a także podczas długoterminowej hodowli hiPSC-CM jest miR-378a. Jak dotąd, jego rola w kardiomiocytach nie została dobrze poznana. Zaangażowany jest on w procesy związane z metabolizmem kwasów tłuszczowych. Reguluje ekspresję IGF, IGF1R oraz kinaz uczestniczących w przekazie sygnału komórkowego m.in. reagującego na wzrost hipertroficzny kardiomiocytów. Wykonane w poniższej pracy badania wskazują na zaangażowanie miR-378a w regulację procesów związanych zarówno z metabolizmu hiPSC-CM jak i ich wzrostem hipertroficznym. Zaobserwowano, że komórki z delecją miR-378a po wprowadzeniu pożywki z kwasami tłuszczowymi wykazują wzrost ekspresji IGF1R, zarówno na poziomie białka jak i mRNA, co w połączeniu ze spadkiem fosforylacji kinazy Erk i wzrostem ekspresji p21 prowadzi do senescencji i wzrostu hipertroficznego. Dodatkowo, po podaniu pożywki zawierającej kwasy tłuszczowe zaobserwowano spadek ilości mitochondriów oraz wzrost ekspresji podjednostek enzymów łańcucha oddechowego. Obniżeniu uległa ekspresja PPARα zaangażowanego w regulację metabolizmu kwasów tłuszczowych, co mogło wpłynąć na pobranie kwasu palmitynowego z pożywki i ochronę hiPSC-CM miR-378aKO przed efektem cytotoksycznym. Brak miR-378a w badanych komórkach przyczynił się do zwiększonej hipertrofii charakterystycznej dla dojrzewających kardiomiocytów. Jednak, obniżona ilość mitochondriów, spadek aktywności błon mitochondrialnych oraz niższy stosunek izoform tityny N2B/ N2BA świadczy o ich charakterze płodowym. Wyniki wskazują na zaangażowanie miR-378a w procesy łączące zmiany metaboliczne wynikające z obecności kwasów tłuszczowych w pożywce hodowlanej ze wzrostem hipertroficznym oraz regulacją zmian ekspresji genów związanych z procesem dojrzewania.Cardiomyocytes derived from hiPSC (hiPSC-CM) are potent tool for drug screening, cardiac development and disease modelling and are tested in regenerative approaches. However, their phenotype is similar to fetal cardiomyocytes which can limit their potential in regenerative medicine and new drugs testing. Process of cardiomyocytes maturation both in in vivo and in vitro has not be understood so far. It is highly probable that this process is induced by higher fatty acids availability after a birth. In in vitro experiments it was noticed that applying fatty acid into culture medium improves maturation of hiPSC-CM. Nevertheless, pathways which drives this process has not been investigated. miR-378a is one of the most highly expressed microRNAs in heart. Its expression increases with life time and in long term hiPSC-CM culture. What is more, it is expressed only in cardiomyocytes and not in any other cells in heart. miR-378a is involved in hypertrophic growth via regulation of IGF1R and MAPK kinases pathways and in regulation of metabolism. Despite all of those functions, role of miR-378a in the heart has not been well investigated so far. Generated isogenic hiPSC miR-378a lines did not show differences in pluripotency markers expression or in ability to differentiate into cells derived from all three germ layers. Cardiac differentiation efficiency was more than 85% in all hiPSC-CM miR-378aKO lines and controls. Hypertrophic growth is promoted in miR-378a-deficient cardiomyocytes in media containing either glucose (GLCM) or palmitic acid (FAM) as an energy source. Titin N2B/ Titin N2BA ratio is decreased in hiPSC-CM miR-378aKO in GLCM and FAM. The mitochondrial content and mitochondrial membrane activity are decreased in hiPSC-CM FAM in comparison to control. These Data show that hiPSC-CM miR-378a are less mature than control ones, which suggests that miR-378a connects cardiac metabolism and hypertrophic growth during maturation process

Construction of lentiviral vectors for doxycycline inducible overexpression of heme oxygenase-1

Jedną z metod terapii genowej jest wprowadzanie pewnych sekwencji za pomocą konstrukcji plazmidów lentiwirusowych. Korzystne jest wprowadzenie systemu umożliwiającego włączanie i wyłączanie ekspresji transgenu za pomocą różnych substancji jak np. doksycyklina. Umożliwia to kontrolowaną ekspresję danego białka lub innych cząsteczek regulatorowych. Oksygenaza Hemowa- 1 została wybrana ze względu na swoje rozległe działanie wpływające na organizm człowieka.One of the methods of gene therapy is to introduce certain sequences by constructing lentiviral plasmids. It is preferable to introduce a system that allows the transgene to be switched on and off by various substances such as doxycycline. This enables the controlled expression of a particular protein or other regulatory molecule. Ho-1 was selected as a transgene because of its extensive effect on the human body

Derivation of human pluripotent stem cell line via CRISPR/Cas9 mediated deletion of exon 3 LAMA2 gene (DMBi001-A-1)

LAMA2-related muscular dystrophy (LAMA2-MD) results from mutations in LAMA2 gene, encoding laminin α-2. It is a congenital disease characterized by muscle wasting, with the most severe version being diagnosed within first few months after birth. To generate LAMA2-DM in vitro model, we excised exon 3 from the LAMA2 gene in our previously derived healthy human induced pluripotent stem cells (hiPSCs). Obtained hiPSCs show expression of pluripotency markers, differentiation capacity into all three germ layers, normal karyotype and lack of LAMA2 expression on mRNA and protein level after differentiation into skeletal myocytes. Accordingly, it may provide novel insight into the molecular basis of LAMA2-MD

Generation of DMBi002-A human induced pluripotent stem cell line from patient with Spinal muscular atrophy type 3

Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disease caused by mutations in SMN1 gene encoding survival motor neuron (SMN) protein. Lack of this protein leads to progressive loss of motor neurons and therefore to gradual loss of signal transmission between motor neurons and skeletal muscle cells. As a consequence, patients develop muscle atrophy and lose the ability to move independently, what is also related to problems with breathing and swallowing. Here, we describe the generation of human induced pluripotent stem cells (hiPSC) from peripheral blood mononuclear cells (PBMC) of adult SMA type 3 patient with a use of Sendai virus vectors