Engineered Human Cardiac Tissue from Muscle Derived Stem Cells

Heart failure results in significant cardiomyocyte (CM) loss, and post-natal mammalian heart has limited regenerative capacity. Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function. A number of cell types illicit functional benefits through paracrine mechanisms, but cardiac stem cells are unique in their ability to preferentially differentiate down a cardiac lineage to replace lost CMs. However, cardiac stem cell isolation is highly invasive. Alternatively, skeletal myoblasts can be safely isolated and showed some benefits in clinical trials as donor muscle cells, but arrhythmias occurred due to lack of electric coupling with host cells. This limitation could be overcome by differentiating cells toward a cardiomyogenic lineage. Multipotent muscle derived stem cells (MDSC) are different from skeletal myoblasts and possess greater phenotypic plasticity. Our studies showed that cardiac and skeletal muscle share major genes/proteins during development in rodents, so it may be possible for human MDSCs to differentiate into CM-like cells under the appropriate conditions. My dissertation aims to develop approaches to differentiate human MDSCs into CM-like cells. Specifically, my work focuses on three aims: (I) to characterize the biochemical and functional properties of human MDSCs cultured in a 3-dimensional engineered muscle tissue (EMT) and examine whether it recapitulates properties of developing striated muscle; (II) to determine the potential for further CM differentiation under defined biophysical and chemical conditions; (III) to evaluate the potential of human MDSC derived cardiac progenitors to improve cardiac function in a human-rat xenograft model. The results of my studies showed that human MDSCs in EMT beat spontaneously, displayed calcium transients, expressed cardiac-specific genes/proteins, and exhibited pharmacological responses similar to iPS cell-derived CMs. They also possessed characteristics of skeletal muscle including expression of MyoD, myogenin, and sk-fMHC. Their electrical coupling also remained immature. By temporally treating EMT with 4 chemical factors (4CF: miR-206 inhibitor, IWR-1, BMP4, and LiCl) and improving aggregation conditions, 4F-AEMT showed better muscle tissue formation and cardiac-like morphology with improved contractility, pharmacological responses, and electrical coupling. Although 4F-AEMT expressed MyoD and myogenin, it exhibited more cardiac-like function. Finally, human MDSC-aggregates showed evidence of survival and improved cardiac function in vivo

Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle. © 2012 Clause et al

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a usefulin vitrofunctioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.</jats:p

Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

Abstract Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a “safety switch” based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is “turned on” with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies. </jats:sec

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.</jats:p

Éclosion d’Escherichia coli O121 associée à un fromage au lait cru de type Gouda en Colombie- Britannique, au Canada, 2018

Contexte : En 2018, une éclosion d’Escherichia coli O121 produisant la toxine de Shiga avec sept cas a été associée à la production de fromage au lait cru de type Gouda en Colombie-Britannique, au Canada. Objectifs : Décrire l’enquête sur l’éclosion d’E. coli O121 et recommander des mesures de contrôle plus strictes pour les fromages au lait cru de type Gouda. Méthodes : Les cas d’E. coli O121 ont été identifiés grâce aux résultats d’analyses en laboratoire et aux données de surveillance épidémiologique. Ils ont ensuite été interrogés en fonction de l’exposition d’intérêt, puis analysés en fonction des valeurs du Rapport Foodbook pour la Colombie-Britannique. Une investigation a été effectuée dans l’usine laitière et des prélèvements ont été effectués dans les produits de fromage afin de déterminer la source de contamination. Le typage de séquence multilocus pour le génome entier a été effectué sur tous les isolats cliniques et alimentaires positifs pour E. coli O121 au laboratoire provincial. Résultats Quatre des sept cas avaient consommé le même fromage au lait cru de type Gouda entre août et octobre 2018. Ce fromage avait été affiné pendant une période supérieure au minimum requis de 60 jours, et aucun défaut n’a été constaté dans sa production. Un échantillon du fromage visé a cependant obtenu un résultat positif à l’analyse de dépistage de la bactérie E. coli O121. Sept isolats cliniques et un isolat de fromage ont été jugés identiques par typage de séquence multilocus pour le génome entier avec une différence de 0 à 6,5 allèles. Conclusion Le Gouda au lait cru et les fromages de type Gouda au lait cru avaient déjà été impliqués dans trois éclosions antérieures d’E. coli producteur de toxines Shiga en Amérique du Nord. Il a donc été recommandé d’étiqueter les produits afin de sensibiliser les consommateurs au risque et de thermiser le lait afin de réduire le risque de maladie associé au Gouda au lait cru et au fromage de type Gouda au lait cru.</jats:p

Escherichia coli O121 outbreak associated with raw milk Gouda-like cheese in British Columbia, Canada, 2018

Background: In 2018, a Shiga toxin–producing Escherichia coli O121 outbreak that affected seven individuals was associated with raw milk Gouda-like cheese produced in British Columbia, Canada. Objectives: To describe the E. coli O121 outbreak investigation and recommend greater control measures for raw milk Gouda-like cheese. Methods: Cases of E. coli O121 were identified through laboratory testing results and epidemiologic surveillance data. The cases were interviewed on exposures of interest, which were analyzed against Foodbook Report values for British Columbia. Environmental inspection of the dairy plant and the cheese products was conducted to ascertain a source of contamination. Whole genome multi-locus sequence typing (wgMLST) was performed on all positive E. coli O121 clinical and food isolates at the provincial laboratory. Results: Four out of the seven cases consumed the same raw milk Gouda-like cheese between August and October 2018. The implicated cheese was aged longer than the required minimum of 60 days, and no production deficiencies were noted. One sample of the implicated cheese tested positive for E. coli O121. The seven clinical isolates and one cheese isolate matched by wgMLST within 6.5 alleles. Conclusion: Raw milk Gouda and Gouda-like cheese has been implicated in three previous Shiga toxin–producing E. coli outbreaks in North America. It was recommended product labelling to increase consumer awareness and thermization of milk to decrease the risk of illness associated with raw milk Gouda and Gouda-like cheese.</jats:p

RT-PCR analysis of cardiac and skeletal muscle transcription factors, sk-fMHC, cTn-I mRNA expression.

<p><b>Lane 1</b>: ED13 ventricle, <b>Lane 2</b>: ED20 ventricle; <b>Lane 3</b>: ND0 ventricle; <b>Lane 4</b>: ND4 ventricle; <b>Lane 5</b>: PND10 ventricle; <b>Lane 6</b>: Adult ventricle; <b>Lane 7</b>: ED13 hind limbs; <b>Lane 8</b>: ED20 hind limbs, <b>Lane 9</b>: ND0 hind limbs; <b>Lane 10</b>: ND4 hind-limbs; <b>Lane 11</b>: PND10 gastrocnemius muscle; <b>Lane 12</b>: Adult gastrocnemius muscle. We note that ED20 skeletal muscle (hind limbs) mRNA expression patterns are similar to ED20 ventricular myocardium.</p

sk-fMHC (green color) and cardiac troponin-I (red color) expression (Panel A) and gene expression (Panel B) of skeletal muscle derived stem cell 3D collagen gel bioreactor (MDSC-3DGB).

<p>MDSC-3DGB showed co-localized expression of sk-fMHC and cardiac troponin I. Transcription factor and structural gene expression was increased compared to 2D undifferentiated MDSCs. Scale in panel A indicates 20 µm.</p