Generation and Cultivation of Transgene-Free Macaque and Baboon iPSCs Under Chemically Defined Conditions.

Non-human primates (NHP), and in particular Old World monkeys including macaques and baboons, are key animal models for the late preclinical testing of novel stem cell-based therapies and other advanced therapy medical products (ATMP) for the treatment of degenerative diseases. These pathologies are characterized by the loss of functional cells in an organ, as in Parkinson's disease, age-related macular degeneration, or after myocardial infarction. For preclinically relevant testing of induced pluripotent stem cell (iPSC)-based therapies, robust, and standardized protocols for the generation, characterization, and differentiation of NHP-iPSCs are required. Since the discovery of iPSCs by Takahashi and Yamanaka in 2006, human reprogramming protocols have been continuously refined. However, the generation of integration-free NHP-iPSC lines and a stable feeder- and serum-free long-term culture turned out to be difficult or even impossible with the current protocols established for human iPSCs. Here, we provide a robust protocol for the generation of transgene-free Old World monkey (and human) iPSCs and long-term cultivation under chemically defined conditions. This protocol was successfully applied to generate human, baboon (Papio anubis), rhesus (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis) iPSCs from skin fibroblasts. The resulting NHP-iPSCs provide a valuable resource for the preclinical testing of regenerative therapies in NHP.

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of I Na density, a 69.5% reduction of NaV1.5 expression, and the impaired localization of NaV1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The I to in BrS-CMs was significantly augmented, and the I CaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of I to in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

Baboon induced pluripotent stem cell generation by piggyBac transposition of reprogramming factors.

Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.

Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes.

Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.

hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities.

Brugada syndrome (BrS) is a rare cardiac rhythm disorder associated with sudden cardiac death. Mutations in the sodium channel gene SCN5A are found in ~20% of cases while mutations in other genes collectively account for <5%. In the remaining patients the genetic defect and the underlying pathogenic mechanism remain obscure. To provide insight into the mechanism of BrS in individuals without identified mutations, we here studied electrophysiological properties of cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) from 3 BrS patients who tested negative for mutations in the known BrS-associated genes. Patch clamp studies revealed no differences in sodium current (INa) in hiPSC-CMs from the 3 BrS patients compared to 2 unrelated controls. Moreover, action potential upstroke velocity (Vmax), reflecting INa, was not different between hiPSC-CMs from the BrS patients and the controls. hiPSC-CMs harboring the BrS-associated SCN5A-1795insD mutation exhibited a reduction in both INa and Vmax, demonstrating our ability to detect reduced sodium channel function. hiPSC-CMs from one of the BrS lines demonstrated a mildly reduced action potential duration, however, the transient outward potassium current (Ito) and the L-type calcium current (ICa,L), both implicated in BrS, were not different compared to the controls. Our findings indicate that ion channel dysfunction, in particular in the cardiac sodium channel, may not be a prerequisite for BrS.

Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

AIMS: Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. METHODS AND RESULTS: We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. CONCLUSIONS: Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.