Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome.

Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.

Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Coordinated release of calcium (Ca(2+) ) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress-induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca(2+) release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca(2+) release events (Ca(2+) sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.

Characterization of a naturally-occurring p27 mutation predisposing to multiple endocrine tumors.

BACKGROUND: p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in Cdkn1b (encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that Cdkn1b is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated Cdkn1b mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) in vitro and in vivo. RESULTS: In vitro, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 in vivo which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues. CONCLUSION: Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.

Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.

Neuropotent self-renewing neural stem (NS) cells derived from mouse induced pluripotent stem (iPS) cells.

Neural stem (NS) cells are a homogenous population of stem cells that expands in monolayer under serum-free conditions while remaining highly neuropotent. Here, we generated NS cells from induced pluripotent stem (iPS) cells that were previously derived from mouse fibroblasts (NS-(f)iPS). We showed that NS-(f)iPS cells exhibit long-term expansion and express markers of neurogenic radial glia. Analyses of the regional markers expressed in NS-(f)iPS cells suggested a ventral-rhombencephalic identity. Upon exposure to differentiation protocols, NS-(f)iPS cells produce neurons, astrocytes, and oligodendrocytes with an efficiency similar to ES-derived NS cells. NS-(f)iPS cells represent a new tool for studying neural cell fate determination and terminal differentiation, providing an interesting resource for experimental transplantation. Comparative studies between NS cells derived from iPS cells, reprogrammed from different somatic sources, and from authentic ES cells are necessary to identify critical elements for multipotency acquisition.