The lipodystrophic hotspot lamin A p.R482W mutation deregulates the mesodermal inducer T/Brachyury and early vascular differentiation gene networks.

The p.R482W hotspot mutation in A-type nuclear lamins causes familial partial lipodystrophy of Dunnigan-type (FPLD2), a lipodystrophic syndrome complicated by early onset atherosclerosis. Molecular mechanisms underlying endothelial cell dysfunction conferred by the lamin A mutation remain elusive. However, lamin A regulates epigenetic developmental pathways and mutations could perturb these functions. Here, we demonstrate that lamin A R482W elicits endothelial differentiation defects in a developmental model of FPLD2. Genome modeling in fibroblasts from patients with FPLD2 caused by the lamin A R482W mutation reveals repositioning of the mesodermal regulator T/Brachyury locus towards the nuclear center relative to normal fibroblasts, suggesting enhanced activation propensity of the locus in a developmental model of FPLD2. Addressing this issue, we report phenotypic and transcriptional alterations in mesodermal and endothelial differentiation of induced pluripotent stem cells we generated from a patient with R482W-associated FPLD2. Correction of the LMNA mutation ameliorates R482W-associated phenotypes and gene expression. Transcriptomics links endothelial differentiation defects to decreased Polycomb-mediated repression of the T/Brachyury locus and over-activation of T target genes. Binding of the Polycomb repressor complex 2 to T/Brachyury is impaired by the mutated lamin A network, which is unable to properly associate with the locus. This leads to a deregulation of vascular gene expression over time. By connecting a lipodystrophic hotspot lamin A mutation to a disruption of early mesodermal gene expression and defective endothelial differentiation, we propose that the mutation rewires the fate of several lineages, resulting in multi-tissue pathogenic phenotypes.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols.

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.

Prognostic impact of high ABC transporter activity in 111 adult acute myeloid leukemia patients with normal cytogenetics when compared to FLT3, NPM1, CEBPA and BAALC.

ATP-binding cassette transporter (and specially P-glycoprotein) activity is a well known prognostic factor in acute myeloid leukemia, but when compared to other molecular markers its prognostic value has not been well studied. Here we study relationships between this activity, fms-like tyro-sine kinase 3(FLT3/ITD), nucleophosmin(NPM1), CAAT-enhancer binding protein alpha(CEBPα), and brain and acute leukemia cytoplasmic protein (BAALC), in 111 patients with normal cytogenetics who underwent the same treatment, and evaluate its prognostic impact. Independent factors for survival were age (P=0.0126), ATP-binding cassette transporter activity (P=0.018) and duplications in the fms-like tyrosine kinase 3 (P=0.0273). In the 66 patients without fms-like tyrosine kinase 3 duplication and without nucleophosmin mutation, independent prognostic factors for complete remission achievement and survival were age and ATP-binding cassette transporter activity. In conclusion, ATP-binding cassette transporter activity remains an independent prognostic factor, and could assist treatment decisions in patients with no nucleophosmin mutation and no fms-like tyrosine kinase 3 duplication.

Functional Human Beige Adipocytes From Induced Pluripotent Stem Cells.

Activation of thermogenic beige adipocytes has recently emerged as a promising therapeutic target in obesity and diabetes. Relevant human models for beige adipocyte differentiation are essential to implement such therapeutic strategies. We report a straightforward and efficient protocol to generate functional human beige adipocytes from human induced pluripotent stem cells (hiPSCs). Without overexpression of exogenous adipogenic genes, our method recapitulates an adipogenic developmental pathway through successive mesodermal and adipogenic progenitor stages. hiPSC-derived adipocytes are insulin sensitive and display beige-specific markers and functional properties, including upregulation of thermogenic genes, increased mitochondrial content, and increased oxygen consumption upon activation with cAMP analogs. Engraftment of hiPSC-derived adipocytes in mice produces well-organized and vascularized adipose tissue, capable of ß-adrenergic-responsive glucose uptake. Our model of human beige adipocyte development provides a new and scalable tool for disease modeling and therapeutic screening.

Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells.

A large number of drugs can induce prolongation of cardiac repolarization and life-threatening cardiac arrhythmias. The prediction of this side effect is however challenging as it usually develops in some genetically predisposed individuals with normal cardiac repolarization at baseline. Here, we describe a platform based on a genetically diverse panel of induced pluripotent stem cells (iPSCs) that reproduces susceptibility to develop a cardiotoxic drug response. We generated iPSC-derived cardiomyocytes from patients presenting in vivo with extremely low or high changes in cardiac repolarization in response to a pharmacological challenge with sotalol. In vitro, the responses to sotalol were highly variable but strongly correlated to the inter-individual differences observed in vivo. Transcriptomic profiling identified dysregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization machinery as underlying high sensitivity to sotalol. Our findings offer novel insights for the development of iPSC-based screening assays for testing individual drug reactions.

Generating patient-specific induced pluripotent stem cells-derived cardiomyocytes for the treatment of cardiac diseases.

INTRODUCTION: Induced pluripotent stem cells (iPSC) represent an appealing cell source to develop disease-modeling assays, drug testing assays and cell-based replacement therapies especially for cardiac disorders. AREAS COVERED: The development of efficient protocols to generate pure populations of cardiac myocytes is a prerequisite to provide reproducible, robust and valid assays. Different techniques have been recently proposed that allow production of high-yield high-quality cardiomyocytes. In addition, the newly developed genome-editing techniques offer multiple opportunities to manipulate the genome of patient-specific iPSC thus generating syngeneic iPSC lines. Genome-editing techniques will also allow engineering of iPSC to make them suitable for replacement therapies. EXPERT OPINION: Since their discovery, iPSCs have shown promise to revolutionize the way human diseases are studied. During the last years, different protocols have been developed to achieve reproducible and efficient differentiation of iPSCs including in cardiac and vascular cells. The recent introduction of the genome-editing techniques now allow targeted manipulation of the genome of patient-specific and control iPSCs lines. This approach would help to address a couple of current limitations, including the generation of isogenic lines for disease modeling and of clinical-grade lines for replacement therapy.

A homozygous mutation of prelamin-A preventing its farnesylation and maturation leads to a severe lipodystrophic phenotype: new insights into the pathogenicity of nonfarnesylated prelamin-A.

CONTEXT: Mutations in LMNA, encoding A-type lamins, lead to multiple laminopathies, including lipodystrophies, progeroid syndromes, and cardiomyopathies. Alterations in the prelamin-A posttranslational maturation, resulting in accumulation of farnesylated isoforms, cause human progeroid syndromes. Accumulation of mutant nonfarnesylated prelamin-A leads to cardiomyopathy or progeria in mice, but no data have been provided in humans. OBJECTIVE, DESIGN, SETTING, AND PATIENTS: We searched for LMNA mutations in seven women originating from Reunion Island who were referred for a severe lipodystrophic syndrome. Clinical, molecular, genealogical, and cellular studies were performed in probands and relatives. RESULTS: The seven probands showed a severe partial lipodystrophic syndrome with diabetes and/or acanthosis nigricans, liver steatosis, hypertriglyceridemia, and low serum leptin and adiponectin levels. Three probands also had severe cardiac rhythm and conduction disturbances. We identified in all probands a homozygous LMNA p.T655fsX49 mutation leading to expression of a mutated prelamin-A with 48 aberrant C-terminal amino acids, preventing its physiological posttranslational farnesylation and maturation. Genealogical and haplotype analyses were consistent with a founder mutation transmitted from a common ancestor in the 17th century. In probands' cultured fibroblasts, mutated prelamin-A was associated with typical laminopathic nuclear dysmorphies, increased oxidative stress, and premature senescence. Heterozygous relatives were asymptomatic or partially affected, in favor of a codominant transmission of the disease with incomplete penetrance in heterozygotes. CONCLUSIONS: We reveal that a homozygous mutation of prelamin-A preventing its farnesylation leads to a severe lipodystrophic laminopathy in humans, which can be associated with cardiac conduction disturbances, stressing the pathogenicity of nonfarnesylated prelamin-A in human laminopathies.

Molecular aspects of myeloproliferative neoplasms.

During these past 5 years several studies have provided major genetic insights into the pathogenesis of the so-called classical myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The discovery of the JAK2V617F mutation first, then of the JAK2 exon 12 and MPLW515 mutations, have modified the understanding of these diseases, their diagnosis, and management. Now it is established that almost 100% of PV patients present a JAK2 mutation. Nearly 60% of ET patients and 50% of patients with PMF have the JAK2V617F mutation. The MPLW515 mutations are also present in a small proportion of ET and PMF patients. These mutations are oncogenic events that cause these disorders; however, they do not explain the heterogeneity of the entities in which they occur. Genetic defects have not been yet identified in around 40% of ET and PMF. There are likely additional somatic genetic factors important for the MPN phenotype like the recently described TET2, ASXL1, and CBL mutations. Moreover, polymorphisms in the JAK2 gene have been recently described as associated with MPN. Additional studies of large cohorts are required to dissect the genetic events in MPNs and the mechanisms of these oncogenic cooperations.