Ronit Shtrichman

Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation.

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca2+ handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine‐induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT‐derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs‐CMs), which expressed the mutant CASQ2 protein. The major findings were that the β‐adrenergic agonist isoproterenol caused in CPVT iPSCs‐CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after‐contractions and diastolic [Ca2+]i rise. Electron microscopy analysis revealed that compared with control iPSCs‐CMs, CPVT iPSCs‐CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient‐specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.

Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS‐derived cardiomyocytes), in the present study we investigated in iPS‐derived cardiomyocytes, the functional properties related to [Ca2+]i handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca2+ release to contraction and the b‐adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C‐Myc. Our major findings showed that iPS‐derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force–frequency relations and mild (compared to adult) post‐rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR‐Ca2+ handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF‐derived iPS, the functional properties related to excitation–contraction coupling, resemble in part those of adult cardiomyocytes.

Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus.

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.

Alu Sequences in Undifferentiated Human Embryonic Stem Cells Display High Levels of A-to-I RNA Editing

Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

LifeMap Discovery™: the embryonic development, stem cells, and regenerative medicine research portal.

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com

Cell-based therapy approaches: the hope for incurable diseases

Cell therapies aim to repair the mechanisms underlying disease initiation and progression, achieved through trophic effect or by cell replacement. Multiple cell types can be utilized in such therapies, including stem, progenitor or primary cells. This review covers the current state of cell therapies designed for the prominent disorders, including cardiovascular, neurological (Parkinsons disease, amyotrophic lateral sclerosis, stroke, spinal cord injury), autoimmune (Type 1 diabetes, multiple sclerosis, Crohns disease), ophthalmologic, renal, liver and skeletal (osteoarthritis) diseases. Various cell therapies have reached advanced clinical trial phases with potential marketing approvals in the near future, many of which are based on mesenchymal stem cells. Advances in pluripotent stem cell research hold great promise for regenerative medicine. The information presented in this review is based on the analysis of the cell therapy collection detailed in LifeMap Discovery(®) (LifeMap Sciences Inc., USA) the database of embryonic development, stem cell research and regenerative medicine.

Altered A-to-I RNA Editing in Human Embryogenesis

Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.

ADAR1 is involved in the regulation of reprogramming human fibroblasts to induced pluripotent stem cells.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional, site-specific modification process that is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) gene family members. Since ADARs act on double-stranded RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, as the result of the inherent property of these sequences to fold and form double strands. ADAR1-mediated A-to-I RNA editing was recently implicated in the regulation of human embryonic stem cells (hESCs). Spontaneous and neuronal differentiation of hESC was shown to result in a decrease in A-to-I editing levels. Knockdown of ADAR1 in hESCs results in an elevation of the expression of differentiation-related genes. In addition, we found that hESCs over-expressing ADAR1 could not be generated. The current study shows that the editing levels of induced pluripotent stem cells (iPSCs) change throughout reprogramming, from a source cell level to a level similar to that of hESCs. Up- or down-regulation of the ADAR1 level in human foreskin fibroblast (HFF) cells before induction of reprogramming results in varied reprogramming efficiencies. Furthermore, HFF-iPSC early clones derived from source cells in which the ADAR1 level was down-regulated lose their iPSC properties shortly after iPSC colony formation and instead exhibit characteristics of cancer cells. Taken together, our results imply a role for ADAR1 in the regulation of pluripotency induction as well as in the maintenance of early iPSC properties.