SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells.

Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.

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 [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) 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-Ca(2+) 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.

Vascular cells.

Embryonic stem (ES) cells are cells derived from the inner cell mass of a blastocyst stage embryo. These self-renewing multipotent cells are able to differentiate to the three embryonic germ layers, the endoderm, ectoderm, and mesoderm, and are thus able to produce virtually all cell types. The ES cell capacity to generate various cell types has been studied extensively, and exploitation of ES cell characteristics allowed the production of several differentiated cell types of multiple tissues. Moreover, the process of ES cell differentiation provides a unique opportunity to observe early embryonic developmental events that are unattainable in the embryo itself. This chapter addresses the in vitro differentiation procedure of endothelial and vascular smooth muscle cells from human ES cells, with reference to similar studies performed in mouse and nonhuman primate ES cells, and provides several tools for the detailed characterization of differentiated cells.

Human embryonic stem cells for vascular development and repair.

Embryonic stem cells, derived from the inner cell mass of embryos in the blastocyst stage, are cells capable of perpetual self-renewal and long-term propagation and hold the potential to differentiate to progeny of the three embryonic germ layers. Since their derivation approximately two decades ago, exploration of mouse ES cells made major advances in ES cell differentiation research and in the successful development and propagation of various cell types. The subsequent derivation of ES cells from human embryos allows detailed study of early developmental events practically unreachable in early human embryos, and the potential derivation of a variety of adult cell types differentiated from the ES cells holds immense therapeutic promise. Recently, the study of ES cell-derived teratomas identified the partial presence of human ES cell-derived premature vessels within the teratoma, and a preliminary protocol for the in vitro derivation of a vascular progenitor was developed based on the study with the mouse ES cells. Furthermore, genetic profiling identified a pattern of expression of various endothelial and vascular smooth muscle cell genes that provide additional information on the degree of vascular development that ES cells undergo. Finally, the clinical application of ES cells in transplantation medicine is closer than ever following the affirmation that human ES cell-derived endothelial progenitors conferred increased neovascularization in transplanted engineered skeletal muscle. This review summarizes these recent advances in vascular development from human ES cells and their potential clinical applications.

Pharmacogenomic interaction between the Haptoglobin genotype and vitamin E on atherosclerotic plaque progression and stability.

OBJECTIVE: Homozygosity for a 1.7 kb intragenic duplication of the Haptoglobin (Hp) gene (Hp 2-2 genotype), present in 36% of the population, has been associated with a 2-3 fold increased incidence of atherothrombosis in individuals with Diabetes (DM) in 10 longitudinal studies compared to DM individuals not homozygous for this duplication (Hp 1-1/2-1). The increased CVD risk associated with the Hp 2-2 genotype has been shown to be prevented with vitamin E supplementation in man. We sought to determine if there was an interaction between the Hp genotype and vitamin E on atherosclerotic plaque growth and stability in a transgenic model of the Hp polymorphism. METHODS AND RESULTS: Brachiocephalic artery atherosclerotic plaque volume was serially assessed by high resolution ultrasound in 28 Hp 1-1 and 26 Hp 2-2 mice in a C57Bl/6 ApoE(-/-) background. Hp 2-2 mice had more rapid plaque growth and an increased incidence of plaque hemorrhage and rupture. Vitamin E significantly reduced plaque growth in Hp 2-2 but not in Hp 1-1 mice with a significant pharmacogenomic interaction between the Hp genotype and vitamin E on plaque growth. CONCLUSIONS: These results may help explain why vitamin E supplementation in man can prevent CVD in Hp 2-2 DM but not in non Hp 2-2 DM individuals.

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.

The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

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.

Human embryonic stem cell transplantation to repair the infarcted myocardium.

OBJECTIVE: To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI). METHODS: Undifferentiated hESCs (0.5-1x10(6)), human embryoid bodies (hEBs) (4-8 days; 0.5-1x10(6)), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2-4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining. RESULTS: Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p<0.05). The transplanted hESCs and hEBs rarely created new vessels and did not form new myocardium. Transplantation of hESCM tissue into normal heart produced islands of disorganised myofibres, fibrosis and, in a single case, a teratoma. However, transplantation of hESCMs into the infarcted myocardium did prevent post-MI dysfunction and scar thinning. CONCLUSIONS: Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.

Molecular analysis of cardiomyocytes derived from human embryonic stem cells.

During early embryogenesis, the cardiovascular system is the first system to be established and is initiated by a process involving the hypoblastic cells of the primitive endoderm. Human embryonic stem (hES) cells provide a model to investigate the early developmental stages of this system. When removed from their feeder layer, hESC create embryoid bodies (EB) which, when plated, develop areas of beating cells in 21.5% of the EB. These spontaneously contracting cells were demonstrated using histology, immunostaining and reverse transcription-polymerase chain reaction (RT-PCR), to possess morphological and molecular characteristics consistent with cardiomyocytic phenotypes. In addition, the expression pattern of specific cardiomyocytic genes in human EB (hEB) was demonstrated and analyzed for the first time. GATA-4 is the first gene to be expressed in 6-day-old EB. Alpha cardiac actin and atrial natriuretic factor are expressed in older hEB at 10 and 20 days, respectively. Light chain ventricular myosin (MLC-2V) was expressed only in EB with beating areas and its expression increased with time. Alpha heavy chain myosin (alpha-MHC) expression declined in the pulsating hEB with time, in contrast to events in EB derived from mice. We conclude that human embryonic stem cells can provide a useful tool for research on embryogenesis in general and cardiovascular development in particular.

Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation.

Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development.

Differentiation of human embryonic stem cells into insulin-producing clusters.

Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing beta cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin-producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of beta cells for transplantation. We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells. The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers. Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells. Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells.

During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4-8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.