Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of i) high-quality hESCs for expansion, and ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

Background Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated. Methods and Results Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation. Conclusions By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs.

BackgroundMicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive.Methods and ResultsWe hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased Ito, IKs and IKr, and decreased If (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca2+ transient amplitude and kinetics. Molecular pathway analyses were performed for further insights.ConclusionWe conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.

Mouse (m) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are known to exhibit immature Ca 2+ dynamics such as small whole-cell peak amplitude and slower kinetics relative to those of adult. In this study, we examined the maturity and effi ciency of Ca 2+ -induced Ca 2+ release in m and hESC-CMs, the presence of transverse (t) tubules and its effects on the regional Ca 2+ dynamics. In m and hESC-CMs, fl uorescent staining and atomic force microscopy (AFM) were used to detect the presence of t-tubules, caveolin-3, amphiphysin-2 and colocalization of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs). To avoid ambiguities, regional electrically-stimulated Ca 2+ dynamics of single ESC-CMs, rather than spontaneously beating clusters, were measured using confocal microscopy. m and hESC-CMs showed absence of dyads, with neither t-tubules nor colocalization of DHPRs and RyRs. Caveolin-3 and amphiphysin-2, crucial for the biogenesis of t-tubules with robust expression in adult CMs, were also absent. Single m and hESC-CMs displayed non-uniform Ca 2+ dynamics across the cell that is typical of CMs defi cient of t-tubules. Local Ca 2+ transients exhibited greater peak amplitude at the peripheral than at the central region for m (3.50 ± 0.42 vs. 3.05 ± 0.38) and hESC-CMs (2.96 ± 0.25 vs. 2.72 ± 0.25). Kinetically, both the rates of rise to peak amplitude and transient decay were faster for the peripheral relative to the central region. Immature m and hESC-CMs display unsynchronized Ca 2+ transients due to the absence of t-tubules and gene products crucial for their biogenesis. Our results provide insights for driving the maturation of ESC-CMs.

The generation of human ventricular cardiomyocytes from human embryonic stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and growth factors, we developed a fully chemically defined, directed differentiation system to generate ventricular-like cardiomyocytes (VCMs) from human embryonic stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the differentiation recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-1 plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies. STEM CELLS

Kurzrock EA, Lieu DK, deGraffenried LA, Chan CW, Isseroff RR. Label-retaining cells of the bladder: candidate urothelial stem cells. Am J Physiol Renal Physiol 294: F1415-F1421, 2008. First published March 26, 2008 doi:10.1152/ajprenal.00533.2007.-Adult tissue stem cells replicate infrequently, retaining DNA nucleotide label (BrdU) for much longer periods than mature, dividing cells in which the label is diluted during a chase period. Those "labelretaining cells" (LRCs) have been identified as the tissue stem cells in skin, cornea, intestine, and prostate. However, in the urinary tract uroepithelial stem cells have not yet been identified. In this study, BrdU administration identified urothelial LRCs in the rat bladder with 9% of the epithelial basal cells retaining BrdU label 1 yr after its administration. Markers for stem cells in other tissues, Bcl, p63, cytokeratin 14, and ␤1 integrin, were immunolocalized in the basal bladder epithelium in or near urothelial LRCs, but not uniquely limited to these cells. Flow cytometry demonstrated that urothelial LRCs were small, had low granularity, and were uniquely ␤4 integrin bright. Urothelium from long-term labeled bladders was cultured and LRCs were found to be significantly more clonogenic and proliferative, characteristics of stem cells, than unlabeled urothelial cells. Thus, this work demonstrates that LRCs in the bladder localize to the basal layer, are small, low granularity, uniquely ␤4 integrin rich, slowly cycling and demonstrate superior clonogenic and proliferative ability compared with unlabeled epithelial cells. We propose that LRCs represent putative urothelial stem cells.

Raman and infrared (IR) spectroscopy are two complementary vibrational spectroscopic techniques that have experienced a tremendous growth in their use in biological and biomedical research. This is, in large part, due to their unique capability of providing label‐free intrinsic chemical information of living biological samples at tissue, cellular, or sub‐cellular resolutions. This article reviews recent developments in applying these techniques for the characterization of stem cells. A discussion of the potential for these methods to address some of the major challenges in stem cell research is presented, as well as the technological and scientific advancements that are needed to progress the knowledge in the field. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Early atherosclerotic lesions localize preferentially in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.