Abstract-Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. ␤-Adrenergic stimulation with 1.0 mol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology recorded from different cells, suggesting that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for I Kr based on E-4031 induced prolongation of AP duration as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially therapeutic applications. R ecent studies have demonstrated that human embryonic stem (hES) cells in vitro can form embryoid bodies (EBs), some of which begin to spontaneously contract. [1][2][3] These beating EBs contain cardiac myocytes (CMs) based on the expression of cardiac-specific genes, cellular ultrastructure, and extracellular electrical activity. 1,3 Progress has been made in isolating cardiac myocytes from the mixed population of cells in the EB, 2 but no studies have yet defined what types of cardiac myocytes exist in the differentiating human EBs. In comparison, multiple types of mouse embryonic stem (mES) cell-derived CMs have been identified and characterized including nodal, atrial, ventricular, and Purkinje cells. However, mES cells have significant differences from hES cells such as variations in the stage-specific antigens and in the ability of leukemia inhibitory factor (LIF) to maintain the undifferentiated state. 4 Thus, differentiation of CMs from ES cells from the different species may be significantly different.Defining the types of CMs that can be obtained from hES cells is essential for further research using this model system and for any potential utilization of these cells for cell-based therapies. A var...

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ± s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca 2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial-and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1α α promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-β β gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5′ ′ end of the chicken β β-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34 + hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.

Molecularly imprinted polymers (MIPs) have now earned the reputation as "artificial receptors" or "plastic antibodies". As the mimics of natural receptors, MIPs are reminiscent of some basic functions of natural receptors in living systems, e.g., the ability to interact with or recognize cells. The latest decade has witnessed a great advance in MIPs from simple molecular extraction to efficient cell recognition, implying that MIP-based synthetic receptors are approaching to be perfectly functioning replicates of their natural counterparts. With the most emerging development in molecular imprinting, MIP-mediated cell recognition has now shown great promise in cell biology research, theranostics and regenerative medicine. This tutorial review provides a panoramic view of current MIPs for both microorganism and mammalian cell recognition. The most representative developments of MIP-mediated cell recognition, from initial imprinting strategies to eventual bio-related applications, are highlighted.

To achieve on‐demand drug release, mesoporous silica nanocarriers as antitumor platforms generally need to be gated with stimuli‐responsive capping agents. Herein, a “smart” mesoporous nanocarrier that is gated by the drug itself through a pH‐sensitive dynamic benzoic–imine covalent bond is demonstrated. The new system, which tactfully bypasses the use of auxiliary capping agents, could also exhibit desirable drug release at tumor tissues/cells and enhanced tumor inhibition. Moreover, a facile dynamic PEGylation via benzoic–imine bond further endows the drug‐self‐gated nanocarrier with tumor extracellular pH‐triggered cell uptake and improves therapeutic efficiency in vivo. In short, the paradigm shift in capping agents here will simplify mesoporous nanomaterials as intelligent drug carriers for cancer therapy. Moreover, the self‐gated strategy in this work also shows general potential for self‐controlled delivery of natural biomolecules, for example, DNA/RNA, peptides, and proteins, due to their intrinsic amino groups.

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