Gisela F. Wilson

Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell–derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell–derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell–derived cardiomyocytes exhibited responsiveness to β-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells: The Matrix Sandwich Method

Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Neurons in the cat's primary auditory cortex distinguished by their responses to tones and wide-spectrum noise.

In the cortex of barbiturate-anesthetized cats, area AI was identified by its tonotopic organization, and single neurons in that field were examined with regard to the shapes of their spike count-versus-intensity functions, the organization of their frequency-intensity response areas, and their responses to wide-spectrum noise, using calibrated sealed stimulating systems. Neurons whose pure tone rate intensity functions were monotonic in shape displayed V-shaped response areas that were open-ended at high tone intensities. In contrast, cells displaying nonmonotonic tone intensity functions tended to have circumscribed response areas; these cells were responsive to tones over limited ranges of both frequency and intensity. Monotonic neurons almost always responded to wide-spectrum noise stimuli, while nonmonotonic neurons often did not. The mean minimum latent period of monotonic cells (14.0 ms) was significantly shorter than that for nonmonotonic neurons (19.1 ms). For those cells that responded to both tones and noise, minimum latent periods for the two stimuli were similar or identical. Monotonic neurons tended to be horizontally segregated from nonmonotonic neurons across AIs middle cortical layers. The implications of these data for the nature of some neural mechanisms underlying the stimulus selectivity of cortical cells are discussed.

The eag family of K+ channels in Drosophila and mammals.

ABSTRACT: Mutations of eag, first identified in Drosophila on the basis of their leg‐shaking phenotype, cause repetitive firing and enhanced transmitter release in motor neurons. The encoded EAG polypeptide is related both to voltage‐gated K+ channels and to cyclic nucleotide‐gated cation channels. Homology screens identified a family of eag‐related channel polypeptides, highly conserved from nematodes to humans, comprising three subfamilies: EAG, ELK, and ERG. When expressed in frog oocytes, EAG channels behave as voltage‐dependent, outwardly rectifying K+‐selective channels. Mutations of the human eag‐related gene (HERG) result in a form of cardiac arrhythmia that can lead to ventricular fibrillation and sudden death. Electrophysiological and pharmacological studies have provided evidence that HERG channels specify one component of the delayed rectifier, IKr, that contributes to the repolarization phase of cardiac action potentials. An important role or HERG channels in neuronal excitability is also suggested by the expression of these channels in brain tissue. Moreover, mutations of ERG‐type channels in the Drosophila sei mutant cause temperature‐induced convulsive seizures associated with aberrant bursting activity in the flight motor pathway. The in vivo function of ELK channels has not yet been established, but when these channels are expressed in frog oocytes, they display properties intermediate between those of EAG‐ and ERG‐type channels. Coexpression of the K+‐channel b subunit encoded by Hk with EAG in oocytes dramatically increases current amplitude and also affects the gating and modulation of these currents. Biochemical evidence indicates a direct physical interaction between EAG and HK proteins. Overall, these studies highlight the diverse properties of the eag family of K+ channels, which are likely to subserve diverse functions in vivo.

Interaction of the K channel beta subunit, Hyperkinetic, with eag family members.

Assembly of K channel α subunits of theShaker (Sh) family occurs in a subfamily specific manner. It has been suggested that subfamily specificity also applies in the association of β subunits with Sh channels (Rhodes, K. J., Keilbaugh, S. A., Barrezueta, N. X., Lopez, K. L., and Trimmer, J. S. (1995) J. Neurosci. 15, 5360–5371; Sewing, S., Roeper, J. and Pongs, O. (1996) Neuron 16, 455–463; Yu, W., Xu, J., and Li, M. (1996) Neuron 16, 441–453). Here we show that theDrosophila β subunit homologue Hyperkinetic(Hk) associates with members of the ether àgo-go (eag), as well as Sh, families. Anti-EAG antibody coprecipitates EAG and HK indicating a physical association between proteins. Heterologously expressed Hkdramatically increases the amplitudes of eag currents and also affects gating and modulation by progesterone. Through their ability to interact with a range of α subunits, the β subunits of voltage-gated K channels are likely to have a much broader impact on the signaling properties of neurons and muscle fibers than previously suggested.

Sensitivity of auditory cortical neurons of kittens to monaural and binaural high frequency sound

The experiments reported here describe the abilities of young auditory cortical neurons to encode information about tone bursts having frequencies above 2.5 kHz. The studies were carried out in anesthetized kittens ranging from 8 to 44 days of age. At all ages studied, stimulation of the contralateral ear was most effective in evoking spikes. Typically the response was confined to stimulus onset. Thresholds were comparatively high and response latencies were comparatively long in the youngest kittens studied. The time course of threshold development was very similar to that of the auditory nerve and cochlear nuclei indicating that most, if not all, age related thresholds and threshold changes at the cortical level are accounted for by mechanisms operating at the level of the cochlea and auditory nerve. Response latency shortened progressively over the first month of postnatal life and while the absolute change in response latency differed considerably from that of cells in the cochlear nuclei the proportional changes were very similar. These data indicate that the comparatively long response latency and latency changes recorded at the cortex are imposed by underdeveloped central auditory processes. Response areas of kitten cortical neurons resembled those of the adult. At all ages studied, binaural interactions were robust and similar in kind to those recorded in adult cats. We conclude that cortical neurons of kittens preserve the results of interactions occurring at lower brainstem levels and that the development of the circuits of which these neurons are a part develop as a functional unit.

Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells

Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem CellsNovelty and Significance: The Matrix Sandwich Method

Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem CellsNovelty and Significance

Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.