Transformation of jaw muscle satellite cells to cardiomyocytes.

In the embryo a population of progenitor cells known as the second heart field forms not just parts of the heart but also the jaw muscles of the head. Here we show that it is possible to take skeletal muscle satellite cells from jaw muscles of the adult mouse and to direct their differentiation to become heart muscle cells (cardiomyocytes). This is done by exposing the cells to extracellular factors similar to those which heart progenitors would experience during normal embryonic development. By contrast, cardiac differentiation does not occur at all from satellite cells isolated from trunk and limb muscles, which originate from the somites of the embryo. The cardiomyocytes arising from jaw muscle satellite cells express a range of specific marker proteins, beat spontaneously, display long action potentials with appropriate responses to nifedipine, norepinephrine and carbachol, and show synchronized calcium transients. Our results show the existence of a persistent cardiac developmental competence in satellite cells of the adult jaw muscles, associated with their origin from the second heart field of the embryo, and suggest a possible method of obtaining cardiomyocytes from individual patients without the need for a heart biopsy.

Pax3 and Tbx5 specify whether PDGFRα+ cells assume skeletal or cardiac muscle fate in differentiating embryonic stem cells.

Embryonic stem cells (ESCs) represent an ideal model to study how lineage decisions are established during embryonic development. Using a doxycycline-inducible mouse ESC line, we have previously shown that expression of the transcriptional activator Pax3 in early mesodermal cells leads to the robust generation of paraxial mesoderm progenitors that ultimately differentiate into skeletal muscle precursors. Here, we show that the ability of this transcription factor to induce the skeletal myogenic cell fate occurs at the expenses of the cardiac lineage. Our results show that the PDGFRα+FLK1--subfraction represents the main population affected by Pax3, through downregulation of several transcripts encoding for proteins involved in cardiac development. We demonstrate that although Nkx2-5, Tbx5, and Gata4 negatively affect Pax3 skeletal myogenic activity, the cardiac potential of embryoid body-derived cultures is restored solely by forced expression of Tbx5. Taking advantage of this model, we used an unbiased genome-wide approach to identify genes whose expression is rescued by Tbx5, and which could represent important regulators of cardiac development. These findings elucidate mechanisms regulating the commitment of mesodermal cells in the early embryo and identify the Tbx5 cardiac transcriptome.

Micromagnetic stimulation (µMS) dose-response of the rat sciatic nerve.

Objective.The objective of this study was to investigate the effects of micromagnetic stimuli strength and frequency from theMagneticPen(MagPen) on the rat right sciatic nerve. The nerve's response was measured by recording muscle activity and movement of the right hind limb.Approach.The MagPen was custom-built to be stably held over the sciatic nerve. Rat leg muscle twitches were captured on video, and movements were extracted using image processing algorithms. EMG recordings were also used to measure muscle activity.Main results.The MagPen prototype, when driven by an alternating current, generates a time-varying magnetic field, which, according to Faraday's law of electromagnetic induction, induces an electric field for neuromodulation. The orientation-dependent spatial contour maps of the induced electric field from the MagPen prototype have been numerically simulated. Furthermore, in thisin vivowork onµMS, a dose-response relationship has been reported by experimentally studying how varying the amplitude (Range: 25 mVp-pthrough 6Vp-p) and frequency (range: 100 Hz through 5 kHz) of the MagPen stimuli alters hind limb movement. The primary highlight of this dose-response relationship (repeated overnrats, wheren= 7) is that for aµMS stimuli of higher frequency, significantly smaller amplitudes can trigger hind limb muscle twitch. This frequency-dependent activation can be justified by Faraday's Law, which states that the magnitude of the induced electric field is directly proportional to the frequency.Significance.This work reports thatµMS can successfully activate the sciatic nerve in a dose-dependent manner. The impact of this dose-response curve addresses the controversy in this research community about whether the stimulation from theseµcoils arise from a thermal effect or micromagnetic stimulation. MagPen probes do not have a direct electrochemical interface with tissue and therefore do not experience electrode degradation, biofouling, and irreversible redox reactions like traditional direct contact electrodes. Magnetic fields from theµcoils create more precise activation than electrodes because they apply more focused and localized stimulation. Finally, unique features ofµMS, such as the orientation dependence, directionality, and spatial specificity, have been discussed.

Strength-frequency curve for micromagnetic neurostimulation through excitatory postsynaptic potentials (EPSPs) on rat hippocampal neurons and numerical modeling of magnetic microcoil (µcoil).

Objective.The objective of this study was to measure the effect of micromagnetic stimulation (µMS) on hippocampal neurons, by using single microcoil (µcoil) prototype, magnetic pen (MagPen). MagPen will be used to stimulate the CA3 region magnetically and excitatory post synaptic potential (EPSP) response measurements will be made from the CA1 region. The threshold for micromagnetic neurostimulation as a function of stimulation frequency of the current driving theµcoil will be demonstrated. Finally, the optimal stimulation frequency of the current driving theµcoil to minimize power will be estimated.Approach.A biocompatible, watertight, non-corrosive prototype, MagPen was built, and customized such that it is easy to adjust the orientation of theµcoil and its distance over the hippocampal tissue in anin vitrorecording setting. Finite element modeling of theµcoil design was performed to estimate the spatial profiles of the magnetic flux density (in T) and the induced electric fields (in V m-1). The induced electric field profiles generated at different values of current applied to theµcoil can elicit a neuronal response, which was validated by numerical modeling. The modeling settings for theµcoil were replicated in experiments on rat hippocampal neurons.Main results.The preferred orientation of MagPen over the Schaffer Collateral fibers was demonstrated such that they elicit a neuron response. The recorded EPSPs from CA1 region due toµMS at CA3 region were validated by applying tetrodotoxin (TTX). Application of TTX to the hippocampal slice blocked the EPSPs fromµMS while after prolonged TTX washout, a partial recovery of the EPSP fromµMS was observed. Finally, it was interpreted through numerical analysis that increasing frequency of the current driving theµcoil, led to a decrease in the current amplitude threshold for micromagnetic neurostimulation.Significance.This work reports that micromagnetic neurostimulation can be used to evoke population EPSP responses in the CA1 region of the hippocampus. It demonstrates the strength-frequency curve forµMS and its unique features related to orientation dependence of theµcoils, spatial selectivity and stimulation threshold related to distance dependence. Finally, the challenges related toµMS experiments were studied including ways to overcome them.

Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells.

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Defined Culture Conditions Accelerate Small-molecule-assisted Neural Induction for the Production of Neural Progenitors from Human-induced Pluripotent Stem Cells.

The use of defined conditions for derivation, maintenance, and differentiation of human-induced pluripotent stem cells (hiPSCs) provides a superior experimental platform to discover culture responses to differentiation cues and elucidate the basic requirements for cell differentiation and fate restriction. Adoption of defined systems for reprogramming, undifferentiated growth, and differentiation of hiPSCs was found to significantly influence early stage differentiation signaling requirements and temporal kinetics for the production of primitive neuroectoderm. The bone morphogenic protein receptor agonist LDN-193189 was found to be necessary and sufficient for neural induction in a monolayer system with landmark antigens paired box 6 and sex-determining region Y-box 1 appearing within 72 h. Preliminary evidence suggests this neuroepithelium was further differentiated to generate ventral spinal neural progenitors that produced electrophysiologically active neurons in vitro, maintaining viability posttransplantation in an immunocompromised host. Our findings support current developments in the field, demonstrating that adoption of defined reagents for the culture and manipulation of pluripotent stem cells is advantages in terms of simplification and acceleration of differentiation protocols, which will be critical for future clinical translation.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

UNLABELLED: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable. SIGNIFICANCE: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain.

AIMS: Fibroblasts can be directly reprogrammed to cardiomyocyte-like cells by introducing defined genes. However, the reprogramming efficiency remains low, delaying the clinical application of this strategy to regenerative cardiology. We previously showed that fusion of the MyoD transactivation domain to the pluripotency transcription factor Oct4 facilitated the transcriptional activity of Oct4, resulting in highly efficient production of induced pluripotent stem cells. We examined whether the same approach can be applied to cardiac transcription factors to facilitate cardiac reprogramming. METHODS AND RESULTS: We fused the MyoD domain to Mef2c, Gata4, Hand2, and Tbx5 and transduced these genes in various combinations into mouse non-cardiac fibroblasts. Transduction of the chimeric Mef2c with the wild-types of the other three genes produced much larger beating clusters of cardiomyocyte-like cells faster than the combination of the four wild-type genes, with an efficiency of 3.5%, >15-fold greater than the wild-type genes. CONCLUSION: Fusion of a powerful transactivation domain to heterologous factors can increase the efficiency of direct reprogramming of fibroblasts to cardiomyocytes.

Effective cardiac myocyte differentiation of human induced pluripotent stem cells requires VEGF.

Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs), which have paved the way for regeneration therapy using patients' own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines, which inhibit the application of this technology significantly. Here, we demonstrate a novel myocyte differentiation protocol that can yield a significant, high percentage of cardiac myocyte differentiation (>85%) in 2 hiPSC lines, which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts, but also from blood mononuclear cells.

Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function.

OBJECTIVE: Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34(+) cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization. MATERIALS AND METHODS: hESCs are cocultured on M2-10B4 stromal cells or Wnt1-expressing M2-10B4 for 13 to 15 days to generate a CD34(+) cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin-coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24 to 48 hours. RESULTS: CD34(+) vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell-specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Human umbilical vein endothelial cells cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1-overexpressing stromal cells produced an increased number of progenitor cells. CONCLUSIONS: The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The stepwise differentiation outlined here is an efficient, reproducible method with potential for large-scale cultures suitable for clinical applications.

Pharmacological characterization of P2Y receptor subtypes on isolated tiger salamander Müller cells.

Müller cells express a variety of neurotransmitter receptors that permit them to "sense" the extracellular environment within the retina. We have used a battery of agonists and antagonists to characterize the purinergic receptor subtypes expressed on isolated tiger salamander Müller cells. Changes in intracellular calcium ion concentration ([Ca(2+)](i)) in Müller cells were measured using the Ca(2+) indicator dye Fura-2 and digital imaging microscopy. ATP, 2-methylthio-ATP, 2-methylthio-ADP, ADP, UTP, UDP, deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP evoked increases in [Ca(2+)](i) in both the presence and absence of extracellular Ca(2+). Therefore, the increases we observed were likely due to intracellular Ca(2+) release mediated by G-protein-coupled P2Y receptor activation, rather than Ca(2+) influx via P2X receptor channels. The P2Y(1) receptor agonists 2-methylthio-ATP, 2-methylthio-ADP, and ADP evoked increases in [Ca(2+)](i) that were inhibited by the P2Y(1) receptor antagonists adenosine 3'-phosphate 5'-phosphosulfate and 2'-deoxy-N(6)-methyleneadenosine-3',5'-bisphosphate. Responses to ADP were not completely inhibited by the P2Y(1) receptor antagonists. The residual response to ADP could be mediated by P2Y(13) receptors. UTP evoked an increase in [Ca(2+)](i) that was partially inhibited by suramin, suggesting that Müller cells express P2Y(2) and P2Y(4) receptors. The P2Y(6) receptor agonist UDP, and the P2Y(11) receptor agonists deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP, evoked increases in [Ca(2+)](i) in Müller cells. We conclude that isolated tiger salamander Müller cells express P2Y(1), P2Y(2), P2Y(6), P2Y(11), and possibly P2Y(4) and P2Y(13) receptors. Therefore, the physiological release of ATP, ADP, UTP, and UDP and/or their accumulation in the retina under pathological conditions could stimulate increases in [Ca(2+)](i) in Müller cells.