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.

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.

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.

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.

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.