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Ng K.-M.,University of Hong Kong | Lee Y.-K.,University of Hong Kong | Chan Y.-C.,University of Hong Kong | Lai W.-H.,University of Hong Kong | And 7 more authors.
Journal of Molecular and Cellular Cardiology | Year: 2010

Hypoxia plays an important role in the proliferation, differentiation and maintenance of the cardiovascular system during development. While low oxygen tension appears to direct the cultured embryonic stem cells (ESCs) to differentiate into cardiomyocytes, the underlying molecular mechanism remains unclear. At a molecular level, hypoxia inducible factor-1 (HIF-1) plays an important role in handling the hypoxia signal. In the present study, we demonstrated that expression of exogenous HIF-1α cDNA into murine ESCs significantly promoted cardiogenesis as indicated by a higher percentage of beating embryoid body and troponin-T positive cell counts as well as increased expression of early and late cardiac markers, such as GATA-binding protein 4 and 6, NK2 transcription factor related locus 5, α-myosin heavy chain, β-myosin heavy chain and myosin light chain 2 ventricular transcripts. In addition, the transduced cells exhibited increased mRNA levels of cardiotrophin-1 and vascular endothelial growth factor, along with phosphorylation of eNOS [p-eNOS (ser1171)]. Application of NOS inhibitors, diphenyleneiodonium chloride (DPI), Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or Nω-Nitro-l-arginine (l-NNA) abolished the HIF-1α stimulated cardiac differentiation. With the clues of upregulated mRNA expression of calcium handling proteins, ryanodine receptor 2, sodium calcium exchanger and sarcoplasmic/endoplasmic reticulum calcium ATPase, in the transduced HIF-1α ESCs, further study indicated that the maximum upstroke and decay velocity was significantly increased in both non-caffeine and caffeine-induced calcium transient in ESCs-derived cardiomyocytes. This suggests a well developed function of the sarcoplasmic reticulum in ESC-derived cardiomyocytes. Electrophysiological study also indicated that a portion of the HIF-1α-transduced cells exhibited prominent phase-4 depolarization. These findings suggest that keen activation of the HIF-1 pathway enhances differentiation and maturation of cardiomyocytes derived from ESCs. © 2010 Elsevier Ltd.

Barrilleaux B.L.,Tulane University | Barrilleaux B.L.,Institute of Pediatric Regenerative Medicine | Fischer-Valuck B.W.,Tulane University | Fischer-Valuck B.W.,Louisiana State University Health Sciences Center | And 3 more authors.
In Vitro Cellular and Developmental Biology - Animal | Year: 2010

Therapeutic administration of mesenchymal stem cells (MSCs) by systemic delivery utilizes the innate ability of the cells to home to damaged tissues, but it can be an inefficient process due to a limited knowledge of cellular cues that regulate migration and homing. Our lab recently discovered that a potent pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), inhibits MSC migration. Because MIF may act on multiple cellular targets, an activating antibody (CD74Ab) was employed in this study to examine the effect of one MIF receptor, CD74 (major histocompatibility complex class II-associated invariant chain), on MSC motility. CD74 activation inhibits in a dose- dependent manner up to 90% of in vitro migration of MSCs at 40 μg/ml CD74Ab (p<0.001), with consistent effects observed among three MSC donor preparations. A blocking peptide from the C-terminus of CD74 eliminates the effect of CD74Ab on MSCs. This suggests that MIF may act on MSCs, at least in part, through CD74. Late-passage MSCs exhibit less chemokinesis than those at passage 2. However, MSCs remain responsive to CD74 activation during ex vivo expansion: MSC migration is inhibited -2-fold in the presence of 5 μg/ml CD74Ab at passage 9 vs. -3-fold at passage 2 (p<0.001). Consistent with this result, there were no significant differences in CD74 expression at all tested passages or after CD74Ab exposure. Targeting CD74 to regulate migration and homing potentially may be a useful strategy to improve the efficacy of a variety of MSC therapies, including those that require ex vivo expansion. © The Society for In Vitro Biology 2010.

Selvaraj V.,Cornell University | Jiang P.,University of California at Davis | Chechneva O.,University of California at Davis | Lo U.-G.,University of California at Davis | And 2 more authors.
Frontiers in Bioscience | Year: 2012

Research on the biology of adult stem cells, embryonic stem cells and induced pluripotent stem cells, as well as cell-based strategies for treating nervous system disorders has begun to create the hope that these cells may be used for therapy in humans after injury or disease. In animal models of neurological diseases, transplantation of stem cells or their derivatives can improve function not only due to direct replacement of lost neurons or glia, but also by providing trophic support. Despite intense research efforts to translate these studies from the bench to bedside, critical problems remain at several steps in this process. Recent technological advancements in both the derivation of stem cells and their directed differentiation to lineagecommitted progenitors have brought us closer to therapeutic applications. Several preclinical studies have already explored the behavior of transplanted cells with respect to proliferation, migration, differentiation and survival, especially in complex pathological disease environments. In this review, we examine the current status, progress, pitfalls, and potential of these stem cell technologies, focusing on directed differentiation of human stem cells into various neural lineages, including dopaminergic neurons, motor neurons, oligodendroglia, microglia, and astroglia, and on advancements in cell-based regenerative strategies for neural repair and criteria for successful therapeutic applications.

Fu J.-D.,California Stem Cell | Fu J.-D.,University of California at Davis | Jiang P.,California Stem Cell | Jiang P.,University of California at Davis | And 9 more authors.
Stem Cells and Development | Year: 2010

In adult cardiomyocytes (CMs), the Na+/Ca2+ exchanger (NCX) is a well-defined determinant of Ca2+ homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofibrillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2vdsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While ICa-L, If, and I Kr were robustly expressed, IKs and IK1 were absent in dsRed+ ventricular hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca2+]i, Ca2+ transient amplitude, maximum upstroke, and decay velocities significantly increased (P<0.05). The ICa-L antagonizer nifedipine (1μM) decreased the Ca2+ transient amplitude (to ∼30%) and slowed the kinetics (by ∼2-fold), but Ca2+ transients could still be elicited even after complete ICa-L blockade, suggesting the presence of additional Ca2+ influx(es). Indeed, Ni2+-sensitive INCX could be recorded in 7+40- and +90-day dsRed+ hESC-CMs, and its densities increased from -1.2±0.6 pA/pF at -120 mV and 3.6±1.0 pA/pF at 60 mV by 6- and 2-folds, respectively. With higher [Ca2+]i, 7+90-day ventricular hESC-CMs spontaneously but irregularly fired transients upon a single stimulus under an external Na+-free condition; however, without extracellular Na +, nifedipine could completely inhibit Ca2+ transients. We conclude that INCX is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation-contraction coupling. © Copyright 2010, Mary Ann Liebert, Inc.

Jiang P.,Institute of Pediatric Regenerative Medicine | Jiang P.,California Stem Cell | Rushing S.N.,Institute of Pediatric Regenerative Medicine | Rushing S.N.,California Stem Cell | And 14 more authors.
American Journal of Physiology - Cell Physiology | Year: 2010

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC50 = 3.3 ± 2.7 mM) delayed rectifier K + currents (IKDR) in 105 of 110 single iPSCs (15.4 ± 0.9 pF). IKDR in iPSCs displayed a current density of 7.6 ± 3.8 pA/pF at +40 mV. The voltage for 50% activation (V1/2) was -7.9 ± 2.0 mV, slope factor k = 9.1 ± 1.5. However, Ca 2+-activated K+ current (IKCa), hyperpolarization-activated pacemaker current (If), and voltage-gated sodium channel (NaV) and voltage-gated calcium channel (Ca V) currents could not be measured. TEA inhibited iPSC proliferation (EC50 = 7.8 ± 1.2 mM) and viability (EC50 = 5.5 ± 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC50 = 4.5 ± 0.5 mM) but had less effect on proliferation (EC50 = 0.9 ± 0.5 mM). Cell cycle analysis further revealed that K+ channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of IKCa in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application. Copyright © 2010 the American Physiological Society.

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