Tracking Stem Cell Differentiation in the Setting of Automated Optogenetic Stimulation

Department of Bioengineering, Behavioral Sciences, Stanford University, Stanford, USA.
Stem Cells (Impact Factor: 6.52). 01/2011; 29(1):78-88. DOI: 10.1002/stem.558
Source: PubMed


Membrane depolarization has been shown to play an important role in the neural differentiation of stem cells and in the survival and function of mature neurons. Here, we introduce a microbial opsin into ESCs and develop optogenetic technology for stem cell engineering applications, with an automated system for noninvasive modulation of ESC differentiation employing fast optogenetic control of ion flux. Mouse ESCs were stably transduced with channelrhodopsin-2 (ChR2)-yellow fluorescent protein and purified by fluorescence activated cell sorting (FACS). Illumination of resulting ChR2-ESCs with pulses of blue light triggered inward currents. These labeled ESCs retained the capability to differentiate into functional mature neurons, assessed by the presence of voltage-gated sodium currents, action potentials, fast excitatory synaptic transmission, and expression of mature neuronal proteins and neuronal morphology. We designed and tested an apparatus for optically stimulating ChR2-ESCs during chronic neuronal differentiation, with high-speed optical switching on a custom robotic stage with environmental chamber for automated stimulation and imaging over days, with tracking for increased expression of neural and neuronal markers. These data point to potential uses of ChR2 technology for chronic and temporally precise noninvasive optical control of ESCs both in vitro and in vivo, ranging from noninvasive control of stem cell differentiation to causal assessment of the specific contribution of transplanted cells to tissue and network function.

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    • "Pathways to specification of neurons expressing particular transmitters have been identified (Lee et al., 2000; Bibel et al., 2004), mirroring the initial process of transmitter specification in early development. Whether these transmitter phenotypes can be altered by activity or protein factors can now be tested (Stroh et al., 2011; Sun et al., 2013). Is transmitter switching regulated epigenetically? "
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    ABSTRACT: Among the many forms of brain plasticity, changes in synaptic strength and changes in synapse number are particularly prominent. However, evidence for neurotransmitter respecification or switching has been accumulating steadily, both in the developing nervous system and in the adult brain, with observations of transmitter addition, loss, or replacement of one transmitter with another. Natural stimuli can drive these changes in transmitter identity, with matching changes in postsynaptic transmitter receptors. Strikingly, they often convert the synapse from excitatory to inhibitory or vice versa, providing a basis for changes in behavior in those cases in which it has been examined. Progress has been made in identifying the factors that induce transmitter switching and in understanding the molecular mechanisms by which it is achieved. There are many intriguing questions to be addressed. Copyright © 2015 Elsevier Inc. All rights reserved.
    Neuron 06/2015; 86(5):1131-1144. DOI:10.1016/j.neuron.2015.05.028 · 15.05 Impact Factor
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    • "V mem arises from the combined action of ion channels and ion pumps, as well as of gap junctions (GJs) – highly versatile aqueous connections between the cytoplasm of adjacent cells that allow voltage and current-mediated signals to be propagated and regionalized across cell groups. Cellular V mem regulates cell-autonomous properties such as proliferation, differentiation and apoptosis (Blackiston et al. 2009; Sundelacruz et al. 2009; Aprea & Calegari, 2012), in mature somatic cells (Cone & Tongier, 1971; Stillwell et al. 1973) as well as stem cells (Stroh et al. 2011; Sundelacruz et al. 2013) and cancer cells (Yang & Brackenbury, 2013). Moreover, spatio-temporal patterns of differential V mem levels across the body are now known to be instructive cues during embryogenesis, regeneration and cancer (Adams, 2008; Levin, 2012a; Tseng & Levin, 2013). "
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    ABSTRACT: Pattern formation, as occurs during embryogenesis or regeneration, is the crucial link between genotype and the functions upon which selection operates. Even cancer and aging can be seen as challenges to the continuous physiological processes that orchestrate individual cell activities toward the anatomical needs of an organism. Thus, the origin and maintenance of complex biological shape is a fundamental question for cell, developmental, and evolutionary biology, as well as for biomedicine. It has long been recognized that slow bioelectrical gradients can control cell behaviors and morphogenesis. Here, I review recent molecular data that implicate endogenous spatio-temporal patterns of resting potentials among non-excitable cells as instructive cues in embryogenesis, regeneration, and cancer. Functional data have implicated gradients of resting potential in processes such as limb regeneration, eye induction, craniofacial patterning, and head-tail polarity, as well as in metastatic transformation and tumorigenesis. The genome is tightly linked to bioelectric signaling, via ion channel proteins that shape the gradients, downstream genes whose transcription is regulated by voltage, and transduction machinery that converts changes in bioelectric state to second-messenger cascades. However, the data clearly indicate that bioelectric signaling is an autonomous layer of control not reducible to a biochemical or genetic account of cell state. The real-time dynamics of bioelectric communication among cells are not fully captured by transcriptomic or proteomic analyses, and the necessary-and-sufficient triggers for specific changes in growth and form can be physiological states, while the underlying gene loci are free to diverge. The next steps in this exciting new field include the development of novel conceptual tools for understanding the anatomical semantics encoded in non-neural bioelectrical networks, and of improved biophysical tools for reading and writing electrical state information into somatic tissues. Cracking the bioelectric code will have transformative implications for developmental biology, regenerative medicine, and synthetic bioengineering.
    The Journal of Physiology 06/2014; 592(Pt 11):2295-2305. DOI:10.1113/jphysiol.2014.271940 · 5.04 Impact Factor
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    • "Our data extend the previous analyses of cell membrane voltage to neural stem cells by using the new optogenetic technique. Neural stem cells or ESCs can be made photosensitive by genetic modification with ChRs and enabled to analyze proliferation or differentiate change by depolarization with rhythmic photo-stimulation (Stroh et al. 2011; Tonnesen et al. 2011; Weick et al. 2010). Optical stimulation-induced depolarization inhibited proliferation of neural stem cells in our study. "
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    ABSTRACT: Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G1 phase and reduced the percentage in S phase. It is likely that the influence of blue light on cell proliferation and the cell cycle was mediated by membrane depolarization, which induced accumulation of p21 and p27 proteins. Our data provide additional specific evidence that membrane depolarization may inhibit neural stem cell proliferation.
    Journal of Membrane Biology 04/2014; 247(6). DOI:10.1007/s00232-014-9659-7 · 2.46 Impact Factor
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