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After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the β and γ ranges (10–100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.
The ability of transplanted stem cell-derived neurons to receive and send functional excitatory and inhibitory inputs is a key indicator of successful functional integration into the CNS(Kim et al., 2002; Benninger et al., 2003; Wernig et al., 2004; Lee et al., 2007; Li et al., 2008; Weick et al., 2010; Weick et al., 2011); however, it remains to be shown that whole populations of transplanted human embryonic stem cell (hESC)-derived neurons can drive the activity of local neural networks in the brain. Since local neural circuits in organotypic hippocampal slices retain the ability to generate synchronized high-frequency oscillations(Fischer et al., 2002), and they can support the maturation and integration of transplanted stem cell-derived neurons(Benninger et al., 2003; Scheffler et al., 2003; Opitz et al., 2007), we used this system to ask whether transplanted hESC-derived neurons can integrate and drive neuronal network activity in the CNS. By combining the high spatial resolution provided by optogenetics (via expression of genetically-encoded photosensitive membrane proteins (Zhang et al., 2006) in transplanted hESC-derived neurons) with electrophysiological recording of local neuronal network activity (using multielectrode array or MEA techniques (Taketani and Baudry, 2006)), we were able to elicit high-frequency oscillations in the β/γ range (10–100 Hz) in the host hippocampal network. Since light-stimulation specifically activated the transplanted hESC-derived neurons, we could show that these new neurons behaved not only as conductors of electrical impulses along a network but could also actively engage local neuronal networks into firing at frequency rates commonly observed during normal processing of neural information in the hippocampus(Traub et al., 1999a).
Differentiation of hESCs into neurons was performed using a modification of previously described methods (Elkabetz and Studer, 2008; Li et al., 2008; Cho et al., 2011). Briefly, undifferentiated H9 hESCs were grown on a monolayer of fibroblasts from human foreskin (Hs27, ATCC). Cell culture medium was composed of DMEM/F12, knockout serum replacement (20%), basic fibroblast growth factor (8 ng/ml), β-mercaptoethanol (0.1 mM), and non-essential amino acids (1 mM). Media changes were performed daily and cells were subcultured on a weekly basis. For neural stem/progenitor cell (NPC) induction, hESCs were incubated for 24 hours in cell culture medium composed of DMEM/F12:Neurobasal (1:1), B27 serum-free supplement (2%), and N2 serum-free supplement (1%). They were then mechanically dissociated until small clusters of cells were obtained. These cell clusters were cultured for up to 72 hours in the same medium. For neural expansion, cells were grown for up to 6 days in cell culture medium composed of DMEM/F12:Neurobasal (1:1), B27 serum-free supplement (1%), N2 serum-free supplement (0.5%), basic fibroblast growth factor (20 ng/ml), and epidermal growth factor (20 ng/ml). Cells were then plated on dishes coated with 10 μg/ml laminin and grown for up to 48 hours until they took a rosette-like appearance. These rosette-neural stem cells (R-NSCs) were retrieved using a needle and transferred to a dish where they were cultured for 3 to 30 days in the same medium.
Epifluorescence microscopy for immunocytochemical analysis and gel electrophoresis followed by immunoblot analysis were performed as previously described (Li et al., 2008; Cho et al., 2011). Primary antibodies included: β-tubulin III (TuJ1; mouse, 1:1000, Covance), microtubule-associated protein 2 (MAP2; mouse, 1:1000, Sigma), synaptophysin (SYPH; rabbit, 1:1000, Dako), tyrosine hydroxylase (TH; mouse, 1:1000, Pel-Freez; rabbit, 1:1000, Chemicon), glutamate transporter 1 protein (vGAT; mouse, 1:1000), and glutamic acid decarboxylase (GAD65/67; rabbit, 1:500, Abcam). Alexa 488, 555 or 647-conjugated goat anti-mouse or anti-rabbit IgGs (1:1000, Invitrogen) were used as secondary antibodies.
Cells at the R-NSC stage were transduced with lentiviral expression vectors. As previously described, to enhance neurogenesis we expressed a lentivirus containing a constitutively active form of the transcription factor myocyte enhancer factor-2 (MEF2C-CA) (Li et al., 2008; Cho et al., 2011). Additional lentiviral vectors included pSynapsinI-hChR2-EGFP and pEF1-NpHR-mCherry (Zhang et al., 2006). Once infected, R-NSCs were dissociated and transferred to plates coated with a mixture of 10 μg/ml poly-L-ornithine and 1 μg/ml laminin. These R-NSCs were grown to become hNPCs as a monolayer in the medium for neural expansion. At this point, hNPCs were enzymatically dissociated into a single cell suspension using Accutase. For transplantation, hNPCs were washed in Ca2+/Mg2+-free Hanks’ buffered salt solution and concentrated. Cells were placed onto the CA3 region using a glass micropipette attached to a nanoliter injector (Nanoject, Drummond). Each slice received a single injection of a single-cell suspension containing 50,000–100,000 cells. Slices were placed back in a 37 °C/5% CO2 incubator and analyzed for electrophysiological activity every week for at least 4 weeks post injection.
Organotypic slices were prepared as described elsewhere (Stoppini et al., 1991; Scheffler et al., 2003; Opitz et al., 2007). Briefly, 400 μm-thick transverse hippocampal slices were cut from P6 male and female rats pups (Sprague-Dawley, Harlan), in ice-cold calcium-free MEM. Immediately after sectioning, the slices were transferred on Millicell-CM membrane inserts (Millipore) in wells containing culture medium of the following composition: 50% Basal Medium Eagle, 25% horse serum, 19% Earle’s Balanced Salt Solution, 25 mM Hepes, 32 mM glucose; 2 mM glutamine, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B, pH 7.20. Slices were kept in a humidified incubator at 37 °C in 5% CO2. After 24 hours, slices were transferred to a maintenance culture medium of similar composition with a lower concentration of horse serum (5%). Media was changed every 3–4 days.
For cultures, whole-cell recordings were performed at room temperature (22 °C) using a patch-clamp amplifier (Axopatch 200B, Molecular Devices, Union City, CA). Cells on coverslips were placed in a 150 μl recording chamber mounted on the stage of a Zeiss Axiovert inverted microscope. Cells were continuously superfused with Hepes-buffered external solution of the following composition (in mM): 137 NaCl, 1 NaHCO3, 0.34 Na2HPO4, 2.5 KCl, 0.44 KH2PO4, 2.5 CaCl2, 5 HEPES, 22.2 glucose, pH adjusted to 7.3 with NaOH. Patch pipettes were pulled from borosilicate glass capillaries (GC150F-10, Warner Instruments) using a Flaming/Brown micropipette puller (P80/PC, Sutter Instruments, Novato, CA). Patch pipettes had open tip resistances of 4–10 MΩ. For organotypic slices, extracellular field potentials were recorded at 35 °C using a multielectrode array (MEA60, Multi Channel Systems, Reutlingen, Germany). The MEA chamber contained 60 electrodes, each 30 μm in diameter at a distance apart of 200 μm. Slices were held down against the electrodes by a ring with a fine mesh and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 D-glucose; pH 7.4, osmolarity 310 mOsm. Photostimulation of optogenetic constructs was controlled via a xenon lamp in a Lambda DG-4 High Speed Filter Changer (Sutter, Novato, CA), equipped with appropriate excitation filters (ChR2: FF01-475/35-25; NpHR: FF01-585/40-25, Semrock, Rochester, NY).
To confer the ability for optogenetic stimulation to our transplanted cells, neural progenitor cells (hESC-derived NPCs) were transduced with lentiviral expression vectors containing channelrhodopsin (pSynI-ChR2-EYFP), as an excitatory ion channel, and halorhodopsin (pEF1-NpHR-mCherry), as an inhibitory transporter (Fig. 1A) (Zhang et al., 2006). When assayed electrophysiologically, hESC-derived neurons displayed resting membrane potentials of approximately −50 mV and spontaneous action potentials that were sensitive to the selective sodium channel blocker tetrodotoxin (TTX, 1 μM; Fig. 1B). Next, we recorded from these hESC-derived neurons with a patch electrode in voltage-clamp mode to obtain evidence for the expression of the optogenetic constructs by observing both depolarizing and hyperpolarizing light-activated responses (Fig. 1C, n = 4 cells). The hESC-derived NPCs had differentiated to the neuronal phenotype as evidenced by electrophysiological criteria, including firing action potentials (Fig. 1B), and immunocytochemical evidence based on multiple markers (Fig. 1D) (Li et al., 2008; Cho et al., 2011).
MEF2CA-driven neuronal differentiation (Li et al., 2008; Cho et al., 2011) produced neurons with typical neuronal phenotype, as seen with immunocytochemical staining for the specific neuronal markers synapsin 1 and MAP2, synaptophysin,, tau protein (Fig. 1D), and TuJ1 (Fig. 1E). When MEF2CA-programmed (compared to vector-infected control) hESC-derived neurons were analyzed for the presence of neurotransmitter specific markers, they showed high levels of expression of the synthetic enzymes tyrosine hydroxylase and glutamic acid decarboxylase (Fig. 1E), indicating an abundance of dopaminergic and GABAergic neurons, respectively. At the same time, hESC-derived neurons manifested relatively low levels of vesicular glutamate transporter 1 protein, indicating generation of a lower number of glutamatergic neurons than under control conditions (Fig. 1E).
To study the integration of hESC-derived NPCs into CNS tissue, we transplanted these cells, after viral infection, onto hippocampal slices and cultured them for up to 7 weeks at 37 °C and 5% CO2. Slices were assayed using pulses of light at wavelengths of 475 and 589 nm in order to activate ChR2 and NHpR2, respectively. Starting two weeks post-transplantation, cells with characteristic neuronal morphology and long processes that extended for hundreds of microns could be seen on the slices (Fig. 1F). Control slices without transplanted hESC-derived neurons or with transplanted hESC-derived neurons expressing GFP only (without optogenetic constructs) did not respond to light stimulation (Fig. 1G). In contrast, photostimulation of slices transplanted with hESC-derived neurons expressing optogenetic constructs elicited oscillations several millimeters distal to the site of the transplanted cells, whose location could be monitored by the presence of fluorescent markers (Fig. 2A, B). This network activity was characterized by a transient initial phase of high-frequency oscillations in the γ-band range followed by persistent lower frequency oscillations in the β range (Figs. 2C–F and and3;3; n = 18 slices analyzed). The low frequency of spikes associated with these oscillations suggested that this network phenomenon did not represent seizure activity (Khazipov and Holmes, 2003). Thus, the transplanted hESC-derived NPCs developed into neurons capable of driving an endogenous neuronal network in the hippocampus at some distance from the site of transplantation.
This study supports the use of slice preparations (Scheffler et al., 2003; Opitz et al., 2007) with optogenetic technology (Zhang et al., 2006), not only to assess single cell activity as previously reported (Weick et al., 2010; Weick et al., 2011), but also to monitor network events associated with the functional integration of engrafted stem cell-derived neuronal populations into CNS tissue. Importantly, this is the first demonstration that a population of transplanted stem cell-derived neurons can integrate into and participate in endogenous neuronal network activity. We show that MEF2CA-driven neuronal differentiation produces a neuronal population enriched in dopaminergic and GABAergic neurons, which are known to a play role in the generation and modulation of high-frequency oscillations in the hippocampus (Mann and Paulsen, 2007; Andersson et al., 2012). Hence, the light-induced high-frequency oscillations that we observed in organotypic slices may be explained at least in part by the ability of the newly-formed, optogenetically competent hESC-derived GABAergic and dopaminergic neurons to synchronize local neuronal networks within the hippocampus. The need for relatively long (10–20 s) periods of light stimulation for the induction of oscillations may relate to the relatively immature state of the hESC-derived neurons at the time of the experiments, as shown by their somewhat depolarized resting membrane potential. An interesting network behavior that we observed during optogenetic stimulation of engrafted hESC-derived neurons was a shift from gamma to slower beta oscillations, which has been previously described in human brain electroencephalograms (EEG) during sensory stimulation (Haenschel et al., 2000) and in acute hippocampal slices during electrical stimulation (Traub et al., 1999b). Because fast neuronal oscillations in the β/γ range (10–100 Hz) are linked to the performance of sensory-motor and cognitive tasks (Gray, 1994; Wang, 2010) and disruption of such network activity has been shown to accompany neuronal dysfunction in neurological disorders (Uhlhaas and Singer, 2006; Barth and Mody, 2011), transplantation of optogenetically-controllable hESC-derived NPCs can potentially be used to restore neuronal network activity, and thus reestablish motor and cognitive function (Traub et al., 1999a; De Feo et al., 2012). Similarly, the use of combined optogenetic and MEA techniques should prove useful in the assessment of functional integration of transplanted stem cell-derived neurons in a variety of in vivo paradigms.
This work was supported in part by CIRM Comprehensive Grant RC1-00125-1, and NIH grants P01 HD29587, P01 ES016738, P30 NS076411, R01 EY05477, and R01 EY09024 (to S.A.L.). We thank Traci Fang Newmeyer for excellent technical assistance.
Author Contributions: J.C.P.-C., M.T., E.-G.C., W.S., S.M. and S.A.L. designed research. K.D. supplied optogenetic constructs and commented on the manuscript. J.C.P.-C., M.T., E.-G.C., W.S., S.D.R, R.A., and S.M. performed research and analyzed data. J.C.P.-C. M.T., E.-G.C., and S.A.L. wrote the paper.