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Keywords:

  • Human embryonic stem cells;
  • Differentiation;
  • Cytokines;
  • Leukemia inhibitory factor;
  • Neural differentiation;
  • Stem cell transplantation;
  • Progenitor cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC-derived NP cells. In NP cells, as well as NP-derived neurons, LIF reduced caspase-mediated apoptosis and reduced both spontaneous and H2O2-induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self-renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3-kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF+ media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell-based therapeutic transplant studies. STEM CELLS2012;30:2387–2399


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Human embryonic stem cells (hESC), by definition, can generate every cell type found in the body [1]. We have previously derived a self-renewing but fate-restricted neural progenitor (NP) cell line from hESC that can generate highly enriched neuronal populations under specific conditions [2, 3], thus serving as a potentially unlimited source of human neurons for both basic [4] and applied research [5]. This progenitor line has previously been used to study neurotoxicity, differentiation to specific neuronal phenotypes, drug screening, and transplantation experiments in rodent models of stroke [4, 5]. However, the ultimate success of NP cell-based therapy in transplantation experiments may depend on identifying factors that enhance the capacity of these cells to expand in vitro and resisting suboptimal conditions such as the generation of reactive oxygen species (ROS) [6–8] and to survive and function in a diseased or injured niche.

In the mouse, leukemia inhibitory factor (LIF, a member of the IL-6 family of cytokines) promotes cell survival and influences cell fate both in vivo and in vitro; LIF has pleiotropic effects on cell function in different biological contexts. Upon binding to its heterodimeric receptor formed by gp130 (IL6ST) and LIFR, LIF elicits a cascade of signaling events mediated through JAK-STAT3 (Janus kinase-Signal transducer and activator of transcription 3), MEK (MAPK/ERK kinase), and PI3K (phosphoinositide 3-kinases) pathways [9]. These pathways are primarily associated with cell survival, differentiation, and regulation of apoptosis, respectively, in non-neural stem cells, such as mouse ESCs [10]. In the rodent nervous system, LIF promotes regeneration of transected/axotomized nerves [11–13] and protects neurons during development [14] and in disease [15, 16], but similar human studies are not available.

Little is known about the role of LIF or its effects on growth and differentiation of hESC-derived neural cells in vitro or in vivo. Although human fetal neural stem cells are responsive to LIF signaling [17], no study has elucidated the role of LIF in neural cells derived from hESC. Also, hESC-derived NP cells and fetal NP cells differ in many ways including their use of critical pathways like wingless-type MMTV integration site family, member (WNT), Fibroblast growth factor (FGF), and LIF signaling [18]. With the added potential of hESC-derived NPs over fetal NPs as an unlimited source of therapeutic cells, it is essential to specifically address LIF-induced effects in these cells. However, since hESC are not LIF dependent, the involvement of LIF signaling in hESC neural differentiation and in vivo transplantation has largely been ignored. In this study, the effects of LIF on hESC-derived NP cell apoptosis, ROS production, and neuronal differentiation were investigated. LIF prevented apoptotic NP cell death, protected NP cells against ROS, enhanced proliferation, and played a role in neuronal differentiation in vitro, promoting the extension of neurites in NP-derived neurons. We also show that in hESC-derived NP cells, LIF specifically modulates expression of one of the LIF receptor components, gp130 (IL6ST), without changing the expression of the other component, LIFR. Here, we demonstrate that the modulation of LIF signaling has multiple beneficial effects on human NP cells and NP-derived neuronal cultures, and LIF-treated NP cells, maintained their therapeutic potential after transplantation into the infarcted brain. As pluripotent stem cell-derived cells move closer to clinical trials, enhanced culture conditions such as activation of LIF pathways should increase yield and improve viability in bioprocessing systems and ultimately lead to reduced costs and improved product consistency.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

NP Propagation and Derivation of Neurons

NPs were derived from WA09 hESC and cryopreserved as described previously [2]. They were subcultured on polyornithine- and laminin-coated dishes using neurobasal medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 2 mM L-glutamine (Gibco), 1× penicillin/ streptomycin (Gibco), 1× B27 (Gibco), 20 ng/ml of FGF2 (Sigma-Aldrich, St Louis, MO, http://www.sigmaaldrich.com), and 10 ng/ml LIF (Chemicon-Millipore, Billerica, MA, http://www.millipore.com/). Cells were passaged by mechanical dissociation via trituration and replating on polyornithine- (20 μg/ml) and laminin (5 μg/ml)-coated plates or on Permanox slides (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com). Neurons were generated by differentiating NP cells for 2 weeks in medium lacking FGF2. NP cells were plated overnight on polyornithine- and laminin-coated dishes in medium containing FGF2 as described above. The next day cells were switched to medium lacking FGF2, and culture was replenished with fresh medium twice a week.

TUNEL Assay and Caspase Glo 3/7 Assay for Apoptosis Detection

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay is commonly used for detecting DNA fragmentation caused by apoptosis [19, 20]. For detection of apoptosis in adherent culture, cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature, then in 70% ethanol at −20°C overnight. TUNEL assay was performed using the APO-BrdU TUNEL assay kit (Molecular Probes-Invitrogen) following manufacturer's instructions. Photomicrographs of the stained cells were taken using Olympus IX81 with Disc-Spinning Unit and Slide Book Software (Intelligent Imaging Innovations). Activities of Caspase3/7 were determined from homogenous cell lysates by measuring luminescence using the Caspase Glo 3/7 assay (Promega, Madison, WI, http://www.promega.com) as per manufacturer's instructions. Cells exposed to actinomycin-D were used as positive control and used to normalize other corresponding treatments.

Semiquantitative Reverse Transcription Polymerase Chain Reaction and Quantitative Real-Time PCR

RNA was isolated from NP cells and neurons using RNeasy plus kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) [21]. One microgram RNA was reverse transcribed using Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA, http://www.clontech.com). Polymerase chain reaction (PCR) was performed using GoTaq Green Master Mix (Promega). Target genes were amplified using primers listed in Supporting Information Table 1. For real-time PCR, Taqman assays were used: LIFR (Hs00158730_m1), IL6ST (Hs00174360_m1), PCNA (Hs00427214_g1) and endogenous control, GAPDH (Hs99999905_m1).

Detection of ROS

ROS were detected using either hydrocyanine dye, Hydro-Cy3 [22] or CM-H2DCFDA dyes (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). Epifluorescence of cells plated on Permanox slides were photographed using a Nikon inverted microscope at ×200 magnification. For quantification of ROS, cells were grown on a black wall clear bottom Costar 96-well plate (Corning, Acton, MA, http://www.corning.com/lifesciences), and fluorescence intensity was detected using a FlexStation 3 plate reader (Molecular Devices, Union City, CA, http://www.moleculardevices.com). For generation of ROS by hydrogen peroxide (H2O2), cells were plated in 96-well plates for 24 hours in the absence of LIF. Cells were exposed to H2O2 at final concentrations of 0, 10, 50, 250, and 500 μM 1 hour prior to measurement. HydroCy3 was added at 50 μM final concentration for the last 30 minutes, and adjustments were made to keep the H2O2 concentrations constant. For LIF dose-response experiments, LIF was added back to the appropriate wells at 0, 10, and 50 ng/ml 1 hour prior to the addition of H2O2. All wells were rinsed once in PBS containing Ca2+/Mg2+ (PBS+/+) and readings were taken in PBS+/+.

EdU Assay for Detection of Proliferating Cells

NP cells were cultured in media containing LIF (LIF+) and without LIF (LIF) for 14 days. Cells were exposed to EdU for 12 hours before harvesting and stained for EdU using the Click-iT EdU cell proliferation assay kit (Life Technologies, New York, NY, http://www.lifetech.com) as per manufacturer's protocol. Flow cytometry was performed to detect percent of EdU+ proliferating cells on a BD FACSCalibur flow cytometer (BD Biosciences).

Immunocytochemistry

Immunocytochemistry was performed on 4% PFA-fixed cells as described previously [3, 4]. Antibodies used were: SOX2 1:100 MAB2018 (R&D Systems, Minneapolis, MN, http://www.rndsystems.com), Nestin 1:200 MO15012 (Neuromics), HuC/D 1:40 A21271 (Invitrogen Corp., Carlsbad, CA, http://www.invitrogen.com), and MAP2 (microtubule-associated protein 2) 1:400 AB5622 (Chemicon, Temecula, CA, http://www.chemicon.com).

Flow Cytometry

Cells were harvested in PBS lacking Ca2+/Mg2+ (PBS−/−), fixed using 4% PFA at room temperature for 5 minutes, washed twice with PBS−/−, and centrifuged at 1,000g at 23°C for 4 minutes. Fixed cells were stained as described previously [3] and data were acquired using FACSCalibur system (BD Biosciences). Analysis was done with FlowJo analysis software (Tree Star, Inc., Ashland, OR, http://www.flowjo.com/). Forward- and side-scatter plots were used to exclude dead cells and debris from the histogram analysis. Antibodies used for flow cytometry experiment were the same as above.

Cellomics High Content Analysis

This assay was performed as described previously [23]. Briefly, cryopreserved NP cells differentiated for 2 weeks in the presence (LIF+) or absence (LIF) of LIF were thawed and plated at a density of 10,000 viable cells per well (3.33 × 104 cells per cm2) in Costar 96-well polystyrene cell culture plates (Corning) coated with polyornithine and laminin. Average live cell yields post-thawing were 29% ± 4% and 43% ± 11% for the culture without or with LIF, respectively. Cells were allowed to grow for 2, 6, 24, or 48 hours in vitro prior to sampling and stained with mouse monoclonal antibody against βIII-tubulin (1:800) and Hoechst 33258 nuclear dye as provided in Cellomics Neurite Outgrowth HitKit (ThermoFisher Scientific, Inc., Waltham, MA, http://www.thermofisher.com/). Time course experiments were performed in triplicate using three independently cultured frozen stocks. Stained cells were imaged using a Cellomics ArrayScan VTI HCS reader high-content imaging system (ThermoFisher Scientific, Waltham, MA) and analyzed with the Neuronal Profiler BioApplication.

Image analysis algorithm optimization, including determination of nucleus selection, cell body masking, and neurite tracing parameters, was performed a priori using representative images from cultures differentiated with or without LIF 48 hours after plating. Output from the image analysis (HCA) included indices of cell health (% viable nuclei per well) and measurements of neurite outgrowth (% of viable cells with ≥1 neurite, average number of neurites per viable cell, total neurite length per viable cell, and total neurite length per neurite). Data were collected on a cell-by-cell basis, and values were averaged to obtain population means within each well. These well-level data were treated as the statistical unit for analysis of neurite outgrowth. At ×20 magnification, the Cellomics ArrayScan VTI can sample 81 individual fields within each well. In this study, a sufficient number of fields were sampled within each well so that at least 350 individual neurons were measured. Image analyses data were analyzed using two-way ANOVA with time and cell type as the independent variables (significance threshold, p < .05). Data were also analyzed for differences across time within each treatment (LIF+ or LIF) and differences across treatments within each time point using Bonferonni post hoc t tests at a significance level of p < .05. All data are presented as well-level population averages ± SD. Analyses were performed using GraphPad Prism v 5.

Western Blotting

Cells were harvested in PBS, and total proteins were extracted using phosphosafe lysis buffer (Novagen, Madison, WI). The samples were sonicated at 3 W for 15 seconds using a VirSonic 100 VirTis Sonicator (Gardiner, NY). Approximately 30–50 μg proteins were electrophoresed on 4%–20% SDS-PAGE (Criterion Precast gel, Biorad, Hercules, CA, http://www.bio-rad.com) gels. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA in TBS-Tween-20 (0.05% Tween-20) and incubated at 4°C overnight with appropriate primary antibodies: Phospho-AKT (Cell Signaling, Danvers, MA, , http://www.cellsignal.com; dilution 1:1,000), HXKII (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com; 1:200), Phospho-ERK1/2 (Cell Signaling, 1:1,000), Phospho-STAT3 (Signalway Antibody, Pearland, TX, 1:1,000), Phospho-c-Myc (Thr58/Ser62) (Cell Signaling; 1:1,000), or anti-β-actin (Santa Cruz Biotechnology, 1:1,000). After washing, the blots were incubated for 1 hour with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (1:2,000, Invitrogen, San Diego, CA) and visualized by enhanced chemiluminescence.

Stroke Model, Cell Transplantation, and Measurement of Cell Survival and Infarct Volume

The animal protocol was approved by the Emory University Institutional Animal Care and Use Committee. For assessing cell survival, nonstroke animals (C57BL/6 mice) were transplanted with 400,000 cells. Animals were fixed on a stereotaxic frame (David Kopf Instruments, CA), and an incision was made exposing the skull. Cells were dissociated with Accutase, counted and resuspended in culture media without LIF or FGF2, and injected in the barrel cortex 500–600 μm deep at a rate of 0.5 μl/minute. Animals were sacrificed 48 hours after transplantation, and their brains immediately frozen on dry ice. Brain coronal sections were cut at 10 μm thickness using a cryostat (Leica CM 1950). TUNEL staining was performed using the DeadEnd Fluorometric TUNEL system (Promega) following previously described procedures [24]. SC-121 (SC121-StemCells, Inc.; 1:500) is a human specific antibody used to stain the human NP cells after transplantation in mice. Cell counting was performed following a modification of the principles of design-based stereology [25]. Systematic random sampling was used to ensure accurate and nonredundant cell counting. Every section under analysis was at least a 100 μm away from the next. A total of six 10-μm-thick sections spanning the entire regions of interest were randomly selected for cell counting from each animal. Counting was performed on six nonoverlapping randomly selected ×20 fields per section. Sections from different animals represent the same area in the anterior posterior direction.

Focal ischemic stroke was induced by occluding the right middle cerebral artery (MCA) in C57BL/6 male mice (8–12 weeks) as previously described [26]. In brief, animals were anesthetized with chloryl hydrate. The right MCA was permanently ligated together with a 7-minute bilateral common carotid artery ligation. Body temperature was maintained at 37°C during surgery, using a heating pad controlled by a homeothermic control unit (Harvard Apparatus, Holliston, MA). Control animals were injected with media without LIF or FGF2, and 2 million cells were injected in each experimental animal (LIF+ or LIF). After transplantation, animals were placed in an incubator until they regained mobility. For infarct volume measurement, animals were sacrificed 24 hours after transplantation and their brains were removed and placed in a coronal brain matrix. Brains were sliced into 1-mm-thick sections, and infarct volume was determined using 2,3,5-triphenyltetrazolium chloride staining as detailed in [27].

Statistical Analysis

Student's t test was used to compare data from the in vivo study with p < .05 for significance. ANOVA followed by Tukey's test was conducted using SAS 8.01 for all data except data collected from Cellomics high content experiments.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Presence of LIF Increased IL6ST (gp130) Transcription in NP Cells Undergoing Neuronal Differentiation

Cytokine signaling can modulate the expression of cytokine receptor components [28]. LIF binds to a heterodimeric receptor complex formed by two proteins, LIFR and gp130. To determine if transcription of these receptor components were affected in NP cells by LIF signaling while in neuronal transition cultures, both semiquantitative and real-time PCR for LIFR and gp130 were performed after 2 weeks in differentiation media (without FGF2) with LIF (LIF+) or without LIF (LIF). When compared to LIF+ cultures, IL6ST (the gene encoding gp130) transcription in NP cells in LIF medium was significantly downregulated (p < .01), but LIF did not affect LIFR gene transcription (Fig. 1A). To further validate involvement of LIF in modulating IL6ST (gp130) transcription, dose-response experiments were performed. NP cells were differentiated in increasing concentrations of LIF, and IL6ST transcript levels were assessed using real-time PCR.compared to LIF cultures, IL6ST transcription was significantly upregulated (1.6–2.4-fold) depending on the concentration of LIF (p < .001; Fig. 1B). Our data show that 10 ng/ml of LIF in medium is sufficient to cause significant upregulation of IL6ST (gp130) transcription.

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Figure 1. LIF regulates the expression of IL6ST (gp130). LIF signaling is mediated by binding to its heterodimeric receptor formed by gp130 (IL6ST) and LIFR. (A): Reverse transcription polymerase chain reaction (RT-PCR) to detect transcript levels for LIFR, IL6ST, and GAPDH (endogenous control) in NP cells before differentiation and after 2 weeks of LIF+ and LIF differentiation (left panel). Quantification using real-time PCR (right panel, expressed as quantity relative to NP cells) shows that LIF+ differentiated cells express significantly higher amounts of IL6ST transcripts than LIF differentiated cells (p < .01). Transcription of LIFR, however, was not affected by presence or absence of LIF. (B): IL6ST expression was significantly higher at all LIF concentrations tested (5, 10, 20, 40, and 100 ng/ml) when compared to cultures in LIF medium (p < .01). (C): TUNEL assay to detect apoptosis in neural progenitors differentiated for 2 weeks in LIF+ and LIF medium. Apoptotic cells were BrdU positive (green), nuclei were stained with PI (red). Representative pictures of cells differentiated in LIF+ (left column) and LIF (right column). Magnified view (inset in C) shows several nonapoptotic nuclei (PI+/BrdU) and one apoptotic nucleus (PI+/BrdU+). (D): Quantification of BrdU positive apoptotic cells. Approximately 85% of cells were apoptotic in LIF differentiation. This was reduced to about 35% in LIF+ differentiation medium (p < .01). (E): Caspase Glo 3/7 assay to quantify caspase3 and 7 activity showed significantly higher relative luminescence values indicating higher caspase activity in LIF culture compared to LIF+ differentiation (p < .05). Abbreviations: BrdU, Bromo-2′-deoxyuridine; FGF, Fibroblast growth factor; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; LIFR, LIF receptor; NP, neural progenitor; PI, Propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP (2′-deoxyuridine-5′-O-triphosphate) nick end labeling.

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Presence of LIF Reduces Cell Death in NP Cell-Derived Neurons

NP cells differentiate toward postmitotic neurons upon FGF2 withdrawal, and this event is accompanied by an initial cell death in these cultures [29]. To test the predicted role of LIF in preventing cell death during differentiation, NP cell populations differentiated in the presence of 10 ng/ml (LIF+) or absence (LIF) of LIF for 2 weeks were compared for apoptosis using TUNEL assay. The proportion of TUNEL-positive nuclei was significantly higher in LIF cultures (∼ 85%) than in LIF+ cultures (∼ 35%) (p < .0001; Fig. 1C, 1D). To test if these were caspase-mediated apoptotic events, caspase3 and 7 activities were estimated in these cultures using the Caspase Glo 3/7 assay. Significantly higher amounts of caspases were detected in LIF cultures (p < .05; Fig. 1E).

LIF Decreases ROS in Both NP and NP-Derived Neurons

Since elevated ROS can lead to neuronal cell death and in some cases apoptosis, NP cells were cultured or differentiated in the LIF+ or LIF medium, and intracellular ROS were quantified. LIF NP cell cultures produced significantly higher ROS compared to cells in LIF+ medium (p < .01; Fig. 2A). Similarly, ROS was significantly higher in LIF differentiated neuronal cultures compared to LIF+ medium (p < .005; Fig. 2D). Together, these results suggest that LIF can lower the level of spontaneous ROS in both NP and differentiated neuronal cells.

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Figure 2. LIF reduces production of reactive oxygen species (ROS) in NP cells and NP cell-derived neurons. Intracellular ROS was quantified using fluorescent detectors CM-H2DCFDA or Hydro-Cy3 and expressed as RFU. Addition of LIF in culture significantly reduced ROS in (A) NP cells (p < .05), and (D) in differentiated neurons (p < .005). An optimal dose of H2O2 for generating ROS was determined in NP cells (B), and NP-derived neurons (E). Dose-dependent reduction of ROS was observed in NP cells (C) and derived neurons (F) exposed to 250 μM of H2O2 in presence of 0, 10, and 50 ng/ml LIF. Different superscripts in B, C, E, and F indicate significant differences (p < .05). Abbreviations: LIF, leukemia inhibitory factor; NP, neural progenitor; RFU, relative fluorescent unit.

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LIF Reduces H2O2-Induced ROS in a Dose-Dependent Manner

H2O2 induces ROS in many cell types including human NP cells [30, 31]. First, intracellular ROS were assayed in NP cells and NP-derived neurons using increasing concentrations of H2O2 (0, 10, 50, 100, 250, and 500 μM) in culture media. NP cells exposed to 250 μM H2O2 for an hour showed significantly higher ROS than any of the lower concentrations (Fig. 2B). In subsequent experiments, NP cells cultured in the presence (LIF+) or absence (LIF) of LIF were challenged with H2O2 and assayed for ROS production. NP cells exposed to 250 μM H2O2 had significantly higher ROS (p < .05) than control cells. Addition of LIF (10 ng/ml) to this NP culture reduced the level of H2O2-induced ROS production, while addition of 50 ng/ml of LIF brought the level of ROS down further to that of the control culture without H2O2 or LIF (Fig. 2C). Likewise, for NP-derived neuronal cultures, 250 μM H2O2 was found to generate higher ROS than any of the lower concentration (Fig. 2E). Thus, neuronal cells were exposed to 250 μM H2O2 in media containing varying concentrations (0, 10, 50 ng/ml) of LIF and ROS levels were measured. In the presence of 10 ng/ml LIF, neuronal cultures produced significantly less ROS than LIF cultures, when exposed to H2O2 (p < .05). In the 50 ng/ml LIF+ cultures, ROS levels were roughly equal to those in control cells without H2O2 or LIF (Fig. 2F). Thus, LIF+ has a significant role in reducing H2O2-induced ROS in both NP and neuronal cells.

LIF Enhances Proliferation of Neuronal Cells

To determine if LIF had any significant effect on cell proliferation, LIF+ and LIF neuronal cultures were evaluated for expression of proliferation markers PCNA and SOX2. We also performed EdU-based proliferation assay to detect percent proliferating cells in cultures. The protein PCNA acts as a processivity factor for DNA polymerase-δ in eukaryotic cells and is expressed in the nuclei during the DNA synthesis phase of cell cycle [32]. Real-time PCR quantification showed that PCNA transcript levels were significantly higher in cells differentiated in LIF+ medium compared to LIF medium (p < .001; Fig. 3A). The EdU assay used is claimed to be similar but superior to BrdU assay for detecting proliferating cells in culture. We performed flow cytometry to count percent of EdU+ cells in LIF+ and LIF cultures and detected approximately 18% and 13% EdU+ cells, respectively, in LIF+ and LIF cultures (p < .005) (Fig. 3B). SOX2 is a self-renewal marker expressed in ESCs and NP cells [33]. Immunocytochemistry was performed to detect SOX2 protein expression. SOX2 protein was detected in both LIF+ and LIF neuronal cultures (Fig. 3C shows the SOX2 staining in LIF+ cells). Flow cytometric analysis revealed that about 88.5% NP cells expressed SOX2 before differentiation. However, after 2 weeks of differentiation, there was significant reduction in SOX2+ cells in the population, down to 59.6% (p < .0005) and 41.1% (p < .005) in LIF+ and LIF cultures, respectively. Also, after 2 weeks of differentiation the percentage of remaining SOX2+ cells was significantly higher in LIF+ cultures compared to LIF cultures at the same time point (p < .02; Fig. 3D, 3E).

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Figure 3. LIF increases proliferation in NP cell population during differentiation. NP cells were differentiated in LIF+ and LIF media for 2 weeks. (A): Bar diagram shows transcript levels of proliferation marker PCNA quantified by real-time polymerase chain reaction (PCR). Significantly higher amounts of PCNA transcripts were detected in LIF+ culture than LIF culture (p < .001). LIF+ and LIF cells were treated with EdU for 12 hours before harvesting, and EdU+ cells were quantified using flow cytometry. Bar diagram comparing mean percent of EdU+ cells show that the percent of EdU+ cells was significantly higher in LIF+ cultures than LIF (p < .005) (B). LIF+ and LIF cultures were immunostained for SOX2. SOX2 expression in LIF+ cells (C) and quantification by flow cytometry (D, E). (D): Representative histogram showing expression of SOX2 in NP and differentiated cells. (E): Bar diagram comparing mean percent of SOX2+ cells show that the percentage of SOX2+ cells was significantly lower in both LIF+ and LIF differentiated population compared to undifferentiated NP population (p < .005). When the differentiated populations were compared among themselves, LIF+ cultures had significantly higher SOX2+ cells compared to LIF cultures (p < .02). Abbreviations: EdU, (5-ethynyl-2′-deoxyuridine); FGF, Fibroblast growth factor; LIF, leukemia inhibitory factor; NP, neural progenitor; PCNA, proliferating cell nuclear antigen.

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LIF Enhances Yield of Differentiated Neuronal Cultures

NP cells differentiated in LIF+ and LIF media were quantified by immunocytochemistry and flow cytometry for the expression of three different markers: Nestin (a marker for NPs), HuC/D (an early neuronal marker), and MAP2 (a later neuronal marker). Before differentiation, the majority of NP were Nestin+ (more than 80.0%; Fig. 4A), consistent with their progenitor state. The neuronal markers HuC/D and MAP2 could only be detected in the population upon differentiation (Fig. 4B, 4C). The percentage of Nestin+ cells was significantly reduced after 14 days of differentiation in media without FGF2, with 42.7% ± 2.7% and 42.9% ± 4.3% cells being Nestin+ in LIF and LIF+ differentiation medium, respectively (p < .005; Fig. 4D, 4G). However, only 8.9% ± 0.9% of cells in the LIF neuronal cultures expressed HuC/D, whereas the percentage of HuC/D+ cells was significantly higher (38.9% ± 0.95%) in LIF+ culture (p < .005; Fig. 4E, 4H). Similarly, the percentage of MAP2+ cells was higher in LIF+ cultures (37.9% ±1.3) than in LIF cultures (11.1% ± 1.30%) (p < .005; Fig. 4F, 4I). Thus, LIF promotes the number of NP cell-derived neurons of hESC origin.

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Figure 4. Effect of LIF on neuronal differentiation of NP. (A): Expression of progenitor marker Nestin shown by immunocytochemistry (red). Nuclei are stained with DAPI (blue). Upon differentiation, these cells express neuronal markers (B) HuC/D (red) and (C) MAP2 (green). (D–I): Flow cytometry for above markers in undifferentiated NP cells, and NP cells differentiated in LIF+ and LIF media for 2 weeks. Representative histograms (D–F) and their corresponding cytometry (G–I) for Nestin, HuC/D, and MAP2, respectively. (G): Differentiation significantly reduced the percent Nestin+ cells both in LIF+ and LIF culture compared to undifferentiated NP cells (p < .005). Percent of Nestin+ cells in differentiated cultures did not differ significantly between LIF+ and LIF differentiation. (H): shows percent HuC/D+ cells in differentiated cells. Use of LIF+ differentiation media significantly increased the percent of HuC/D+ cells (p < .005). (I) :Significantly higher percentage of cells expressed MAP2 in LIF+ than in LIF differentiation (p < .005). Abbreviations: FGF, Fibroblast growth factor; LIF, leukemia inhibitory factor; MAP2, microtubule-associated protein 2; NP, neural progenitor.

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LIF Enhances Neurite Outgrowth in NP Cell-Derived Neurons

To study the effect of LIF on neurite outgrowth, cells were differentiated in LIF+ or LIF media. Figure 5A is a representative image of differentiated cells stained with βIII-tubulin/Hoechst. At 2 hours, the percentage of viable nuclei did not differ between LIF+ and LIF cultures. However, the percentage of viable nuclei decreased over time in both culture types (Fig. 5B). The decrease in the percentage of viable nuclei was significantly greater in LIF cultures compared to LIF+ cultures at 6, 24, and 48 hours. Large differences in neurite characteristics were observed between cells differentiated in LIF+ or LIF media. The percentage of neurite-bearing cells significantly increased over time between 2 and 48 hours in LIF+ cultures but not in LIF cultures (Fig. 5C). Similarly, the average number of neurites per neuron significantly increased over time in LIF+ culture but not in LIF cultures (Fig. 5D). Measurements of total neurite length per neuron increased over time in both LIF+ and LIF cultures (Fig. 5E), although the amount of total neurite outgrowth in LIF+ cultures was much greater as compared to LIF cultures. A significant increase in total neurite length was observed earlier in LIF+ cultures (6 hours) compared to LIF cultures (24 hours). Measurements of average neurite length (Fig. 5F) calculated independently of cell numbers demonstrate that neurites present in LIF and LIF+ cultures both increased in length between 6 and 24 hours in vitro. However, individual neurites in LIF+ cultures were comparatively longer than in LIF cultures at 6, 24, and 48 hours in vitro. Thus, LIF promotes neurite outgrowth of differentiating neurons derived from NP cells.

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Figure 5. LIF promotes neurite outgrowth in neural progenitor (NP) cells during differentiation. (A): NP cells differentiated in LIF (left) or LIF+ (right) media were immunostained for βIII-tubulin (green). Nuclei are stained with Hoechst (blue). (B–F): High content image analysis-based morphometric measurements of cells grown in the LIF+ (white bars) or LIF (gray bars) cultures: (B) % viable nuclei, (C) % neurite bearing cells, (D) average number of neurites per neuron, (E) total neurite length per neuron, and (F) average neurite length. For each measurement, a significant interaction of time and cell type was observed following two-way ANOVA (p < .05). Hash marks indicate a significant difference between LIF+ and LIF cells within a particular time point (Bonferroni post hoc t test, p < .05). Abbreviation: LIF, leukemia inhibitory factor.

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LIF Activates PI3K, MEK, and JAK/STAT3 Signaling Cascades

We examined expression of proteins related to PI3K, MEK, and JAK/STAT3 signaling pathways in NP cells grown in LIF+ and LIF culture using Western blotting. Each blot was stained with β-actin antibody to normalize for protein loading in gels (Fig. 6A). Undifferentiated NP cells showed the highest expression levels for each of the proteins tested, and differentiation in both LIF+ and LIF media led to their downregulation. However, the extent of downregulation was greater in the absence of LIF (Fig. 6). LIF activated the PI3K pathway as reflected by higher amount of phosphorylated AKT (PKB) and hexokinase II (HXKII) expression (Fig. 6A). LIF+ media also led to an increase in the level of phosphorylated ERK1/2, a component of the MAPK/MEK signaling pathway (Fig. 6A), when compared to LIF media. Additionally the level of phosphorylated STAT3 (pSTAT3) was higher in LIF+ than in LIF neuronal cultures (Fig. 6A). Further, we detected transcriptional upregulation of Stat3 target genes in LIF+ cultures, namely CCNE1, FAS, JUNB, SOCS3, TGFB, and VEGF (Fig. 6B). Although we did not find upregulation of cMyc transcription, the level of phosphorylated cMyc (active form) was higher in LIF+ neuronal cultures compared to those in LIF cultures (Fig. 6A). Thus, it is evident that in human NP cells, LIF possibly elicits its effects via PI3K, MEK, and JAK/STAT3 signaling pathways (summarized in schematic Fig. 6C)

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Figure 6. LIF activates PI3K, MEK, and JAK/STAT3 signaling in neural progenitors (NP) derived from human embryonic stem cells. NP cells were grown in the presence of LIF and were differentiated in LIF+ or LIF medium for 2 weeks. Signaling proteins or transcripts were detected by Western blotting or reverse transcriptase polymerase chain reaction (RT-PCR). β-Actin was used for normalizing the loading of samples.compared to NP cells, differentiated cells had lower expression of signaling proteins. Expression of (A) phosphorylated AKT and HXKII, phosphorylated ERK1/2, phosphorylated cMyc, and phosphorylated STAT3 in NP cells and differentiated LIF+ and LIF cells. LIF could activate STAT3-target genes (B) CCNE1, FAS, JUNB, cMyc, SOCS3, TGFB, and VEGF (two transcripts) detected by RT-PCR. (C): Schematic diagram showing possible signaling pathways corresponding to antiapoptotic, differentiation and cell survival roles of LIF in NP cells. Abbreviations: AKT, AKT v-akt murine thymoma viral oncogene; FGF, Fibroblast growth factor; ERK, extracellular signal-regulated kinase; HXKII, hexokinase II; LIF, leukemia inhibitory factor; MEK, MAPK/ERK kinase; JAK, Janus kinase; PI3K, phosphoinositide 3-kinase; STAT3, signal transducer and activator of transcription 3.

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LIF-Treated NP Cells Decrease Infarct Volume in an Ischemic Stroke Model

To determine whether LIF pretreatment increases survival in vivo, we counted the total number of TUNEL-positive and SC-121-positive cells in normal animals 48 hours after transplantation (Fig. 7A, 7B). TUNEL-positive cells in the LIF+ group showed a trend of less cell death than that in the LIF group, although the difference was not statistically significant. To measure infarct volume, NP cells cultured in LIF+ or LIF media were injected into the barrel cortex of stroke mice, and the infarct volume was measured 24 hours later (Fig. 7C–7E). Both LIF+ and LIF pretreated cells reduced infarct volume after ischemic stroke (Fig. 7C). The indirect infarct volume (Fig. 7D) and the infarct volume ratio (Fig. 7E) values were significantly reduced in both LIF and LIF+ groups compared to media injected controls. There was no difference between the LIF and LIF+ groups.

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Figure 7. Survival and effects of LIF and LIF+ NP cells in the noninjured and stroke CNS central nervous system. Human ESC-derived neural progenitor (NP) cells were cultured in LIF+ or LIF media and transplanted in the barrel cortex of normal and stroke mice. For measuring survival, we used normal mice. As shown in (A) and (B), LIF+ pretreated cells survived better than the LIF group (n = 10 in each group). 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed 24 hours later on stroke mice. (C): TTC staining of stroke brains injected with media (control), LIF untreated cells (LIF), and LIF treated cells (LIF+). Red shows live mitochondria (tissue) and white shows dead mitochondria (tissue). Indirect infarct volume (D) and infarct volume ratio (E) measurements showed a significant reduction in infarct volume in both LIF and LIF+ groups compared to control animals. There was no difference between LIF and LIF+ groups (n = 8 for all groups; *, p < .05 using one-way ANOVA and Bonferroni's post hoc analysis). Abbreviations: LIF, leukemia inhibitory factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The purpose of this study was to elucidate the role of LIF in hESC-derived NP cells both in vitro and in vivo. It is well-established that hESC, unlike murine ESC, do not require LIF either for maintenance of pluripotency or for differentiation along the neuronal lineage [34–38], although the LIF receptor components are expressed in both hESC [34] and primate ESC [37]. In contrast, LIF affects the in vitro maintenance and differentiation of NP cells isolated from fetal as well as adult human sources [17, 38, 39]. While not required for hESC and initial differentiation to ectodermal lineages, we found that LIF in fact had pronounced effects on multiple facets of early neuronal differentiation, which suggests a role for LIF in hESC-based in vitro neurogenesis and in providing expanded NP cell populations that retain therapeutic potential upon transplantation into the CNS.

Reduced Apoptosis Correlated to Reduction in Caspases and ROS

Apoptosis is an integral part of neural development [40–43] in vivo and a common feature of neural differentiation in vitro [41]. We observed a significant amount of cell death during NP cell-based neuronal differentiation, however, NP cells differentiated in LIF+ medium showed significantly lower number of TUNEL positive apoptotic cells (Fig. 1). Apoptosis during neurogenesis is primarily mediated by activation of caspases 3 and 7, although caspase-independent apoptotic events are also well documented [44, 45]. The addition of LIF during differentiation reduced activation of both caspase 3 and 7 (Fig. 1E), suggesting this as a possible mechanism behind its antiapoptotic role. Also, several studies have implicated caspase3 in nonapoptotic roles in neural development, including promotion of neurogenesis, refinement of neural circuits and synaptic plasticity [46–49]. Thus we cannot rule out the possibility that LIF, via reduced caspase3, could have affected other aspects of NP cell in vitro differentiation as well.

One common trigger for apoptosis in neural stem cells and neurons is the generation of excessive ROS including superoxide [50–52] and in vitro differentiation is accompanied by an increase in ROS in newly born neurons [54]. We found that along with reducing apoptosis, LIF significantly reduced the amounts of spontaneously produced ROS in both NP cells and in NP-derived neurons, suggesting that a potentially neuroprotective mechanism is triggered by LIF. Apart from inducing apoptosis, ROS also modulates neuronal stem cell differentiation [55–57]. Thus by modulating ROS, LIF could also influence differentiation. Given the reduction of ROS in LIF+ cultures, it was important to address whether the observed neuroprotective effect extends to ROS induced by extrinsic factors as may be encountered in a hostile niche upon transplantation or in disease [58–61]. H2O2 is often generated in cells in response to a hostile environment and has widely been used to induce ROS in a variety of neuronal cells [60, 61]. We show that H2O2-mediated ROS production was reduced by LIF, and the effect was dose dependent in both NP and differentiated cell populations (Fig. 2). Although the results do not directly link reduction in ROS with the observed reduction in apoptosis, a correlation exists between reduction of ROS and neuroprotective effects observed with LIF signaling. Both LIF and ciliary neurotrophic factor (CNTF) are members of the Il-6 cytokine family, they are structurally related and share common receptor component subunits [62]. CNTF has neuroprotective properties but cannot prevent oxidative damage in cells exposed to H2O2 [63]. Thus LIF may be effective in controlling damage by in vitro ROS inducers.

LIF Increased Proliferation, STAT3 Signaling, and Yield of Differentiated Neurons

Removal of FGF2, a hESC-derived NP cell mitogen [64] reduced proliferation and promoted exit to postmitotic states, increasing the maturing neuronal cell population in these cultures. Nestin is a type VI intermediate filament marker for NP cells, [65] and FGF2 deprivation drastically reduced the number of Nestin+ cells in the cultures. This indicated that the majority of the cells had exited their progenitor state (Fig. 4D, 4G). Addition of LIF to FGF2-deprived differentiation culture did not affect the proportion of Nestin expressing cells in relation to LIF cultures, suggesting that LIF does not influence exit to postmitotic states. Furthermore, from our experience with immunostaining data, we find that even after exiting the progenitor state, some early neurons continue to express Nestin. This implies that a certain proportion of cells counted as NP cells could be early neurons. HuC/D is a RNA binding protein expressed in the cell bodies of early postmitotic neurons [66–78], and MAP2 marks dendrites of differentiated neurons [69] (Fig. 4). The higher percentages of HuC/D and MAP2 expressing cells in LIF+ neuronal cultures (Fig. 4) imply that a greater number of cells reach a more advanced differentiated stage and express neuronal markers, even though an equal percentage exited the progenitor state. Given that these NP cells do not differentiate to glial cells under the conditions used in this study [70], present results implied a positive role of LIF for increasing the yield of pan-neuronal cells during in vitro differentiation.

NP cells differentiated in LIF+ medium exhibit higher phosphorylated ERK1/2 levels and pSTAT3 (Fig. 6A), suggesting that LIF supports neural proliferation and differentiation. Elevated phosphorylated ERK1/2 is associated with differentiation of mouse ESCs to neuronal cells [71]. Since we also found that LIF acted as a survival factor, it remains to be determined whether the observed percent increase in HuC/D- and MAP2-expressing neurons is due to increased survival of differentiated neuronal cells, apoptosis of differentiating cells in LIF cultures, or enhanced differentiation, or a combination of the above. STAT3 signaling promotes cell proliferation in neural [72] as well as non-neural cells including ESC [73, 74]. The LIF+ cultures had higher levels of pSTAT3 indicating increased STAT3 signaling. Four downstream targets of STAT3 were also upregulated (cyclin E1, cMyc, JUNB, and VEGF) (Fig. 6B, 6C) in LIF+ cultures and all have been implicated in cell proliferation [68, 69]. Thus the observed increase in proliferation of NP cells and NP-derived neurons in LIF+ culture could be due to a portion of the cells responding to LIF by activating STAT3 signaling. The response to LIF in these NP cell cultures suggests that subpopulations of cells may respond differently to LIF in the absence of FGF2. LIF helped maintain the cell proliferation potential (Figs. 3, 6) of a portion of the cultures even after 2 weeks as determined by several in vitro assays. LIF also supported survival of differentiated neuronal cells yielding significantly higher amount (1.4 ± 0.11 vs. 0.6 ± 0.04 million per ml in LIF+ and LIFcultures) of neuronal cells in vitro.

LIF Promotes Neurite Extension in hESC-Derived NP Cells

In primary rodent neurons as well as PC12 cells, LIF promotes neurite outgrowth [75, 76]. In neurons from the auditory cortex of mammals, LIF is a more potent promoter of neurite outgrowth than BDNF [77]. LIF has also been implicated in peripheral nerve regeneration [12] and is induced by axotomy [78] and other conditioning injuries in vivo [79]. In spite of ample evidence implicating LIF in axonal growth in mice, little is known about LIF's effects on neurite extension in human neurons or in hESC-derived neurons. Our results provide clear evidence of enhanced neurite extension in differentiated NP cells in both neurite number and neurite length (Fig. 5). Additionally, we show activation of STAT3 and MAPK signaling in LIF+ differentiation cultures. In previous PC12 cell studies, activation of the STAT3 pathway by pituitary adenylate cyclase-activating polypeptide [80] or STAT3 and MAPK pathways by Interleukin-6 [75, 76] strongly promoted neurite outgrowth. Thus, our observed increase in both STAT3 and MAPK signaling in LIF+ differentiated neurons suggests that LIF could be acting through a similar mechanism to promote neurite outgrowth in hESC-based neurogenesis.

Signaling Pathways Activated by LIF

LIF exerts its effect upon binding to its heterodimeric receptor formed by two protein components: LIFR and gp130. In many cell types, exogenous LIF upregulates LIFR mRNA expression [81, 82]. In contrast, we observed that in response to LIF stimulation in NP cells, LIFR expression remained unchanged, but gp130 mRNA was upregulated in a concentration-dependent fashion (Fig. 1). The activation of gp130 by LIF is accompanied by the recruitment of activated STAT3 (Fig. 6A) and upregulation of SOCS3 in culture as expected based on previous reports [83]. These observations suggest that LIF and other gp130 modulators may potentially be useful to promote outgrowth of human neurons in vivo.

To our knowledge, the activation of all three (PI3K/Akt, JAK/STAT, and ERK) pathways in neural stem cells in response to LIF signaling has not been reported previously although concomitant activation of these three pathways have been demonstrated in carcinoma cells where they are involved in promotion of growth, invasion, and migration [84, 85]. Here, we have shown that, during differentiation of NP cells, LIF activated PI3K/AKT as well as STAT3 signaling (Fig. 6). Previously, it has been shown that activation of JAK/STAT3, but not PI3K or ERK1/2, signaling imparts neuroprotection against retinal degeneration [86]. However, PI3K activation promotes survival of neurons [87]. Also, LIF protects cardiomyocytes from apoptosis through phosphorylation of HXKII in mitochondria and this event is mediated by AKT activation [30]. Therefore, our results suggest that LIF acts as antiapoptotic factor and promotes cell survival of human NP cell and NP-derived neuronal cells in culture, potentially by way of PI3K/AKT and STAT3 signaling.

LIF Pretreatment of NP Cells in Stroke Model

Our in vitro studies indicate that the use of LIF has neurogenic, neuroprotective, and neurotrophic effects, acting through potentially multiple signaling pathways. These data now provide an impetus and opportunity to determine the extended utility of LIF signaling on NP cells to be used in neural cell-based therapies. Although others have used the NP cells we previously generated in animal models, the effect of LIF on these NP cells in vivo was not investigated [5]. Here both LIF and LIF+ treated cells equally reduce infarct volumes in mouse models of stroke in as little as 24 hours postinjection (Fig. 7). These data indicate that human NP cells alone generate a rapid effect in a xenotransplant animal stroke model, and extended studies may be needed to tease out the importance of the LIF pretreatment in vivo. Regardless these are promising early-stage results, and future studies should more exhaustively investigate the effects of LIF on NP cells in multiple and extended duration transplants using neurite outgrowth and functional potential of transplanted NP cells as endpoints.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Taken together, our results indicate that although LIF is not required for culture or differentiation of human NP cells, its inclusion increases proliferation, survival, and ultimately the yield of differentiated neurons from human ES cells. Moreover, in vitro, LIF protects neurons from ROS and helps promote neurite outgrowth. Although extended in vivo functional outcomes of LIF-treated NP need to be determined in depth, this study suggests that human NP cells treated with LIF have significant therapeutic potential in as little as 24 hours post-treatment, compared to nontreated animals in a stroke model. The significant in vitro benefits of LIF should be leveraged to develop enhanced cell bioprocessing systems to more rapidly generate large quantities of robust and viable pluripotent stem cell-derived NP cells for neural cell therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This research is based upon work supported by the National Science Foundation under the Science and Technology Center Emergent Behaviors of Integrated Cellular Systems (EBICS) Grant No. CBET-0939511 to S.L.S., Grant Nos. NS058710 and NS062097 to L.W., and AHA pre-doctoral fellowship 10PRE4430032 to O.M. Authors thank Dr. N Murthy and Dr. K Kundu of Georgia Institute of Technology for providing the ROS detection reagent HydroCy3; Julie Nelson of the Center for Tropical and Emerging Global Diseases Flow Cytometry Facility for her technical expertise; Erin Jordan, Kowser Hasneen, and Anuj Shukla for technical support. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

S.L. Stice is a shareholder in ArunA Biomedical, Inc a manufacturer of hNP cells.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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sc-12-0059_sm_SupplTable1.pdf323KSupplementary Table 1

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