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A simple method for differentiation of H9 cells into neuroectoderm
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A simple method for differentiation of H9 cells into neuroectoderm Annuo Liu a,b,1 , Dijuan Zhang a,1 , Lihua Liu c , Juan Gong b , Chao Liu a,d,∗ a Institute of Stem Cell and Tissue Engineering & Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China b School of Nursing, Anhui Medical University, Hefei, Anhui 230032, China c Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui 230032, China d Central Laboratory of Molecular and Cellular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China
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Article history: Received 12 May 2015 Received in revised form 23 July 2015 Accepted 23 July 2015 Available online xxx Keywords: H9 Neuroectoderm Localized cell density Reaggregation Cell cluster
a b s t r a c t Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Neuroectoderm (NE) is primordium of nervous system (Murry and Keller, 2008; Petros et al., 2011). Human embryonic stem cells (ESCs) can differentiate into NE in vitro, providing a platform for studying NE formation (Lyashenko et al., 2011; Thomson et al., 2011). However, human ESCs can differentiate into any of the three germ layers (endoderm, mesoderm and ectoderm) (Thomson et al., 1998), which makes it complex for researchers to induce NE other than non-NE cells. Directed neural differentiation cannot only provide an in vitro platform for study of human neurodevelopment (Milet and Monsoro-Burq, 2012; Nat et al., 2012), but also hold great promise for cytotherapy of human neurodegenerative
Abbreviations: ESC, embryonic stem cell; NE, neuroectoderm; RT-PCR, reverse transcription with polymerase chain reaction; IF, immunofluorescence; IB, immunoblotting; TH, tyrosine hydroxylase; DA, dopaminergic; PD, Parkinson’s disease; LCD, localized cell density; AA, ascorbic acid; DAPI, 4,6-diamidino-2phenylindole; FGF, fibroblast growth factor. ∗ Corresponding author at: Department of Histology and Embryology, Institute of Stem Cell and Tissue Engineering, School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, China. E-mail address: [email protected] (C. Liu). 1 These authors contributed equally to this work.
diseases (Volarevic et al., 2011). For example, degeneration of dopaminergic (DA) neurons in substantia nigra is a pathological hallmark of Parkinson’s disease (PD) (Mullin and Schapira, 2015). Currently, PD is a primary target for cytotherapy (Athauda and Foltynie, 2015; Kim, 2011; Lindvall, 2012). Because differentiation of human ESCs into NE is prerequisite for differentiation of human ESCs into region-specific neural cells, it is important to understand roles of extrinsic or intrinsic factors involved in NE differentiation. Localized cell density (LCD), a function of the number of neighbors a cell has within a given space, plays a crucial role in the self-renewal and differentiation of human ESCs (Peerani et al., 2007). We recently developed a cell clump-based method for NE differentiation (Liu et al., 2014). This method, in which the cell density is not in control, shows that high LCD promotes specification of NE. To keep plating cell density under control, cells must be dissociated into single cells. However, cell–cell interaction is necessary for NE specification of ESCs (Parekkadan et al., 2008). In addition, a confluent growing of human ESCs normally experiences disrupted lineage specification (Thomson et al., 1998). So far, combined role of above mentioned factors in NE differentiation has not been reported. In this paper, human ESCs, H9 cells, were disassociated into single cells for a cell counting. Then the cells were reaggregated into small cell clusters and cultured in mTeSR. Upon the cell clusters
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were nearly 95% confluent, a NE induction was performed. The induced cells were further subjected to a neural differentiation for at least 10 d. The expression of NE markers and neural markers in the H9-derived cells were analyzed by RT-PCR, immunofluorescence (IF) and immunoblotting assay, respectively. The induced cells are characterized by expression of PAX6 mRNA and PAX6 protein. Expression of GFAP, a glia marker gene, was also identified after 5 d of differentiation. Meanwhile, expression of beta-III-tubulin, a neuron marker protein, was detectable after 5 d of differentiation and reaches to its peak after 10 d of differentiation. Moreover, coexpression of beta-III-tubulin and TH was identified in DA-like neurons after 10 d of the NE differentiation. Our results here may indicate a simple method for a rapid neural induction from H9 cells.
2. Materials and methods 2.1. Cell culture Human ESC line H9, from passage 45 to passage 50, has been previously described (Liu et al., 2014; Yao et al., 2012). A cell clump-based maintaining was performed according to the protocol from StemCell Technologies Inc. Briefly, H9 cells were cultured on Matrigel (BD Biosciences) in mTeSR medium (StemCell Technologies Inc.). The medium was changed every day. The H9 cells were passaged with dispase and re-plated in mTeSR medium at a ratio of 1:5–1:6. Matrigel coating was performed according to the protocol from BD Biosciences. Briefly, Matrigel was thawed on ice and diluted with DMEM-F12 precooled on ice. The volume of one aliquot of Matrigel is typically 160 l. One aliquot of Matrigel was added to 15 ml of DMEM-F12 and mixed well. Then add 1 ml dilution per well of 6-well plates. Allow plate to sit at room temperature for 2 h. Plate may either be used immediately or stored at 4 ◦ C. When ready to use the plate(s), remove the excess liquid.
2.2. NE and neural differentiation A cell cluster-based method for NE differentiation was modified from our previous protocol (Liu et al., 2014). Briefly, H9 cells were dissociated with accutase for 20 min, washed with DMEM-F12 and resuspended using mTeSR medium supplemented with 10 M ROCK inhibitor (Y-27632, TOCRIS). After the cell suspension was subjected to a cell counting, cells with different cell densities (0.4, 0.8 and 2.0 × 104 cells/cm2 ) were reaggregated according to a method modified from previous protocol (Unbekandt and Davies, 2010; Zhang and Zhang, 2010). Briefly, cells were centrifuged at 250 rpm for 15 min first, followed by an incubation at 37 ◦ C for 1 h, which is helpful for cell–cell interaction and formation of small cell clusters. With a 5 ml serological pipette, part of the medium was aspirated and used to push the pellet carefully to detach it. The pellet, detached from the tube walls, was disrupted by aspirating for 2 times gently using a 5 ml pipette and replated on Matrigelcoated coverslips or 6-well plates. The cells were cultured for 3–5 d in mTeSR medium prior to induction. Upon the cell clusters were nearly 95% confluent, NE induction medium, which is composed of KSR medium (DMEM/F12, 20% KSR, 0.1 mM -mercaptoethanol, 10 ng/ml of FGF-2, 1% non-essential amino acid) and N2 medium (DMEM/F12, 1% N2 supplement, 1% non-essential amino acid with 10 ng/ml of FGF-2), supplemented with or without SB431542 (10 M, TOCRIS) and NOGGIN (200 ng/ml, PEPROTECH), was used for the induction. Increasing amounts of N2 medium (25%, 50%, and 75%) were added to the KSR medium every 2 d. NE medium is a combination of KSR medium and N2 medium. The KSR medium and N2 medium were prepared separately as described and mixed as below: day 0–2 (25% N2 medium: 75% KSR medium), day 3–4 (50% N2 medium: 50% KSR medium), day 5–6 (75% N2 medium:25% KSR medium). We added the fresh medium after the spent medium was removed from the well. For further neural differentiation, the cells were cultured in N2 medium supplemented with AA (0.2 mM) for at least 10 d. The cells were passaged mechanically (Perrier et al., 2004) after 6 d of neural differentiation on Matrigel-coated
Fig. 1. Keep plating cell density and LCD under control at the beginning of NE induction. Schematic representation of the main procedures of the NE induction (A). Comparison of the phase micrographs of the dissociated H9 cells plated as single cells (B) and as small cell clusters after a reaggregation (C) for 24 h, respectively.
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coverslips or 6-well plates cultured in N2 medium supplemented with AA (0.2 mM). 2.3. Microscopy and immunofluorescence (IF) assay The microscopy and IF assay was performed as previously described (Liu et al., 2014). Antibodies used for IF analysis were as follows: anti-OCT4 (Santa Cruz), anti-PAX6 (Millipore), anti-SOX1 (abcam), anti-beta-III-tubulin (Millipore) and anti-TH (Chemicon). A NIKON Eclipse 80I fluorescence microscope and an Olympus IX73 inverted fluorescence microscope were used for microscopy. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
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2.4. Immunoblotting (IB) assay An immunoblotting (IB) assay was performed as previously described (Liu et al., 2012). Antibodies used for IB were as follows: anti-OCT4 (Santa Cruz), anti-PAX6 (Millipore), anti-SOX1 (abcam), anti-beta-III-tubulin (Millipore) and rabbit anti-GAPDH (Ptglab). 2.5. Reverse transcription with polymerase chain reaction (RT-PCR) After 6 d of differentiation, H9-derived cells were harvested for RT-PCR assay. Total RNA was isolated using RNA isolation KIT (Axygen). For RT-PCR analysis of mRNAs, 1 g of the total RNA from each
Fig. 2. Expression of PAX6, a NE marker, is regulated by plating cell densities during the NE induction. (A–C) Expression of OCT4 (A and C, green) and PAX6 (B and C, red) in the H9 cells. (D–F) Expression of OCT4 (D and F, green) and PAX6 (E and F, red) in the induced cells. Nuclei were counterstained with DAPI (C and F, blue). (G–I) Coexpression of PAX6 (G and I, green) and NESTIN (H and I, red) in the induced cells. (J) Effect of different plating cell densities on the ratio of PAX6-positive cells to DAPI-positive cells. *P < 0.05 when compared with the cell density of 0.4 × 104 cells/CM2 . **P < 0.01 when compared with the cell density of 0.4 × 104 cells/CM2 . Error bars represent standard deviation of four to six experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4 Table 1 Primer sequences of markers used for RT-PCR analysis. Primer
Sequence
Size (bp)
OCT4
5 -AGTGAGAGGCAACCTGGAGA-3 5 -GTGAAGTGAGGGCTCCCATA-3 5 -CAATGCGGGGAGGAGAAGTC-3 5 -CTCTGGACCAAACTGTGGCG-3 5 -AACAGACACAGCCCTCACAAACA-3 5 -CGGGAACTTGAACTGGAACTGAC-3 5 -TCATCGCTCAGGAGGTCCTT-3 5 -CTGTTCCCAGAGATGGAGGTT-3 5 -GTACTCAGCGCCAGCATCG-3 5 -AGCCACATCGCTCAGACACC-3 5 -AGAATTCGCTTGAGTATTCTGA-3 5 -GGCTTTCAGGTTATTTGACTGA-3 5 -GTCCCCTGGTTCCCAAGAAAAGT-3 5 -TCCAGCTGGGGGATATTGTCTTC-3 5 -AGCCGGCACCTGTTGTGCAA-3 5 -TGACTTCTCCTGCATGCACT-3
273
SOX1 PAX6 GFAP GAPDH REX1 TH GATA2
464 275 383 302 470 331 242
sample was reverse transcribed with Superscript III (Invitrogen). Next, 10% of the first-strand reaction (2 l) was used for subsequent PCRs for each gene of interest. A GAPDH endogenous control was used for normalization. The forward and reverse primers used for gene expression analysis and the size of the PCR products are shown in Table 1. 2.6. Statistics Image J software was used to evaluate differences in protein levels. The relative expression of PAX6 among the total cells was calculated based on the areas in PAX6 and DAPI channels. Statistical analysis was performed using SPSS 17.0 software, and the data are presented as the mean ± standard deviation. For analysis of the differences in mRNA levels, expression of the indicated genes was examined using Adobe Photoshop software. The relative expression of the targets genes normalized to GAPDH gene was presented as the mean ± standard error. The statistical analysis was also performed using the SPSS 17.0 software. 3. Results 3.1. Keep both plating cell density and high LCD under control at the beginning of NE induction
Fig. 3. Expression of marker genes and proteins in the H9-derived cells treated with and without SB431542 and NOGGIN. (A) Expression of OCT4 and REX1 (markers for pluripotency), PAX6 and SOX1 (markers for NE), GFAP (a marker for glia), TH (a marker for dopaminergic neuron) and GATA2 (a marker for non-NE) at day 6 of the NE induction. Statistic analysis of the relative expression of the indicated marker genes normalized to GAPDH. *P < 0.05 when compared with KSR+N2. Error bars represent standard error of three-independent experiments. (B–E) Expression of some NE marker proteins, such as OCT4, PAX6 and SOX1, in the induced cells with or without the presence of SB431542 and NOGIN at day 6 of the NE induction.
Both plating cell density and LCD play role in NE induction (Liu et al., 2014). Dissociation of cells is required for cell counting and cell density manipulating. However, cell–cell interaction, which is disrupted during dissociation of cells, is crucial for differentiation of ESCs into NE (Parekkadan et al., 2008). Moreover, lineage specification is disrupted by a confluent growing of human ESCs (Thomson et al., 1998). Thus, in our study, H9 cells were disassociated into single cells for a following cell counting and were then reaggregated into many small cell clusters prior to NE induction (Fig. 1A). Moreover, when compared with the single cell-plating method (Fig. 1B), the cell cluster-plating method maintains higher LCD and/or more cell–cell interactions (Fig. 1C).
We also identified cells that are positive for both PAX6 (Fig. 2G and I, green) and NESTIN (Fig. 2H and I, red), another NE marker protein (Bhinge et al., 2014), after 5 d of NE induction. The percentage of PAX6-positive cells among total cells was also examined. When plated at the density of 0.4 × 104 cells/cm2 , the cells shows a percentage of no more than 10% (Fig. 2J, left column). With the density increased to 0.8 × 104 cells/cm2 , the percentage becomes more than 50% (Fig. 2J, middle column). At last, when the density increased to 2.0 × 104 cells/cm2 , the percentage reaches to more than 70% (Fig. 2J, right column), which suggests that high plating cell density promotes expression of NE marker proteins in the induced cells significantly (Fig. 2J, *P < 0.05; **P < 0.01).
3.2. Expression of NE marker proteins in induced cells
3.3. Examination of marker genes and proteins in the H9-derived cells during the NE induction
After 5 d of NE induction supplemented with SB431542 and NOGGIN, expression of OCT4 and PAX6 of induced cells was compared with that of H9 cells. As shown in Fig. 2, results of IF assay indicated that while H9 cells are positive for OCT4 (Fig. 2A and C, green) and devoid of PAX6 (Fig. 2B and C, red), most of the induced cells are devoid of OCT4 (Fig. 2D and F, green) and positive for PAX6, especially in the cells with high LCDs (Fig. 2E and F, red).
We also checked expression of some marker genes related to pluripotency (OCT4 and REX1), NE (PAX6 and SOX1) and neural cells (GFAP for glia and TH for dopaminergic neuron) during the NE induction using RT-PCR assay. The results of RT-PCR assay revealed a downregulation of pluripotent gene, such as OCT4 and REX1, together with an upregulation of PAX6, by SB431542 and NOGGIN at
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Fig. 4. Expression of neuronal marker proteins in the H9-derived neural cells. (A, B) Expression of beta-III-tubulin analyzed by IF at day 5 (A, green) and day 10 (B, green) of the induction, respectively. Nuclei were counterstained with DAPI (A and B, blue). (C) Expression of OCT4, PAX6, SOX1 and beta-III-tubulin at the indicated time points of the neural induction. The GAPDH was used for a loading control. (D–F) Expression of TH (D and F, red) and beta-III-tubulin (E and F, green) in the H9-derived cells. Nuclei were counterstained with DAPI (F, blue). Bar = 200 m. Arrows indicate the robust expression of both TH and beta-III-tubulin in the cell aggregates. (G) The number of cell clusters positive for beta-III-tubulin and TH were shown. Error bars represent standard deviation of ten random areas. (H–J) Colocalization of TH (H and J, red) and beta-III-tubulin (I and J, green) in H9-derived neurons (arrow heads). Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
day 6 of the NE induction. Moreover, expression of GFAP, a glial cell marker, was also upregulated. Few or no specific bands of TH, a DA marker, were identified. While the non-specific bands with wrong size were detected, few or no specific bands of GATA2, a non-NE marker, was identified. Moreover, no specific bands of SOX1, an NE marker which is expressed at a later stage of human NE development (Zhang et al., 2010), was detected (Fig. 3A). A significant upregulation of the relative expression of PAX6 mRNA normalized to GAPDH mRNA by SB431542 and NOGGIN was shown (Fig. 3A, *P < 0.05). We also performed IF assay to show expression of some NE marker proteins, such as OCT4, PAX6 and SOX1, in the induced cells at day 6 of the NE induction. SB431542 and NOGGIN induced a downregulation of OCT4 together with an upregulation of PAX6
(Fig. 3B–E). However, few or no SOX1 expression was identified in the induced cells (Fig. 3B and C). 3.4. Differentiation of H9-derived NE cells into neurons Because ability to differentiate into neurons is a hallmark of NE cells (Bhinge et al., 2014). To examine neuronal differentiation potential of the NE cells, we isolated the NE positive cell clumps mechanically and replaced the NE induction medium with the N2 medium supplemented with AA. The NE-derived cells were subjected to IF assay using anti-beta-III-tubulin antibody after 5 d and 10 d of differentiation, respectively. The expression of betaIII-tubulin in the NE-derived cells showed a time dependent style
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(Fig. 4A and B, green). IB assay also proved the time-dependent up-regulation of PAX6 (Fig. 4C). Besides, the time-dependent upregulation of beta-III-tubulin and down-regulation of OCT4 (Fig. 4C, upper panel), indicates the generation of neuronal cells. However, very weak or no SOX1 signal was identified in the induced cells. We performed IF assay and found cells positive for TH (Fig. 4D and F, red) and beta-III-tubulin (Fig. 4E and F, green) after 10 d of the NE induction. The number of and TH-positive cell aggregates or clusters were counted and shown (Fig. 4G). The percentage of THpositive clusters among beta-III-tubulin positive clusters is about 85%. Images with a higher magnification show the well colocalization of beta-III-tubulin with TH in the DA-like cells (Fig. 4H–J, arrows). This finding may suggest the generation of DA neuron-like cells, which are mainly found in the cell aggregates or clusters.
4. Discussion Previously, Studer lab described a neural induction method, in which the human ESCs maintained on MEF feeder cells were dissociated into single cells and grown on Matrigel in mTeSR until the cells were confluent (Chambers et al., 2009). Because we routinely use a cell clump-based culturing system for H9 cells (Liu et al., 2012), we thus developed a cell-clump based NE differentiation protocol (Liu et al., 2014). When compared with the cell clumpbased protocol, the novelties here is that we tried to keep plating cell density and LCD under control to maintain both high LCD and cell–cell interaction, which determines NE specification (Liu et al., 2014; Parekkadan et al., 2008). The promise for cytotherapy of human neurodegenerative diseases depends on the potential of human ESCs to be induced into diverse neural cell populations (Erceg et al., 2009). However, the permit to generate different neural cell types also makes it difficult to be controlled. A recent report indicated local microenvironment can modulate endogenous parameters, such as colony and aggregate size, and influence differentiation trajectories through regulating PAX6 expression (Bauwens et al., 2008). PAX6 is crucial for neurodevelopment, including brain patterning, neuronal migration, and neural circuit formation (Osumi et al., 2008). Our RTPCR assay revealed robust expression of PAX6 mRNA and the very weak expression or absence of SOX1 mRNA after 6 d of NE induction, which is also supported by the data of the IF assay. Our report here is consistent with previous report that human NE cells express PAX6 in advance of SOX1 so that PAX6 is believed to be NE determinant specifically in humans (Zhang et al., 2010). Besides, absence of GATA2, a non-NE marker gene, suggests our method might be specific for NE differentiation. After 6 d of differentiation, downregulation of mRNA levels of REX1, a marker gene for pluripotency, by SMAD signaling blockade, is consistent with the downregulation of both OCT4 mRNA and OCT4 protein levels. Simultaneously, upregulation of GFAP, a marker gene for glia, together with expression of beta-III-tubulin indicates that our method can generate both neurons and glia. PAX6 is expressed in NE cells at an earlier stage of neural development. Beta-III-tubulin is a neuronal marker protein which is expressed in neuronal cells at a later stage of neural development. The further induction with N2 medium may induce more NE cells to differentiate into neuronal cells, which express less PAX6. However, during the further induction with N2 medium, the proliferation ability of the beta-III-tubulin-positive cells might be decreased as compared to that of the cells at an earlier stage of neural development. Therefore, we speculated that the reduction of beta-III-tubulin protein level after 15 d of differentiation might imply the reduction of beta-III-tubulin positive cells. Interestingly, differentiation of P19 cells into NE cells dependents on cell aggregation, which induces an elevation of FGF8 (Wang et al., 2006),
a member of the fibroblast growth factor (FGF) family, which is important for induction of DA neurons (Perrier et al., 2004; Wu et al., 2012). Degeneration of DA neurons is a pathological hallmark of Parkinson’s disease, which is considered a primary target for cytotherapy (Anisimov, 2009; Kim, 2011). To fulfill this potential, lessons from embryonic development has offered important insights into basic culture models and signaling factors involved in inducing ESC differentiate into DA neurons (Murry and Keller, 2008). Generation of DA neuron from ESCs is complex, including three basic culture models: (1) as embryoid bodies (EBs) (Cho et al., 2008; Iacovitti et al., 2007), (2) as monolayers on extracellular matrix proteins (Chambers et al., 2009), and (3) on supportive stromal cells (Perrier et al., 2004; Sonntag et al., 2007). Optimizing culture condition is one of the strategies for improving efficiency of midbrain DA neuron generation. Both aggregates of human ESCs and the derived neural aggregates contributed to generation of DA neurons (Zhang and Zhang, 2010). Because NE cells are characterized by the potential to differentiate into neurons (Bhinge et al., 2014). In our study, after 10 d of differentiation, TH-positive neurons formed mainly in the cell aggregates, suggesting the NE cells induced by our method might have the potential of true NE cells. However, few or no expression of TH, a marker gene for DA neuron, was detected after 6 d of differentiation, implying generation of DA neurons requires at least more times. Together, our results here provide a simple NE induction method, which may contribute to in vitro neural differentiation of H9 cells. Acknowledgements This project was sponsored by a NSFC grant (31271159), a Technology Foundation for Selected Overseas Chinese Scholar (2012 No. 13) Grant from Anhui HRSS, a Grant for scientific research of BSKY (XJ201105) from Anhui Medical University. We thank Dr. Shengyun Fang for the gift of anti-TH antibodies. References Anisimov, S.V., 2009. Cell-based therapeutic approaches for Parkinson’s disease: progress and perspectives. Rev. Neurosci. 20, 347–381. Athauda, D., Foltynie, T., 2015. The ongoing pursuit of neuroprotective therapies in Parkinson disease. Nat. Rev. Neurol. 11, 25–40. Bauwens, C.L., Peerani, R., Niebruegge, S., Woodhouse, K.A., Kumacheva, E., Husain, M., Zandstra, P.W., 2008. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26, 2300–2310. Bhinge, A., Poschmann, J., Namboori, S.C., Tian, X., Loh, S.J.H., Traczyk, A., Prabhakar, S., Stanton, L.W., 2014. MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-beta/BMP signaling. EMBO J. 33, 1271–1283. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., Studer, L., 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280. Cho, M.S., Lee, Y.E., Kim, J.Y., Chung, S., Cho, Y.H., Kim, D.S., Kang, S.M., Lee, H., Kim, M.H., Kim, J.H., Leem, J.W., Oh, S.K., Choi, Y.M., Hwang, D.Y., Chang, J.W., Kim, D.W., 2008. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 105, 3392–3397. Erceg, S., Ronaghi, M., Stojkovic, M., 2009. Human embryonic stem cell differentiation toward regional specific neural precursors. Stem Cells 27, 78–87. Iacovitti, L., Donaldson, A.E., Marshall, C.E., Suon, S., Yang, M., 2007. A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: studies in vitro and in vivo. Brain Res. 1127, 19–25. Kim, H.J., 2011. Stem cell potential in Parkinson’s disease and molecular factors for the generation of dopamine neurons. Biochim. Biophys. Acta 1812, 1–11. Lindvall, O., 2012. Dopaminergic neurons for Parkinson’s therapy. Nat. Biotechnol. 30, 56–58. Liu, C., Sun, Y., Arnold, J., Lu, B., Guo, S., 2014. Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells. Biochem. Biophys. Res. Commun. 452, 895–900. Liu, L., Liu, C., Zhong, Y., Apostolou, A., Fang, S., 2012. ER stress response during the differentiation of H9 cells induced by retinoic acid. Biochem. Biophys. Res. Commun. 417, 738–743.
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A simple method for differentiation of H9 cells into neuroectoderm Annuo Liu a,b,1 , Dijuan Zhang a,1 , Lihua Liu c , Juan Gong b , Chao Liu a,d,∗ a Institute of Stem Cell and Tissue Engineering & Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China b School of Nursing, Anhui Medical University, Hefei, Anhui 230032, China c Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui 230032, China d Central Laboratory of Molecular and Cellular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China
a r t i c l e
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Article history: Received 12 May 2015 Received in revised form 23 July 2015 Accepted 23 July 2015 Available online xxx Keywords: H9 Neuroectoderm Localized cell density Reaggregation Cell cluster
a b s t r a c t Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Neuroectoderm (NE) is primordium of nervous system (Murry and Keller, 2008; Petros et al., 2011). Human embryonic stem cells (ESCs) can differentiate into NE in vitro, providing a platform for studying NE formation (Lyashenko et al., 2011; Thomson et al., 2011). However, human ESCs can differentiate into any of the three germ layers (endoderm, mesoderm and ectoderm) (Thomson et al., 1998), which makes it complex for researchers to induce NE other than non-NE cells. Directed neural differentiation cannot only provide an in vitro platform for study of human neurodevelopment (Milet and Monsoro-Burq, 2012; Nat et al., 2012), but also hold great promise for cytotherapy of human neurodegenerative
Abbreviations: ESC, embryonic stem cell; NE, neuroectoderm; RT-PCR, reverse transcription with polymerase chain reaction; IF, immunofluorescence; IB, immunoblotting; TH, tyrosine hydroxylase; DA, dopaminergic; PD, Parkinson’s disease; LCD, localized cell density; AA, ascorbic acid; DAPI, 4,6-diamidino-2phenylindole; FGF, fibroblast growth factor. ∗ Corresponding author at: Department of Histology and Embryology, Institute of Stem Cell and Tissue Engineering, School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, China. E-mail address: [email protected] (C. Liu). 1 These authors contributed equally to this work.
diseases (Volarevic et al., 2011). For example, degeneration of dopaminergic (DA) neurons in substantia nigra is a pathological hallmark of Parkinson’s disease (PD) (Mullin and Schapira, 2015). Currently, PD is a primary target for cytotherapy (Athauda and Foltynie, 2015; Kim, 2011; Lindvall, 2012). Because differentiation of human ESCs into NE is prerequisite for differentiation of human ESCs into region-specific neural cells, it is important to understand roles of extrinsic or intrinsic factors involved in NE differentiation. Localized cell density (LCD), a function of the number of neighbors a cell has within a given space, plays a crucial role in the self-renewal and differentiation of human ESCs (Peerani et al., 2007). We recently developed a cell clump-based method for NE differentiation (Liu et al., 2014). This method, in which the cell density is not in control, shows that high LCD promotes specification of NE. To keep plating cell density under control, cells must be dissociated into single cells. However, cell–cell interaction is necessary for NE specification of ESCs (Parekkadan et al., 2008). In addition, a confluent growing of human ESCs normally experiences disrupted lineage specification (Thomson et al., 1998). So far, combined role of above mentioned factors in NE differentiation has not been reported. In this paper, human ESCs, H9 cells, were disassociated into single cells for a cell counting. Then the cells were reaggregated into small cell clusters and cultured in mTeSR. Upon the cell clusters
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were nearly 95% confluent, a NE induction was performed. The induced cells were further subjected to a neural differentiation for at least 10 d. The expression of NE markers and neural markers in the H9-derived cells were analyzed by RT-PCR, immunofluorescence (IF) and immunoblotting assay, respectively. The induced cells are characterized by expression of PAX6 mRNA and PAX6 protein. Expression of GFAP, a glia marker gene, was also identified after 5 d of differentiation. Meanwhile, expression of beta-III-tubulin, a neuron marker protein, was detectable after 5 d of differentiation and reaches to its peak after 10 d of differentiation. Moreover, coexpression of beta-III-tubulin and TH was identified in DA-like neurons after 10 d of the NE differentiation. Our results here may indicate a simple method for a rapid neural induction from H9 cells.
2. Materials and methods 2.1. Cell culture Human ESC line H9, from passage 45 to passage 50, has been previously described (Liu et al., 2014; Yao et al., 2012). A cell clump-based maintaining was performed according to the protocol from StemCell Technologies Inc. Briefly, H9 cells were cultured on Matrigel (BD Biosciences) in mTeSR medium (StemCell Technologies Inc.). The medium was changed every day. The H9 cells were passaged with dispase and re-plated in mTeSR medium at a ratio of 1:5–1:6. Matrigel coating was performed according to the protocol from BD Biosciences. Briefly, Matrigel was thawed on ice and diluted with DMEM-F12 precooled on ice. The volume of one aliquot of Matrigel is typically 160 l. One aliquot of Matrigel was added to 15 ml of DMEM-F12 and mixed well. Then add 1 ml dilution per well of 6-well plates. Allow plate to sit at room temperature for 2 h. Plate may either be used immediately or stored at 4 ◦ C. When ready to use the plate(s), remove the excess liquid.
2.2. NE and neural differentiation A cell cluster-based method for NE differentiation was modified from our previous protocol (Liu et al., 2014). Briefly, H9 cells were dissociated with accutase for 20 min, washed with DMEM-F12 and resuspended using mTeSR medium supplemented with 10 M ROCK inhibitor (Y-27632, TOCRIS). After the cell suspension was subjected to a cell counting, cells with different cell densities (0.4, 0.8 and 2.0 × 104 cells/cm2 ) were reaggregated according to a method modified from previous protocol (Unbekandt and Davies, 2010; Zhang and Zhang, 2010). Briefly, cells were centrifuged at 250 rpm for 15 min first, followed by an incubation at 37 ◦ C for 1 h, which is helpful for cell–cell interaction and formation of small cell clusters. With a 5 ml serological pipette, part of the medium was aspirated and used to push the pellet carefully to detach it. The pellet, detached from the tube walls, was disrupted by aspirating for 2 times gently using a 5 ml pipette and replated on Matrigelcoated coverslips or 6-well plates. The cells were cultured for 3–5 d in mTeSR medium prior to induction. Upon the cell clusters were nearly 95% confluent, NE induction medium, which is composed of KSR medium (DMEM/F12, 20% KSR, 0.1 mM -mercaptoethanol, 10 ng/ml of FGF-2, 1% non-essential amino acid) and N2 medium (DMEM/F12, 1% N2 supplement, 1% non-essential amino acid with 10 ng/ml of FGF-2), supplemented with or without SB431542 (10 M, TOCRIS) and NOGGIN (200 ng/ml, PEPROTECH), was used for the induction. Increasing amounts of N2 medium (25%, 50%, and 75%) were added to the KSR medium every 2 d. NE medium is a combination of KSR medium and N2 medium. The KSR medium and N2 medium were prepared separately as described and mixed as below: day 0–2 (25% N2 medium: 75% KSR medium), day 3–4 (50% N2 medium: 50% KSR medium), day 5–6 (75% N2 medium:25% KSR medium). We added the fresh medium after the spent medium was removed from the well. For further neural differentiation, the cells were cultured in N2 medium supplemented with AA (0.2 mM) for at least 10 d. The cells were passaged mechanically (Perrier et al., 2004) after 6 d of neural differentiation on Matrigel-coated
Fig. 1. Keep plating cell density and LCD under control at the beginning of NE induction. Schematic representation of the main procedures of the NE induction (A). Comparison of the phase micrographs of the dissociated H9 cells plated as single cells (B) and as small cell clusters after a reaggregation (C) for 24 h, respectively.
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coverslips or 6-well plates cultured in N2 medium supplemented with AA (0.2 mM). 2.3. Microscopy and immunofluorescence (IF) assay The microscopy and IF assay was performed as previously described (Liu et al., 2014). Antibodies used for IF analysis were as follows: anti-OCT4 (Santa Cruz), anti-PAX6 (Millipore), anti-SOX1 (abcam), anti-beta-III-tubulin (Millipore) and anti-TH (Chemicon). A NIKON Eclipse 80I fluorescence microscope and an Olympus IX73 inverted fluorescence microscope were used for microscopy. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
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2.4. Immunoblotting (IB) assay An immunoblotting (IB) assay was performed as previously described (Liu et al., 2012). Antibodies used for IB were as follows: anti-OCT4 (Santa Cruz), anti-PAX6 (Millipore), anti-SOX1 (abcam), anti-beta-III-tubulin (Millipore) and rabbit anti-GAPDH (Ptglab). 2.5. Reverse transcription with polymerase chain reaction (RT-PCR) After 6 d of differentiation, H9-derived cells were harvested for RT-PCR assay. Total RNA was isolated using RNA isolation KIT (Axygen). For RT-PCR analysis of mRNAs, 1 g of the total RNA from each
Fig. 2. Expression of PAX6, a NE marker, is regulated by plating cell densities during the NE induction. (A–C) Expression of OCT4 (A and C, green) and PAX6 (B and C, red) in the H9 cells. (D–F) Expression of OCT4 (D and F, green) and PAX6 (E and F, red) in the induced cells. Nuclei were counterstained with DAPI (C and F, blue). (G–I) Coexpression of PAX6 (G and I, green) and NESTIN (H and I, red) in the induced cells. (J) Effect of different plating cell densities on the ratio of PAX6-positive cells to DAPI-positive cells. *P < 0.05 when compared with the cell density of 0.4 × 104 cells/CM2 . **P < 0.01 when compared with the cell density of 0.4 × 104 cells/CM2 . Error bars represent standard deviation of four to six experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4 Table 1 Primer sequences of markers used for RT-PCR analysis. Primer
Sequence
Size (bp)
OCT4
5 -AGTGAGAGGCAACCTGGAGA-3 5 -GTGAAGTGAGGGCTCCCATA-3 5 -CAATGCGGGGAGGAGAAGTC-3 5 -CTCTGGACCAAACTGTGGCG-3 5 -AACAGACACAGCCCTCACAAACA-3 5 -CGGGAACTTGAACTGGAACTGAC-3 5 -TCATCGCTCAGGAGGTCCTT-3 5 -CTGTTCCCAGAGATGGAGGTT-3 5 -GTACTCAGCGCCAGCATCG-3 5 -AGCCACATCGCTCAGACACC-3 5 -AGAATTCGCTTGAGTATTCTGA-3 5 -GGCTTTCAGGTTATTTGACTGA-3 5 -GTCCCCTGGTTCCCAAGAAAAGT-3 5 -TCCAGCTGGGGGATATTGTCTTC-3 5 -AGCCGGCACCTGTTGTGCAA-3 5 -TGACTTCTCCTGCATGCACT-3
273
SOX1 PAX6 GFAP GAPDH REX1 TH GATA2
464 275 383 302 470 331 242
sample was reverse transcribed with Superscript III (Invitrogen). Next, 10% of the first-strand reaction (2 l) was used for subsequent PCRs for each gene of interest. A GAPDH endogenous control was used for normalization. The forward and reverse primers used for gene expression analysis and the size of the PCR products are shown in Table 1. 2.6. Statistics Image J software was used to evaluate differences in protein levels. The relative expression of PAX6 among the total cells was calculated based on the areas in PAX6 and DAPI channels. Statistical analysis was performed using SPSS 17.0 software, and the data are presented as the mean ± standard deviation. For analysis of the differences in mRNA levels, expression of the indicated genes was examined using Adobe Photoshop software. The relative expression of the targets genes normalized to GAPDH gene was presented as the mean ± standard error. The statistical analysis was also performed using the SPSS 17.0 software. 3. Results 3.1. Keep both plating cell density and high LCD under control at the beginning of NE induction
Fig. 3. Expression of marker genes and proteins in the H9-derived cells treated with and without SB431542 and NOGGIN. (A) Expression of OCT4 and REX1 (markers for pluripotency), PAX6 and SOX1 (markers for NE), GFAP (a marker for glia), TH (a marker for dopaminergic neuron) and GATA2 (a marker for non-NE) at day 6 of the NE induction. Statistic analysis of the relative expression of the indicated marker genes normalized to GAPDH. *P < 0.05 when compared with KSR+N2. Error bars represent standard error of three-independent experiments. (B–E) Expression of some NE marker proteins, such as OCT4, PAX6 and SOX1, in the induced cells with or without the presence of SB431542 and NOGIN at day 6 of the NE induction.
Both plating cell density and LCD play role in NE induction (Liu et al., 2014). Dissociation of cells is required for cell counting and cell density manipulating. However, cell–cell interaction, which is disrupted during dissociation of cells, is crucial for differentiation of ESCs into NE (Parekkadan et al., 2008). Moreover, lineage specification is disrupted by a confluent growing of human ESCs (Thomson et al., 1998). Thus, in our study, H9 cells were disassociated into single cells for a following cell counting and were then reaggregated into many small cell clusters prior to NE induction (Fig. 1A). Moreover, when compared with the single cell-plating method (Fig. 1B), the cell cluster-plating method maintains higher LCD and/or more cell–cell interactions (Fig. 1C).
We also identified cells that are positive for both PAX6 (Fig. 2G and I, green) and NESTIN (Fig. 2H and I, red), another NE marker protein (Bhinge et al., 2014), after 5 d of NE induction. The percentage of PAX6-positive cells among total cells was also examined. When plated at the density of 0.4 × 104 cells/cm2 , the cells shows a percentage of no more than 10% (Fig. 2J, left column). With the density increased to 0.8 × 104 cells/cm2 , the percentage becomes more than 50% (Fig. 2J, middle column). At last, when the density increased to 2.0 × 104 cells/cm2 , the percentage reaches to more than 70% (Fig. 2J, right column), which suggests that high plating cell density promotes expression of NE marker proteins in the induced cells significantly (Fig. 2J, *P < 0.05; **P < 0.01).
3.2. Expression of NE marker proteins in induced cells
3.3. Examination of marker genes and proteins in the H9-derived cells during the NE induction
After 5 d of NE induction supplemented with SB431542 and NOGGIN, expression of OCT4 and PAX6 of induced cells was compared with that of H9 cells. As shown in Fig. 2, results of IF assay indicated that while H9 cells are positive for OCT4 (Fig. 2A and C, green) and devoid of PAX6 (Fig. 2B and C, red), most of the induced cells are devoid of OCT4 (Fig. 2D and F, green) and positive for PAX6, especially in the cells with high LCDs (Fig. 2E and F, red).
We also checked expression of some marker genes related to pluripotency (OCT4 and REX1), NE (PAX6 and SOX1) and neural cells (GFAP for glia and TH for dopaminergic neuron) during the NE induction using RT-PCR assay. The results of RT-PCR assay revealed a downregulation of pluripotent gene, such as OCT4 and REX1, together with an upregulation of PAX6, by SB431542 and NOGGIN at
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Fig. 4. Expression of neuronal marker proteins in the H9-derived neural cells. (A, B) Expression of beta-III-tubulin analyzed by IF at day 5 (A, green) and day 10 (B, green) of the induction, respectively. Nuclei were counterstained with DAPI (A and B, blue). (C) Expression of OCT4, PAX6, SOX1 and beta-III-tubulin at the indicated time points of the neural induction. The GAPDH was used for a loading control. (D–F) Expression of TH (D and F, red) and beta-III-tubulin (E and F, green) in the H9-derived cells. Nuclei were counterstained with DAPI (F, blue). Bar = 200 m. Arrows indicate the robust expression of both TH and beta-III-tubulin in the cell aggregates. (G) The number of cell clusters positive for beta-III-tubulin and TH were shown. Error bars represent standard deviation of ten random areas. (H–J) Colocalization of TH (H and J, red) and beta-III-tubulin (I and J, green) in H9-derived neurons (arrow heads). Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
day 6 of the NE induction. Moreover, expression of GFAP, a glial cell marker, was also upregulated. Few or no specific bands of TH, a DA marker, were identified. While the non-specific bands with wrong size were detected, few or no specific bands of GATA2, a non-NE marker, was identified. Moreover, no specific bands of SOX1, an NE marker which is expressed at a later stage of human NE development (Zhang et al., 2010), was detected (Fig. 3A). A significant upregulation of the relative expression of PAX6 mRNA normalized to GAPDH mRNA by SB431542 and NOGGIN was shown (Fig. 3A, *P < 0.05). We also performed IF assay to show expression of some NE marker proteins, such as OCT4, PAX6 and SOX1, in the induced cells at day 6 of the NE induction. SB431542 and NOGGIN induced a downregulation of OCT4 together with an upregulation of PAX6
(Fig. 3B–E). However, few or no SOX1 expression was identified in the induced cells (Fig. 3B and C). 3.4. Differentiation of H9-derived NE cells into neurons Because ability to differentiate into neurons is a hallmark of NE cells (Bhinge et al., 2014). To examine neuronal differentiation potential of the NE cells, we isolated the NE positive cell clumps mechanically and replaced the NE induction medium with the N2 medium supplemented with AA. The NE-derived cells were subjected to IF assay using anti-beta-III-tubulin antibody after 5 d and 10 d of differentiation, respectively. The expression of betaIII-tubulin in the NE-derived cells showed a time dependent style
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(Fig. 4A and B, green). IB assay also proved the time-dependent up-regulation of PAX6 (Fig. 4C). Besides, the time-dependent upregulation of beta-III-tubulin and down-regulation of OCT4 (Fig. 4C, upper panel), indicates the generation of neuronal cells. However, very weak or no SOX1 signal was identified in the induced cells. We performed IF assay and found cells positive for TH (Fig. 4D and F, red) and beta-III-tubulin (Fig. 4E and F, green) after 10 d of the NE induction. The number of and TH-positive cell aggregates or clusters were counted and shown (Fig. 4G). The percentage of THpositive clusters among beta-III-tubulin positive clusters is about 85%. Images with a higher magnification show the well colocalization of beta-III-tubulin with TH in the DA-like cells (Fig. 4H–J, arrows). This finding may suggest the generation of DA neuron-like cells, which are mainly found in the cell aggregates or clusters.
4. Discussion Previously, Studer lab described a neural induction method, in which the human ESCs maintained on MEF feeder cells were dissociated into single cells and grown on Matrigel in mTeSR until the cells were confluent (Chambers et al., 2009). Because we routinely use a cell clump-based culturing system for H9 cells (Liu et al., 2012), we thus developed a cell-clump based NE differentiation protocol (Liu et al., 2014). When compared with the cell clumpbased protocol, the novelties here is that we tried to keep plating cell density and LCD under control to maintain both high LCD and cell–cell interaction, which determines NE specification (Liu et al., 2014; Parekkadan et al., 2008). The promise for cytotherapy of human neurodegenerative diseases depends on the potential of human ESCs to be induced into diverse neural cell populations (Erceg et al., 2009). However, the permit to generate different neural cell types also makes it difficult to be controlled. A recent report indicated local microenvironment can modulate endogenous parameters, such as colony and aggregate size, and influence differentiation trajectories through regulating PAX6 expression (Bauwens et al., 2008). PAX6 is crucial for neurodevelopment, including brain patterning, neuronal migration, and neural circuit formation (Osumi et al., 2008). Our RTPCR assay revealed robust expression of PAX6 mRNA and the very weak expression or absence of SOX1 mRNA after 6 d of NE induction, which is also supported by the data of the IF assay. Our report here is consistent with previous report that human NE cells express PAX6 in advance of SOX1 so that PAX6 is believed to be NE determinant specifically in humans (Zhang et al., 2010). Besides, absence of GATA2, a non-NE marker gene, suggests our method might be specific for NE differentiation. After 6 d of differentiation, downregulation of mRNA levels of REX1, a marker gene for pluripotency, by SMAD signaling blockade, is consistent with the downregulation of both OCT4 mRNA and OCT4 protein levels. Simultaneously, upregulation of GFAP, a marker gene for glia, together with expression of beta-III-tubulin indicates that our method can generate both neurons and glia. PAX6 is expressed in NE cells at an earlier stage of neural development. Beta-III-tubulin is a neuronal marker protein which is expressed in neuronal cells at a later stage of neural development. The further induction with N2 medium may induce more NE cells to differentiate into neuronal cells, which express less PAX6. However, during the further induction with N2 medium, the proliferation ability of the beta-III-tubulin-positive cells might be decreased as compared to that of the cells at an earlier stage of neural development. Therefore, we speculated that the reduction of beta-III-tubulin protein level after 15 d of differentiation might imply the reduction of beta-III-tubulin positive cells. Interestingly, differentiation of P19 cells into NE cells dependents on cell aggregation, which induces an elevation of FGF8 (Wang et al., 2006),
a member of the fibroblast growth factor (FGF) family, which is important for induction of DA neurons (Perrier et al., 2004; Wu et al., 2012). Degeneration of DA neurons is a pathological hallmark of Parkinson’s disease, which is considered a primary target for cytotherapy (Anisimov, 2009; Kim, 2011). To fulfill this potential, lessons from embryonic development has offered important insights into basic culture models and signaling factors involved in inducing ESC differentiate into DA neurons (Murry and Keller, 2008). Generation of DA neuron from ESCs is complex, including three basic culture models: (1) as embryoid bodies (EBs) (Cho et al., 2008; Iacovitti et al., 2007), (2) as monolayers on extracellular matrix proteins (Chambers et al., 2009), and (3) on supportive stromal cells (Perrier et al., 2004; Sonntag et al., 2007). Optimizing culture condition is one of the strategies for improving efficiency of midbrain DA neuron generation. Both aggregates of human ESCs and the derived neural aggregates contributed to generation of DA neurons (Zhang and Zhang, 2010). Because NE cells are characterized by the potential to differentiate into neurons (Bhinge et al., 2014). In our study, after 10 d of differentiation, TH-positive neurons formed mainly in the cell aggregates, suggesting the NE cells induced by our method might have the potential of true NE cells. However, few or no expression of TH, a marker gene for DA neuron, was detected after 6 d of differentiation, implying generation of DA neurons requires at least more times. Together, our results here provide a simple NE induction method, which may contribute to in vitro neural differentiation of H9 cells. Acknowledgements This project was sponsored by a NSFC grant (31271159), a Technology Foundation for Selected Overseas Chinese Scholar (2012 No. 13) Grant from Anhui HRSS, a Grant for scientific research of BSKY (XJ201105) from Anhui Medical University. We thank Dr. Shengyun Fang for the gift of anti-TH antibodies. References Anisimov, S.V., 2009. Cell-based therapeutic approaches for Parkinson’s disease: progress and perspectives. Rev. Neurosci. 20, 347–381. Athauda, D., Foltynie, T., 2015. The ongoing pursuit of neuroprotective therapies in Parkinson disease. Nat. Rev. Neurol. 11, 25–40. Bauwens, C.L., Peerani, R., Niebruegge, S., Woodhouse, K.A., Kumacheva, E., Husain, M., Zandstra, P.W., 2008. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26, 2300–2310. Bhinge, A., Poschmann, J., Namboori, S.C., Tian, X., Loh, S.J.H., Traczyk, A., Prabhakar, S., Stanton, L.W., 2014. MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-beta/BMP signaling. EMBO J. 33, 1271–1283. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., Studer, L., 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280. Cho, M.S., Lee, Y.E., Kim, J.Y., Chung, S., Cho, Y.H., Kim, D.S., Kang, S.M., Lee, H., Kim, M.H., Kim, J.H., Leem, J.W., Oh, S.K., Choi, Y.M., Hwang, D.Y., Chang, J.W., Kim, D.W., 2008. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 105, 3392–3397. Erceg, S., Ronaghi, M., Stojkovic, M., 2009. Human embryonic stem cell differentiation toward regional specific neural precursors. Stem Cells 27, 78–87. Iacovitti, L., Donaldson, A.E., Marshall, C.E., Suon, S., Yang, M., 2007. A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: studies in vitro and in vivo. Brain Res. 1127, 19–25. Kim, H.J., 2011. Stem cell potential in Parkinson’s disease and molecular factors for the generation of dopamine neurons. Biochim. Biophys. Acta 1812, 1–11. Lindvall, O., 2012. Dopaminergic neurons for Parkinson’s therapy. Nat. Biotechnol. 30, 56–58. Liu, C., Sun, Y., Arnold, J., Lu, B., Guo, S., 2014. Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells. Biochem. Biophys. Res. Commun. 452, 895–900. Liu, L., Liu, C., Zhong, Y., Apostolou, A., Fang, S., 2012. ER stress response during the differentiation of H9 cells induced by retinoic acid. Biochem. Biophys. Res. Commun. 417, 738–743.
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Please cite this article in press as: Liu, A., et al., A simple method for differentiation of H9 cells into neuroectoderm. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.07.006