CXCR4 ACTIVATION PROMOTES DIFFERENTIATION OF HUMAN
EMBRYONIC STEM CELLS TO NEURAL STEM CELLS
LIJUN ZHANG,aQIUHONG HUA,aKAIYI TANG,a CHANGJIE SHI,aXIN XIEa,b*AND RU ZHANGa* aShanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, Collaborative Innovation Center for Brain Science, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
bCAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Abstract—G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth, death, movement, transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study, we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol, we found that the expression of several chemokine recep-tors changed dramatically during the hESC/NSC transition. Especially, the expression of CXCR4 increased approxi-mately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted, whereas the antagonist AMD3100 delayed the neural induction process. In consis-tence with antagonizing CXCR4, knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together, our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine.Ó2016 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (
http://creative-commons.org/licenses/by-nc-nd/4.0/).
Key words: human embryonic stem cell, neural stem/precur-sor cell, neural differentiation, chemokine receptor, CXCR4, SDF-1.
INTRODUCTION
Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine and disease modeling (Wobus and Boheler, 2005). Directed differenti-ation of hESCs to specific lineages has been a focal point in the field of hESC research. In vitro differentiation of hESCs toward defined neuronal and glial lineages (Dhara and Stice, 2008; Erceg et al., 2009) not only offers a great opportunity to study neurodevelopment and model neurological disease at cellular level, it also holds the potential for the treatment of neurodegenerative diseases and brain injuries (Hedlund and Perlmann, 2009). The seven-transmembrane G protein-coupled receptors (GPCRs) belong to the largest cell surface receptor fam-ily; however, they are poorly investigated in hESC differ-entiation. GPCRs can detect a diversity of ligands and initiate intracellular signaling primarily via activation of intracellular G proteins (Granier and Kobilka, 2012), and they are involved in fundamental cellular responses such as growth, death, movement, transcription and excitation (Zhang and Xie, 2012). Many GPCRs have been found to play important roles in embryonic development (Kobayashi et al., 2010). But detailed information about their expression and functions during the neural special-ization of hESCs is still lacking.
Chemokine receptors are GPCRs that interact with a type of cytokine called chemokines. They play critical roles in trafficking the cells to desired locations, a process named chemotaxis, along the chemokine gradient (Nieto et al., 1997). The involvement of the che-mokine/chemokine receptor family in central nervous sys-tem (CNS) development and neurogenesis has been reported (Bonavia et al., 2003; Doze and Perez, 2012). For example, CXCR4 and its ligand CXCL12, also named stromal cell-derived factor 1 (SDF-1), are highly expressed in the cerebellum, hippocampus, and neocor-tex and also constitutively expressed in the brain during adulthood (Kolodziej et al., 2008). The interaction between CXCR4 and SDF-1 has been strongly suggested to be important in neuronal development and patterning of the cerebellum and hippocampus in the rodent brain (Ma et al., 1998; Lu et al., 2002). CXCR4 is highly expressed in human neural precursor cells (NPCs) (Ni et al., 2004). SDF-1 also increases human NPC proliferation (Wu
http://dx.doi.org/10.1016/j.neuroscience.2016.09.001
0306-4522/Ó2016 The Authors. Published by Elsevier Ltd on behalf of IBRO.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Corresponding authors. Address: 189 Guo Shou Jing Road, Shanghai 201203, China (X. Xie), 1239 Siping Road, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China. Fax: +86-21-65986852 (R. Zhang).
E-mail addresses:xxie@simm.ac.cn(X. Xie),ru.zhang@tongji.edu. cn(R. Zhang).
Abbreviations: AP, alkaline phosphatase; BDNF, brain-derived neurotrophic factor; cAMP, cyclic adenosine monophosphate; CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor type 2; CNS, central nervous system; CXCR4, CXC chemokine receptor type 4; CXCR7, CXC chemokine receptor type 7; EDTA, ethylenediaminetetraacetic acid; FZD1, frizzled homolog 1; FZD10, frizzled homolog 10; FZD2, frizzled homolog 2; GDNF, glial cell line-derived neurotrophic factor; GPCR, G protein-coupled receptor; GPR56, G protein-coupled receptor 56; hESC, human embryonic stem cell; IGF-1, insulin-like growth factor 1; LPA, lysophosphatidic acid; NSC/NPC, neural stem/precursor cell; PFA, paraformaldehyde; RA, retinoic acid; RNAi, RNA interference; S1P, sphingosine-1-phosphate receptor; SDF-1, stromal cell-derived factor 1; shRNA, short hairpin RNA.
et al., 2009) and mediates NPC migration during develop-ment (Li et al., 2010; Merino et al., 2015). Most of the studies so far focused on the chemotaxis effect of chemo-kine receptors to drive cell migration during neural devel-opment, mostly after the neural stem cell (NSC) stage. How chemokine receptors function during the process of neural induction of hESC remains elusive.
Here we sought to identify the role of GPCRs in the neural induction process of hESCs. Using a PCR array, we found significant upregulation of CXCR4 expression upon hESCs to NSCs transition. Activation of CXCR4 by its agonist SDF-1 facilitated NSC generation from hESCs, whereas either antagonizing CXCR4 with its antagonist or downregulation of CXCR4 with RNAi gave the opposite effect. Taken together, we found that CXCR4 plays an important role in hESC neural induction process, and agents activating CXCR4 might be used to facilitate efficient NSC production from hESCs.
EXPERIMENTAL PROCEDURES
hESC culture
hESCs (H9 cell line) (Amit et al., 2000) were cultured under feeder-free conditions in mTeSR1 (STEMCELL Technologies, Vancouver, British Columbia, Canada). hESCs were plated on the hESC-qualified Matrix (BD Bioscience, San Diego, CA, USA)-coated culture vessels and passaged every 3–4 days using 0.5 mM EDTA for 5–10 min at 37°C.
Neural induction and differentiation
When reaching nearly confluent state, hESCs were passaged using accutase (Life Technology, Grand Island, NY, USA) and plated onto Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Life Technology, Grand Island, NY, USA) -coated culture vessels. Cells were treated overnight with the ROCK inhibitor Y-27632 (Sigma– Aldrich, St Louis, MO, USA) to increase cell survival. On day 1 after hESC splitting (about 15–25% confluence), the mTeSR1 medium was aspirated and cells were cultured in Neural Induction Medium (Life Technology, Grand Island, NY, USA) for 7 days to obtain NSCs. Spontaneous differentiation was performed in Neural Differentiation Medium (Neural Basal Medium, 1N2, 1B27 (Life Technology, Grand Island, NY, USA), 10 nM dibutyryl cAMP (Sigma, St Louis, MO, USA), 200 ng/ml Ascorbic acid (Sigma, St Louis, MO, USA), 1 ng/ml BDNF (PeproTech, Rocky Hill, NJ, USA), 1 ng/ ml GDNF (PeproTech, Rocky Hill, NJ, USA), 1 ng/ml IGF-1 (PeproTech, Rocky Hill, NJ, USA)) on laminin-coated surface. 14–16 days later, cells were fixed and stained for neuronal or glial marker.
Real-time PCR
Total RNA was isolated using Trizol (Life Technology, Grand Island, NY, USA) and 2lg RNA was used to synthesize cDNA using the M-MLV Reverse Transcriptase and Recombinant RNasin Ribonuclease
Inhibitor (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Real-time PCR was performed using FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) and analyzed with a Stratagene Mx 3000P thermal cycler (Agilent). A relative standard curve method was used to analyze the relative quantitation of gene expression during hESC differentiation. Two series of real-time PCR reactions were prepared to quantify both the target and the control gene ACTB. Standard curves were generated using a 5 to 10-fold serial dilution of cDNA pool of hESC and NSC samples. Simultaneously, all experimental samples were subjected to PCR amplification and the expression level of target genes or ACTB was determined by interpolating from their standard curve respectively. The expression level of a target gene in one experimental sample was normalized by its ACTB
level first, and then expressed as a relative expression or a fold-change between different samples. The real-time PCR primer sequences were listed inTable 1.
Alkaline phosphatase and immunofluorescent staining
For alkaline phosphatase (AP) staining, hESCs were fixed with 4% paraformaldehyde (PFA) in PBS for 40 s, rinsed once with PBS and stained using a leukocyte alkaline phosphatase kit (Sigma–Aldrich, St Louis, MO, USA) according to the manufacturer’s protocol. For immunofluorescent staining, cells were fixed with 4% PFA for 15 min. The fixed cells were washed with PBS and incubated in 0.2% Triton X-100 for 20 min, then washed with PBS and incubated in blocking buffer containing 20% FBS, 10% Glycerol, 100 nM Glycine and 0.1% Triton X-100 for 30 min at room temperature. The cells were then incubated with primary antibodies against Oct4 (Abcam, ab19857), SSEA-4 (Millipore, MAB4304), Nanog (Abcam, ab21624), Nestin (Millipore, MAB5326), Sox2 (Abcam, ab15830), Sox1 (Abcam, ab22572), Pax6 (DSHB, P3U1), Musashi1 (Abcam, ab21628), Tuj1 (Covance, PRB-435P), GFAP (Invitrogen, 13-0300) or O4 (R&D, MAB1326) overnight at 4°C. The following day cells were washed with PBS and incubated in the appropriate secondary antibodies conjugated to Alexa Fluor 546 or Alexa Fluor 488 (Invitrogen) for one hour at room temperature. Nuclei were counterstained with Hoechst 33342 for 10 min. Images were taken with an Olympus IX71 inverted fluorescent microscope or an Olympus FV10i confocal microscope.
RNA interference in hESCs
For lentivirus-mediated CXCR4 knockdown, lentiviral vector FG12 (derived from the pFUGW vector, Addgene) and packaging plasmids pRSV/REV, pMDLG/ pRRE and pHCMVG were used. To construct the shRNA expression cassette, complementary DNA oligonucleotides were synthesized, annealed and inserted immediately downstream of the U6 promoter of the pBS/U6 plasmid, and the derived cassette was
subcloned into the lentiviral vector FG12 carrying simultaneously a GFP reporter gene. Recombinant lentiviruses were produced in HEK-293T cells. hESCs were infected overnight with lentiviral supernatants, and GFP+ clones representing infected cells were selected 48–72 h after infection for analyzing the mRNA expression by real-time PCR or the protein expression by western blotting using an anti-CXCR4 antibody (abcam, ab124824). The RNAi sequence targeting CXCR4 was 50-GGCAGTCCATGTCATCTAC-30 and 50-CTGGTCATGGGTTACCAGA-30. The scrambled shRNA sequence was 50-GAAGCAGCACGACTTCTTC-30.
Statistical analysis
Values are reported as mean ± SEM. Statistical significance (P value) was determined using the paired Student’s t-test. All graphs were plotted using the GraphPad Prism 5 software.
RESULTS
Neural induction of hESCs and characterization of hESC-derived NSCs
In order to investigate the role of GPCRs in the neural induction of hESCs, we first set up a neural induction and differentiation system using feeder-free hESCs. hESCs cultured under the feeder-free condition grew into large compact colonies with clear and smooth edge, and showed strong alkaline phosphatase staining (Fig. 1A). Immunostaining of the pluripotency markers (Klimanskaya et al., 2006) revealed that all colonies expressed surface antigen SSEA4, as well as the
transcription factors Oct4 and Nanog (Fig. 1B). These cells were then seeded onto matrigel-coated vessels and subjected to neural specification for the next 7 days in neural induction medium. Under such conditions, hESCs were converted into cells exhibited homogenous neuronal epithelial morphology (Fig. 1C). The derived cells were characterized by real-time PCR analysis for the expression of NSC markers (Gage, 2000; Yuan et al., 2011). Compared to hESCs, several NSC genes, includingNestin,Musashi1,Sox1,Sox2 andPax6, were significantly upregulated in the derived cells, whereas the pluripotency genes Oct4, Nanog and Rex1 were downregulated (Fig. 1D). Consistently, immunostaining revealed that the derived NSCs homogenously expressed Nestin, Sox2, Musashi1 and Pax6 (Fig. 1E). These NSCs can further spontaneously differentiate into Tuj1-positive neurons and GFAP-positive astrocytes (Fig. 1F, G) once cultured for 14 days in neural differentiation medium. O4-positive Oligodendrocytes can be detected on day 16 under such conditions (Fig. 1G). These data suggest successful transition from hESCs to NSCs.
Expression pattern of chemokine receptors in hESCs and NSCs
To investigate whether GPCRs are involved in the neural induction of hESCs, we compared the expression profiles of GPCRs in hESCs and hESC-derived NSCs using a TaqmanÒ PCR array. The array results showed GPR56, CD97, and the frizzled receptors, such as FZD1, FZD2 and FZD10 were upregulated upon transition from hESCs to NSCs (data not shown). These results are in consistence with previous publications which showed GPR56 was highly expressed in neural
Table 1.Primers used in real-time PCR analysis.
Gene Forward primer Reverse primer
ACTB 50-TGAAGTGTGACGTGGACATC-30 50-GGAGGAGCAATGATCTTGAT-30
Oct4 50-ATGTGGTCCGAGTGTGGTTC-30 50-GAGACAGGGGGAAAGGCTTC-30
Nanog 50-CAGAAGGCCTCAGCACCTAC-30 50-GGCCTGATTGTTCCAGGATT-30
Sox2 50-AGCTACAGCATGATGCAGGA-30 50-GGTCATGGAGTTGTACTGCA-30
Nestin 50-GCGTTGGAACAGAGGTTGGA-30 50-TGGGAGCAAAGATCCAAGAC-30
Sox1 50-CCTCCGTCCATCCTCTG-30 50-AAAGCATCAAACAACCTCAAG-30
Pax6 50-CTGAAGCGGAAGCTGCAAAG-30 50-TTGCTGGCCTGTCTTCTCTG-30
Musashi1 50-GCTCAGCCAAAGGAGGTGAT-30 50-GCGTAGGTTGAGGCTTGGAA-30 CXCR1 50-CGCCTATGCCCTAGTGTTCC-30 50-AAAATCCAGCCATTCACCTT-30 CXCR2 50-GACCCAGGTGATCCAGGAGA-30 50-GCCCTGAAGAAGAGCCAACA-30 CXCR3 50-CCCTCTACAGCCTCCTCTTTC-30 50-ACTATGTTCAGGTAGCGGTCAAA-30 CXCR4 50-CCCTCCTGCTGACTATTCCC-30 50-TAAGGCCAACCATGATGTGC-30 CXCR5 50-GTCACCCTACCACATCGTCATC-30 50-GTACAGCCCAGCTTCGTCAG-30 CXCR6 50-ACAGAGGCCATCGCATACC-30 50-GAGGCAACCAATGTCCTTCAC-30 CXCR7 50-CAACCTCTTCGGCAGCATTT-30 50-AGACGACACGGCGTACCAT-30 CCR1 50-CAGCCTTCACTTTCCTCACG-30 50-AACGGACAGCTTTGGATTTCTT-30 CCR2 50-TACGGTGCTCCCTGTCATAAA-30 50-CATTCCCAAAGACCCACTCAT-30 CCR3 50-TCTCCCTCTGCTCGTTATGG-30 50-GATGCTTGCTCCGCTCACA-30 CCR4 50-GAAGAAGAACAAGGCGGTGAA-30 50-TGATGGGATTAAGGCAGCAGT-30 CCR5 50-GAGACTCTTGGGATGACGCAC-30 50-CATTTGCAGAAGCGTTTGG-30 CCR6 50-TCCTCATAACATGGTCCTGCTT-30 50-CTCACTGGTCTGCCGAGAAAT-30 CCR7 50-CCATGACCGATACCTACCTGC-30 50-AAAGTGGACACCGAAGACCC-30 CCR8 50-TGGGTGTTTGGGACTGTAATG-30 50-ATCTTCAGAGGCCACTTGGTA-30 CCR9 50-ACAGCCAAATCAAGGAGGAAT-30 50-TAGGGAAACTGAGACAAGACAAAGA-30 CCR10 50-AGGGCTGGAGTCTGGGAAGT-30 50-AGCGGTCGGCGCTGATA-30 SDF-1 50-ACTCCAAACTGTGCCCTTCA-30 50-CCACTTTAGCTTCGGGTCAAT-30
precursors (Bai et al., 2009) and the frizzled were functional in neural induction (Yan et al., 2009; Yu et al., 2010), indicating the reliability of our assay. We also found that CXCR4, a chemokine receptor, was significantly upregulated after differentiation of hESCs to NSCs. Given the well-known roles of chemokines in cell migration during embryo development (Raz and Mahabaleshwar, 2009), we wondered how the other chemokine receptors behave in the neural differentiation
process. Therefore, we examined the expression of 17 chemokine receptors (CCR1 to CCR10, CXCR1 to CXCR7) and SDF-1 by PCR. The semi quantitative RT-PCR results demonstrated a significantly increased expression of CCR1, CCR6, CCR7, CXCR4, CXCR5
andCXCR7upon transition from hESCs to NSCs. In con-trast, CCR9, CXCR2 and SDF-1 were downregulated (Fig. 2A). Similar results were obtained with quantitative real-time PCR analysis (Fig. 2B). Interestingly, the most
Fig. 1.Neural induction of hESCs and characterization of hESC-derived NSCs. (A) Morphology and alkaline phosphatase staining of hESCs (Scale bar = 100lm). (B) Immunofluorescent staining of pluripotency markers (Oct4, Nanog and SSEA4) in hESCs (Scale bar = 10lm). (C) Morphology of cells on day 1, day 3, day 5 and day 7 during neural induction. (Scale bar = 100lm). (D) Quantitative RT-PCR analysis of gene expression of neural markers and pluripotency markers in hESCs and NSCs derived from hESCs. The data are mean ± SEM (n= 6).**P< 0.01,***P< 0.001, versus hESCs. (E) Immunofluorescent staining of neural markers (Nestin, Sox2, Musashi1 and Pax6) in NSCs derived from hESCs (Scale bar = 10lm). (F) Morphology of spontaneously differentiated NSCs (Scale bar = 100lm). (G) Immunofluorescent staining of markers for neuron (Tuj-1), astrocyte (GFAP) and oligodendrocyte (O4) in spontaneously differentiated NSCs (Scale bar = 10lm).
dramatic changes were observed inCXCR4, CXCR7and
SDF-1. CXCR4 and CXCR7 are two closely related che-mokine receptors and their expression increased approx-imately 50 and 20 folds, respectively, in NSCs compared to the original hESCs (Fig. 2B). SDF-1, the ligand for both CXCR4 and CXCR7, decreased around 7 folds. It has been reported that SDF-1 and CXCR4 were involved in neurogenesis processes such as NPC migration, proliferation, and differentiation (Peng et al., 2007; Miller et al., 2008). Whether and how CXCR4 is involved in hESC-NSC transition process remains elusive. Our observation suggested that CXCR4 might be an important regulator in this process. Therefore, we focused on CXCR4 in our subsequent studies.
Activation of CXCR4 accelerates neural induction of hESCs
To demonstrate the functional significance of increased expression of CXCR4 during neural induction, we tested whether activation of CXCR4 can influence the hESC to NSC transition. We monitored the progression of neural induction by immunostaining of Nestin, the most well-defined marker for NSCs. In vehicle-treated cells, almost no Nestin-positive cells could be observed on day 2 (data not shown) and very few Nestin-positive areas (less than 3%) could be seen on day 3 of induction (Fig. 3A, C). The percentage of Nestin-positive area increased to
about 15% on day 4 and reached a plateau on day 7 (Fig. 3A, C, Ctl). When adding CXCR4 agonist SDF-1 in the neural induction medium, 3% of the cell area could already be detected as Nestin-positive on day 2 (data not shown) and the percentage reached 8% and 20% on day 3 and day 4, respectively (Fig. 3A, C, SDF-1). Conversely, in the presence of AMD3100, a specific antagonist of CXCR4, the neural induction process was significantly delayed. Almost no Nestin-positive area appeared on day 3 and only about 3% of the cell area appeared Nestin-positive on day 4 of the neural induction (Fig. 3A, C, AMD3100). Alternatively, cells were stained for Sox1, another common marker for NSCs (Li et al., 2005; Suter et al., 2009), and similar results were obtained (Fig. 3B, D). In vehicle-treated cells, less than 5% Sox1-positive cells can be seen on day 3 of induction. The per-centage of Sox1-positive cells increased to about 10% on day 4 and reached 80% on day 7 (Fig. 3B, D, Ctl). Cells treated with SDF-1 showed accelerated protein expres-sion of Sox1 and 10% of the cells can already be detected as Sox1-positive on day 3 and the percentage reached about 20% and 40% on day 4 and day 5 respectively (Fig. 3B, D, SDF-1). In contrast, blocking CXCR4 with AMD3100 retarded the Sox1 protein expression during neural induction of hESCs. Almost no Sox1-postive cells appeared on day 3 and only 5–10% of the cells appeared Sox1-positive on day 4 and 5 during the neural induction (Fig. 3B, D, AMD3100).
Fig. 2.Expression pattern of chemokine receptors in hESCs and hESC-derived NSCs. (A) RT-PCR analysis for chemokine receptor gene expression in hESCs and hESC-derived NSCs. (B) Real-time PCR analysis of chemokine receptor in hESCs and hESC-derived NSCs. The data are mean ± SEM (n= 3).*P< 0.05,**P< 0.01,***P< 0.001, versus hESCs.
Knockdown of CXCR4 is adverse to neural induction of hESCs
Since activation of CXCR4 promotes neural induction of hESCs, it’s conceivable that downregulation of CXCR4 expression might inhibit neural induction. A shRNA-based technique was employed to specifically knockdown the CXCR4 gene in hESCs and we monitored the progression of neural induction by immunostaining of Nestin. The transfection efficiency was monitored by a GFP reporter gene embedded in the shRNA expression vectors. The knockdown efficiency of two independent shRNAs targeting CXCR4 was tested by real-time PCR and both led to at least 60% downregulation in mRNA level (Fig. 4A). The downregulation of CXCR4 protein was also approved by western blot analysis (Fig. 4B). Interestingly, downregulation of CXCR4 changed the morphology of hESCs remarkably; however, the pluripotent marker Oct4 remained unchanged in these cells (Fig. 4C). In the scramble shRNA transfected cells, around 3%
Nestin-positive cell area could be seen on day 3 of induction, and the percentage of Nestin-positive area increased to nearly 10% on day 4 and reached more than 30% on day 6 (Fig. 5A, C, scramble). In contrast, the progression of neural induction was significantly delayed in CXCR4-knockdown cells and the percentage of Nestin-positive area remained only half of that in the scramble shRNA transfected cells until day 6 (Fig. 5A, C, shRNA-1 and -2). Very interestingly, when using GFP fluorescence to identify CXCR4 shRNA-transfected cells, we found almost none of the GFP-positive cells were Nestin-positive (Fig. 5B, shRNA-1 and -2, cells in white cycles), whereas in cells transfected with scramble shRNA, much more cells are Nestin and GFP double positive (Fig. 5B, scramble). We also did statistic analysis of the cell percentage expressing Nestin in GFP-positive cells during neural induction process. As shown in Fig. 5D, in CXCR4 shRNA transfected cells, less than 20% of GFP-positive cells expressed Nestin, whereas in the scramble group
Fig. 3. Ligands of CXCR4 affect neural induction of hESCs. (A, B) Immunofluorescent staining of Nestin (A) or Sox1 (B) on day 3–7 of neural induction of hESCs in control condition (Ctl), or in the presence of CXCR4 agonist SDF-1 (100 nM), or CXCR4 antagonist AMD3100 (100 nM) (Scale bar = 10lm). (C, D) Statistical analysis of the percent of nestin-positive area (C) presented in (A) or Sox1-positive cell number (D) presented in (B). The data are mean ± SEM (n= 10).*P< 0.05,**P< 0.01,***P< 0.001, versus Ctl.
the percentage reached 80% on day 7. In parallel, Sox1 was stained in these cells (Fig. 6). In consistent with the Nestin data, knock-down of CXCR4 suppressed Sox1 expression (Fig. 6A, C) and much less GFP/Sox1 double-positive cells can be detected (Fig. 6B, D, cells in white dashed-line cycles are mostly GFP-positive but Sox1-negative). Taken together, our results support that CXCR4 plays an important role in neural induction of hESCs.
DISCUSSION
GPCRs belonging to one of the most important drug target families have always been the focus of research in life science. Their functions in stem cell biology (Callihan et al., 2011) and neurogenesis (Doze and Perez, 2012) have attracted great attention recently. Involvement of several GPCRs in neural development has been reported. For example, LPA inhibits neuronal differentiation of NSCs derived form hESCs via LPA receptors (Dottori et al., 2008). S1P induces proliferation and a morphological change of NSCs via S1P receptors (Harada et al., 2004). GPR56 is highly expressed in NSCs and regulates NSC migration, but is downregulated during differentiation (Iguchi et al., 2008; Bai et al., 2009). Both cannabinoid (CB) receptors have been reported to play a role in neural development. Activation of CB2 promotes NSC proliferation (Molina-Holgado et al., 2007), whereas knockout of CB1 in mice results in ineffective adult neuro-genesis (Jin et al., 2004). Alpha-1 adrenergic receptors stimulation induces the proliferation and migration of NPCs (Hiramoto et al., 2006), and dopamine D1 receptor mainly regulates NPC migration (Hiramoto et al., 2008). In addition, activation ofl- andj-opioids improves the per-centage of ESC-derived RA-induced NPCs (Kim et al., 2006). In our study, we also found the dramatic changes in expression levels of GPCRs when hESCs differentiate into NSCs, supporting the regulatory roles of GPCRs in neurogenesis.
Chemokines, the most important and best known group of chemotactic proteins, participate in many physiological processes, including immunosurveillance (Chong et al., 2004), immune cell differentiation (Nieto et al., 1997), inflammation (Koelink et al., 2012), angio-genesis (Rudolph and Woods, 2005), neurogenesis (Doze and Perez, 2012) and so on. Chemokines act on multiple cell targets via their receptors that belong to the rhodopsin-like receptor family of GPCRs. Chemokine receptors include CXC chemokine receptors, CC chemo-kine receptors, CX3C chemochemo-kine receptors and XC che-mokine receptors (Zlotnik and Yoshie, 2000). Among them, CXCR4 and its ligand SDF-1 are relatively well-studied mediators involved in human neuronal develop-ment and play critical roles in human NPC proliferation and migration (Wu et al., 2009). In consistence with previ-ous reports (Ni et al., 2004), we confirmed that CXCR4 is highly expressed in human NSCs. Besides, we observed a dramatic increase in CXCR4 expression during hESC to NSC transition. To rule out the possibility that the increased expression is solely the consequence of neural differentiation, we performed the knockdown experi-ments. Indeed, cells with downregulated CXCR4 is much harder to be induced to express the NSC marker Nestin, supporting that CXCR4 is important for neural differentia-tion of hESCs. Interestingly, we observed dramatic mor-phology changes in CXCR4-knockdown hESCs, although without causing the loss of pluripotent genes, suggesting CXCR4 may also be important in maintaining the properties of hESCs. Further cell culture showed diffi-culty in clonal expansion of CXCR4 shRNA transfected hESCs (data not shown). According to the literatures, we found that CXCR4 regulates neuron survival via affect-ing cell-cycle proteins in neurons (Khan et al., 2003), and CXCR4-mediated endocytotic signaling pathway is essential to human neural progenitor cell survival (Zhu et al., 2012). Whether CXCR4 plays an important role in hESC survival or self-renewal is still unclear and worthy of further investigation in the future.
Fig. 4.Knockdown of CXCR4 in hESCs. (A) Real-time PCR analysis of knockdown efficiency of shRNA targetingCXCR4. The data are mean ± SEM (n= 3),***P< 0.001, versus scramble. (B) Representative western blot analysis of CXCR4 protein level after shRNA expression in hESCs. (C) Morphology of hESCs expressing scramble orCXCR4shRNA (left panel) (Scale bar = 100lm). Immunofluorescent staining of pluripotent marker Oct4 in hESCs expressing scramble orCXCR4shRNA (right panel) (Scale bar = 10lm).
In addition to CXCR4, we found that CXCR7, a receptor closely related to CXCR4, and their common ligand SDF-1 changed dramatically. It was reported that CXCR7 binds to SDF-1 with higher affinity than CXCR4; therefore, CXCR7 may function as an antagonizer for CXCR4 via ligand competition (Naumann et al., 2010). Kalatskaya et al. found that AMD3100, a known CXCR4 antagonist, increases SDF-1 binding to CXCR7 and SDF-1-induced conformational rearrangements in the receptor dimer (Kalatskaya et al., 2009). However, another study demonstrated that SDF-1 promotes human NPC survival in the events of camptothecin-induced apoptosis or growth factor deprivation, which required both CXCR7 and CXCR4 through CXCR7- and
CXCR4-mediated endocytotic signaling (Zhu et al., 2012). In our study, we saw increased expression of both receptors during hESC to NSC transition and we con-firmed that adding SDF-1 in the culture medium promotes the transition and CXCR4 is indispensible for such a pro-cess. Since CXCR7 expression also increased signifi-cantly, whether CXCR7 is also required as a coregulator for the transition needs to be further investigated. Besides, we saw a decreased expression of SDF-1 in NSCs compared to hESCs. It has been reported that the spatial and cellular expression patterns of SDF-1 and CXCR4 in the brain are different and this differential expression of CXCR4 and SDF-1 may be important for the temporal regulation of neuronal migration and circuit
Fig. 5. Knockdown of CXCR4 retarded Nestin expression and blocks neural induction of hESCs. (A) Immunofluorescent staining of Nestin on day 3–7 of neural induction (Scale bar = 10lm). (B) Representative images of cells expressingCXCR4or scramble shRNA (both with GFP reporter) on day 6 of neural induction. (Scale bar = 10lm), cells that are GFP-positive but almost Nestin-negative are cycled with white dashed line. (C) Statistical analysis of the percent of Nestin-positive area presented in (A). The data are mean ± SEM (n= 10). *P< 0.05, **P< 0.01, ***
P< 0.001, versus scramble. (D) Statistical analysis of the percentage of Nestin and GFP double positive cells in shRNA transfected cells (GFP positive). The data are mean ± SEM (n= 3).**P< 0.01,***P< 0.001, versus scramble.
formation during development and possibly in adult neurogenesis and repair (Peng et al., 2007). Therefore, during hESC neural differentiation, the different expres-sion pattern of CXCR4 and SDF-1 can be considered as a way to fulfill their distinct temporal and spatial functions. Taken together, we found that CXCR4 plays an important role in hESC neural induction process and activation of CXCR4 promotes hESC to NSC transition. Therefore, CXCR4 might be considered as a promising target in regenerative medicine to facilitate efficient NSC production from hESCs.
Acknowledgments—This project was supported by grants from the Ministry of Science and Technology of China [2014CB965002 and 2015CB964503], Science and Technology Commission of Shanghai Municipality, China [15JC1400202], the Chinese Academy of Sciences, China [XDA01040301], and the National Natural Science Foundation of China [81425024 and 31371511].
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(Accepted 1 September 2016) (Available online 9 September 2016) L. Zhang et al. / Neuroscience 337 (2016) 88–97
