Biomaterials 39 (2015) 75e84
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells Weiqiang Li a, b, c, 1, Lihua Huang a, 1, Wanyi Lin a, d, 1, Qiong Ke a, e, Rui Chen f, Xingqiang Lai a, Xiaoyu Wang g, Jifeng Zhang h, Meihua Jiang a, i, Weijun Huang a, Tao Wang a, Xuesong Yang g, Yuan Chen h, Wu Song j, **, Andy Peng Xiang a, b, c, * a Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong, PR China b Department of Biochemistry, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China c Key Laboratory of Reproductive Medicine of Guangdong Province, Guangzhou, Guangdong, PR China d Department of Blood Transfusion, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China e Department of Cell Biology, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China f Center for Reproductive Medicine, Key Laboratory for Reproductive Medicine of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, PR China g Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou, PR China h Center for Neurobiology, Zhongshan School of Medicine, Sun-Yat Sen University, Guangzhou, Guangdong, PR China i Department of Anatomy and Neurobiology, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China j Department of Gastrointestinal-Pancreatic Surgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2014 Accepted 19 October 2014 Available online
Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESCderived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2a, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neural crest stem cells Embryonic stem cells Differentiation Non-human primates
1. Introduction
* Corresponding author. Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun YatSen University, Guangzhou, Guangdong, PR China. Tel.: þ86 20 87335822; fax: þ86 20 87335858. ** Corresponding author. Tel./fax: þ86 20 28823389. E-mail addresses: [email protected] (W. Song), [email protected]. cn (A.P. Xiang). 1 These authors made equal contributions to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.056 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
The neural crest comprises a transient population of embryonic multipotent cells that arise from the border between the neural plate and the non-neural ectoderm at the gastrula stage and emerge from the neural tube shortly after the tube closes. Common to all vertebrate embryos, premigratory neural crest stem cells (NCSCs) can be identified by their expression of neural crestspecific genes [1], including the transcription factors Sox10 (SRY box 10), Ap2a (activating enhancer binding protein 2 alpha), Sox9, Snail (Slug), FoxD3 (forkhead box D3), and c-Myc [2]. The NCSCs
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W. Li et al. / Biomaterials 39 (2015) 75e84
then separate from the neural tube and migrate extensively throughout the embryo, a process that involves either a complete or partial epithelial-to-mesenchymal transition (EMT) [2,3]. Early migratory NCSCs migrate along defined pathways and differentiate into a wide array of derivatives, including neurons and glia of the peripheral and enteric nervous systems, bone, cartilage, smooth muscle, melanocytes in the skin, and connective tissue of the head [4]. In addition, neural crest cells are reported to be among the developmental sources of mesenchymal stem cells (MSCs), which, owing to their multipotency, paracrine action and immunomodulatory properties, have been intensively investigated for use in treating different diseases [5,6]. The neural crest also plays important roles in vertebrate developmental processes, and defective growth, differentiation, and migration of neural crest cells usually causes neurocristopathies e a large group of congenital disorders that includes Hirschsprung's disease, DiGeorge syndrome, and family dysautonomia, among others [7]. Major strides in understanding the biology of NCSCs have been made in recent years using numerous model organisms ranging from protochordates to higher vertebrates, including zebrafish, Xenopus, chicken and mouse [8e14]. Rodent NCSCs have been isolated from both fetal and adult tissue, such as gut and sciatic nerve [14,15]. However, access to gastrula-stage human embryos is limited by numerous ethical and legal questions, making studies of neural crest development in humans problematic. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the ability to differentiate into the three germ layers (including NCSCs) both in vitro and in vivo, which makes them powerful in vitro models for developmental biology studies. Derivation of NCSCs from ESCs or iPSCs enables investigation and manipulation of neural crest development and holds promise for mechanistic studies and cell-replacement therapies [16e23]. Understanding the detailed mechanisms of NCSC specification, migration, and differentiation will ultimately contribute to their use in therapeutic applications. Although rodent models play an important role in diverse aspects of biomedical research, they are poor representations of human disease phenotypes. Therefore, large animal models, especially non-human primate models, are invaluable for modeling human embryos. Furthermore, ESCs/iPSCs from non-human primates are similar to human ESCs in terms of morphology, self-renewal capacity, and pluripotency. Although the potential of human ES/iPSderived cells for cell-replacement therapy in patients with degenerative diseases is widely recognized, a thorough understanding of the growth, migration, and differentiation of these cells in vivo is a prerequisite for their clinical application. It is also important to first address questions such as tumor formation, functional capacity, and potential for immune rejection of transplanted human ES/iPSderived cells in a non-human primate model. Cynomolgus monkeys closely resemble humans in terms of anatomy, physiology, pathophysiology, and genetic background. Therefore, studies using cynomolgus monkey ESCs (CmESCs) are expected to provide important insights into the use of human ES/iPS-derived cells for human therapeutic purposes. Although avian, murine and human NCSCs have been identified, NCSCs have not yet been isolated from embryos or pluripotent stem cells of non-human primates. Here, we reported the successful isolation and propagation of NCSCs derived from CmESCs. Our in vitro characterization of CmESC-derived NCSCs showed that these cells express premigratory neural crest markers and are capable of multilineage differentiation towards neural crest lineages in vitro. In vivo, CmNCSCs transplanted into the brains of developing cynomolgus monkeys and chick embryos survived, migrated extensively, and contributed specifically to proper neural crest derivatives.
2. Materials and methods 2.1. Cell culture Undifferentiated CmESCs [24] were maintained on mitotically inactivated mouse embryonic fibroblasts in Knockout DMEM (Life Technologies, Carlsbad, CA, USA) containing 12% KnockOut Serum Replacement (Life Technologies), 8% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% non-essential amino acids (NEAA; Hyclone), 1 mM L-glutamine (Hyclone), 0.1 mM 2-mercaptoethanol (Life Technologies), 10 ng/ml basic fibroblast growth factor (bFGF; Life Technologies), 100 IU/ml penicillin (Hyclone), and 100 mg/ml streptomycin (Hyclone). The medium was changed daily, and CmESCs were passaged every 5e7 days by treating with 1 mg/ml collagenase IV (SigmaeAldrich, St Louis, MO, USA). 2.2. Derivation of NCSCs from CmESCs CmESCs were washed twice with phosphate-buffered saline (PBS) and then disaggregated into small clusters by incubating with 1 mg/ml dispase (Roche, Indianapolis, IN, USA) at 37 C for 5e10 min. For embryoid body formation, clusters were suspended in KSR medium consisting of 80% KnockOut DMEM, 20% KnockOut Serum Replacement (Life Technologies), 1 mM L-glutamine, 1% NEAA, and 0.1 mM 2mercaptoethanol. Embryoid bodies were cultured for 5 days in ultra-lowattachment culture dishes with daily changes of medium. Embryoid bodies were then replated on dishes coated with poly-L-ornithine (15 mg/ml; SigmaeAldrich) and laminin (10 mg/ml; Millipore, Temecula, CA, USA) and cultured in neural crest culture medium (NCM), consisting of DMEM/F-12 and Neurobasal medium (1:1 ratio) (both from Life Technologies), 1% N2, 2% B27, 1 mM L-glutamine, and 0.1 mM 2mercaptoethanol supplemented with 20 ng/ml EGF (epidermal growth factor) and 20 ng/ml bFGF. CmESC-derived NCSCs were labeled with antibodies against HNK1 (also known as CD57/B3GAT1) and p75 (also known as low affinity nerve growth factor receptor, NGFR) (both from BD-Pharmingen, Palo Alto, CA, USA) for fluorescence-activated cell sorting (FACS) using a BD Influx cell sorter (BD-Pharmingen). Sorted cells were replated on polyornithine/laminin-coated dishes for adherent culture (50,000e100,000 cells/cm2) or were cultured in suspension on ultra-low-attachment plates for the formation of neural crest spheres (1000e10,000 cells/cm2). 2.3. Differentiation of CmNCSCs For differentiation of CmNCSCs towards the peripheral nervous system, cells were cultured in NCM without FGF2 and EGF and supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml nerve growth factor (NGF) and 10 ng/ml neurotrophin-3 (NT3) (all from Peprotech, Rocky Hill, New Jersey, USA), as well as 200 mM ascorbic acid (AA) and 0.5 mM dibutyryl-cAMP (db-cAMP) (both from SigmaeAldrich) [23]. Cells were cultured for 2e4 weeks, and media were changed every 2e3 days. Differentiated cells were analyzed by assessing the expression of neural markers by immunocytochemistry. For Schwann cell differentiation, CmNCSCs were first cultured in NCM for at least 4 weeks. Differentiation was then induced by culturing in NCM without FGF2 and EGF and supplemented with 10 ng/ml ciliary neurotrophic factor (CNTF; Peprotech), 20 ng/ml neuregulin (SigmaeAldrich), and 0.5 mM db-cAMP for 3e4 weeks [23]. Media were changed every 2e3 days. Cells were then examined for the expression of Schwann cell protein markers by immunostaining. For mesenchymal differentiation, CmNCSCs were cultured for more than 2 weeks on uncoated tissue culture dishes in a-MEM containing 10% FBS. The differentiation potential of CmNCSC-derived MSCs (CmNCSC-MSCs) was assessed by testing their ability to differentiate into adipocytes, osteoblasts, chondrocytes and smooth muscle cells, as previously described [25]. The phenotype of CmNCSC-MSCs was assessed by FACS analysis and subsequent assay of differentiation into cells of the mesenchymal lineage (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). For chondrogenic differentiation, CmNCSC-MSCs attached in tissue culture plates were cultured in induction medium, consisting of DMEM containing 1% FBS, 10 ng/ml transforming growth factor-b3 (TGF-b3; Peprotech), 6.25 mg/ml insulin (SigmaeAldrich), 6.25 mg/ml transferrin (Life Technologies), 1.25 mg/ml bovine serum albumen (BSA; Life Technologies) and 1 mM pyruvate (SigmaeAldrich) for 3e4 weeks. Media were replaced every 3 days. Cells were then fixed with 4% paraformaldehyde (PFA) and examined by toluidine blue staining and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). For adipogenic differentiation, CmNCSC-MSCs were grown to confluence and exposed to differentiation medium containing 1 mM dexamethasone (SigmaeAldrich), 10 mg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; SigmaeAldrich) for 3 weeks. For osteogenic differentiation, CmNCSC-MSCs were plated at low density (5 103 cells/cm2) in DMEM containing 10 mM b-glycerol phosphate (SigmaeAldrich), 10 nM dexamethasone, and 200 mM AA for 21days. For smooth muscle cell differentiation, CmNCSC-MSCs (2 103 cells/cm2) were seeded onto laminin (10 ng/ml)-coated six-well plates and induced to differentiate by incubating in serum-free medium containing 2.5 ng/ml TGF-b1 (R&D Systems, Minneapolis, MN, USA) for 7 days. Media were changed every 2e3 days.
W. Li et al. / Biomaterials 39 (2015) 75e84 2.4. FACS analysis Expression of cell surface marker in CmNCSCs and CmNCSC-MSCs was determined by flow cytometry. Cells were trypsinized, harvested, and incubated with monoclonal antibodies against human antigens, including p75, HNK1, CD29, CD34, CD44, CD45, CD73, and CD166 (all from BD-Pharmingen). An irrelevant isotypeidentical antibody (BD Biosciences) served as a negative control. Samples were analyzed by collecting 10,000 events using FlowJo software (BD Biosciences). 2.5. Immunocytochemistry Undifferentiated and differentiated CmESCs, CmNCSCs, and CmNCSC-MSCs were fixed with 4% PFA at room temperature for 20 min and rinsed three times with PBS. Cells were then blocked by incubating in PBS containing 0.2% Triton X-100 and 2% BSA or goat serum at room temperature for 1 h. The following antibodies were used at the indicated dilutions (see Supplementary Table 1): anti-p75 (1:200; Promega, San Luis Obispo, CA, USA), anti-HNK1 (1:300; SigmaeAldrich), anti-Sox10 (1:50; Abcam), anti-Ap2a (1:200; DSHB, Iowa City, IA, USA), anti-Slug (1:20; Abcam, Cambridge, UK), anti-bIII Tubulin (Tubb3; 1:100; R&D Systems), anti-peripherin (1:200; SigmaeAldrich), anti-S100b (1:200; Millipore), anti-GFAP (glial fibrillary acidic protein; 1:200; Millipore), anti-Brn3a (1:200; Millipore), anti-Nestin (1:500; Abcam), anti-Pax6 (1:2; DSHB), anti-Sox2 (1:400; Millipore), anti-Vimentin (1:500; Abcam), anti- Smooth Muscle Actin alpha (aSMA). (1:1500; Abcam), anti-Neurofilament-Light (NF-L) (1:500; Millipore), anti-Ki67 (1:200; Abcam) and anti-CD31 (1:50; Abcam). After washing, cells were incubated with Alexa Fluor 488/Alexa Fluor 594-conjugated goat anti-mouse/anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h in the dark. Nuclei were counterstained with 40 ,6-diamidino-2phenylindole (DAPI) (SigmaeAldrich). Undifferentiated cells were used as negative controls. The results were analyzed by direct observation under a fluorescence microscope. Adipogenic-differentiated cells were fixed and incubated with Oil Red O (SigmaeAldrich) for 30 min to detect lipid droplets. Osteogenic-differentiated cells were fixed and incubated with Alizarin Red S (Sigma) for 20 min to detect calcium deposits. For assessment of chondrogenic differentiation, cells were fixed and then incubated with 0.1% toluidine blue for 10 min. The results were analyzed by direct observation under a light microscope. 2.6. qRT-PCR analyses Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. After digestion with DNase I (Fermentas, Glen Burnie, MD, USA), total RNA was reverse transcribed to cDNA using a Quantitect Reverse Transcription kit (Qiagen, Valencia, CA, USA), as described by the manufacturer. qPCR was performed using a DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific, Rutherford, NJ, USA) and the LightCycler 480 Detection System (Roche). DNA was amplified using the following thermocycling conditions: 40 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 10 s, and extension at 72 C for 30 s 18S rRNA served as an internal control. mRNA levels were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and changes in gene expression were calculated as fold changes using the DDCt method. Primer details are provided in Supplementary Table 2. 2.7. Time-lapse analysis and electrophysiology The migration of FACS-purified HNK1þ/p75þ CmNCSCs was monitored using a Zeiss Axio Observer Z1 microscope within 6e24 h after isolation. Cells were cultured at a constant 37 C in an enclosed chamber with CO2 control. Cell migration was quantified at 10-min intervals for 12 h using computer-assisted tracking. The migration distances of individual cells were determined and analyzed [23]. Electrophysiological recordings were performed using an EPC-10 patch-clamp amplifier; results were recorded and analyzed using Patch master V2X69 software (HEKA Electronics Gmbh, Lambrecht, Germany) [26]. The extracellular solution consisted of 128 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 15 mM HEPES (SigmaeAldrich) and15 mM glucose (SigmaeAldrich), adjusted to a pH of 7.2e7.4 and an osmolarity of 340 mOsm. The patch pipettes had resistances of 3e5 Mega Ohm after filling with a solution containing 135 mM KCl, 5 mM Na2-phosphocreatine, 2 mM EGTA, 2 mM MgATP, 0.3 mM Na2GTP and 5 mM HEPES (SigmaeAldrich), adjusted to a pH of 7.2e7.4 and an osmolarity of 315 mOsm. 2.8. In vivo transplantation Neural crest stem cells derived from CmESCs were confirmed to be functional by transplantation into the fetal brain of a non-human primate or chick embryos. CmNCSCs were dissociated using accutase (Life Technologies) and labeled with CellTracker CM-DiI (Life Technologies) according to manufacturer's protocol. For in utero transplantation, maternal cynomolgus monkeys at approximately 8e12 weeks of pregnancy were injected intramuscularly with ketamine and instilled with a 3% isoflurane mixture via an endotracheal tube [27]. Under monitoring with a B-ultrasonic diagnostic apparatus, DiI-labeled CmNCSCs were injected into the fetal brain of monkey embryos through the seam in the skull bone
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connection using laparoscopic techniques. Postnatal cynomolgus monkeys were fixed with 4% PFA by vascular perfusion. For chick embryo transplantation, fertile chicken eggs were incubated at 37 C in a humidified incubator until they reached Hamburger and Hamilton (HH) stage 10e11 [23]. A small window was made and embryos were visualized using India Ink (1:10). DiI-labeled CmNCSCs were then injected into the cranial neural tube and the intersomite space of chick embryos. Eggs were incubated for 3 days posttransplantation, and the embryos were fixed and cryosectioned. All experimental procedures involving animals were approved by the Animal Ethics Committee of Sun Yat-sen University. 2.9. Statistical analysis All results presented representative data collected from at least three independent experiments. Statistical analyses of data employed one-way analyses of variance (ANOVA). A P-value
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Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells Weiqiang Li a, b, c, 1, Lihua Huang a, 1, Wanyi Lin a, d, 1, Qiong Ke a, e, Rui Chen f, Xingqiang Lai a, Xiaoyu Wang g, Jifeng Zhang h, Meihua Jiang a, i, Weijun Huang a, Tao Wang a, Xuesong Yang g, Yuan Chen h, Wu Song j, **, Andy Peng Xiang a, b, c, * a Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong, PR China b Department of Biochemistry, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China c Key Laboratory of Reproductive Medicine of Guangdong Province, Guangzhou, Guangdong, PR China d Department of Blood Transfusion, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China e Department of Cell Biology, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China f Center for Reproductive Medicine, Key Laboratory for Reproductive Medicine of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, PR China g Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou, PR China h Center for Neurobiology, Zhongshan School of Medicine, Sun-Yat Sen University, Guangzhou, Guangdong, PR China i Department of Anatomy and Neurobiology, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong, PR China j Department of Gastrointestinal-Pancreatic Surgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2014 Accepted 19 October 2014 Available online
Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESCderived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2a, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neural crest stem cells Embryonic stem cells Differentiation Non-human primates
1. Introduction
* Corresponding author. Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun YatSen University, Guangzhou, Guangdong, PR China. Tel.: þ86 20 87335822; fax: þ86 20 87335858. ** Corresponding author. Tel./fax: þ86 20 28823389. E-mail addresses: [email protected] (W. Song), [email protected]. cn (A.P. Xiang). 1 These authors made equal contributions to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.056 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
The neural crest comprises a transient population of embryonic multipotent cells that arise from the border between the neural plate and the non-neural ectoderm at the gastrula stage and emerge from the neural tube shortly after the tube closes. Common to all vertebrate embryos, premigratory neural crest stem cells (NCSCs) can be identified by their expression of neural crestspecific genes [1], including the transcription factors Sox10 (SRY box 10), Ap2a (activating enhancer binding protein 2 alpha), Sox9, Snail (Slug), FoxD3 (forkhead box D3), and c-Myc [2]. The NCSCs
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W. Li et al. / Biomaterials 39 (2015) 75e84
then separate from the neural tube and migrate extensively throughout the embryo, a process that involves either a complete or partial epithelial-to-mesenchymal transition (EMT) [2,3]. Early migratory NCSCs migrate along defined pathways and differentiate into a wide array of derivatives, including neurons and glia of the peripheral and enteric nervous systems, bone, cartilage, smooth muscle, melanocytes in the skin, and connective tissue of the head [4]. In addition, neural crest cells are reported to be among the developmental sources of mesenchymal stem cells (MSCs), which, owing to their multipotency, paracrine action and immunomodulatory properties, have been intensively investigated for use in treating different diseases [5,6]. The neural crest also plays important roles in vertebrate developmental processes, and defective growth, differentiation, and migration of neural crest cells usually causes neurocristopathies e a large group of congenital disorders that includes Hirschsprung's disease, DiGeorge syndrome, and family dysautonomia, among others [7]. Major strides in understanding the biology of NCSCs have been made in recent years using numerous model organisms ranging from protochordates to higher vertebrates, including zebrafish, Xenopus, chicken and mouse [8e14]. Rodent NCSCs have been isolated from both fetal and adult tissue, such as gut and sciatic nerve [14,15]. However, access to gastrula-stage human embryos is limited by numerous ethical and legal questions, making studies of neural crest development in humans problematic. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the ability to differentiate into the three germ layers (including NCSCs) both in vitro and in vivo, which makes them powerful in vitro models for developmental biology studies. Derivation of NCSCs from ESCs or iPSCs enables investigation and manipulation of neural crest development and holds promise for mechanistic studies and cell-replacement therapies [16e23]. Understanding the detailed mechanisms of NCSC specification, migration, and differentiation will ultimately contribute to their use in therapeutic applications. Although rodent models play an important role in diverse aspects of biomedical research, they are poor representations of human disease phenotypes. Therefore, large animal models, especially non-human primate models, are invaluable for modeling human embryos. Furthermore, ESCs/iPSCs from non-human primates are similar to human ESCs in terms of morphology, self-renewal capacity, and pluripotency. Although the potential of human ES/iPSderived cells for cell-replacement therapy in patients with degenerative diseases is widely recognized, a thorough understanding of the growth, migration, and differentiation of these cells in vivo is a prerequisite for their clinical application. It is also important to first address questions such as tumor formation, functional capacity, and potential for immune rejection of transplanted human ES/iPSderived cells in a non-human primate model. Cynomolgus monkeys closely resemble humans in terms of anatomy, physiology, pathophysiology, and genetic background. Therefore, studies using cynomolgus monkey ESCs (CmESCs) are expected to provide important insights into the use of human ES/iPS-derived cells for human therapeutic purposes. Although avian, murine and human NCSCs have been identified, NCSCs have not yet been isolated from embryos or pluripotent stem cells of non-human primates. Here, we reported the successful isolation and propagation of NCSCs derived from CmESCs. Our in vitro characterization of CmESC-derived NCSCs showed that these cells express premigratory neural crest markers and are capable of multilineage differentiation towards neural crest lineages in vitro. In vivo, CmNCSCs transplanted into the brains of developing cynomolgus monkeys and chick embryos survived, migrated extensively, and contributed specifically to proper neural crest derivatives.
2. Materials and methods 2.1. Cell culture Undifferentiated CmESCs [24] were maintained on mitotically inactivated mouse embryonic fibroblasts in Knockout DMEM (Life Technologies, Carlsbad, CA, USA) containing 12% KnockOut Serum Replacement (Life Technologies), 8% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% non-essential amino acids (NEAA; Hyclone), 1 mM L-glutamine (Hyclone), 0.1 mM 2-mercaptoethanol (Life Technologies), 10 ng/ml basic fibroblast growth factor (bFGF; Life Technologies), 100 IU/ml penicillin (Hyclone), and 100 mg/ml streptomycin (Hyclone). The medium was changed daily, and CmESCs were passaged every 5e7 days by treating with 1 mg/ml collagenase IV (SigmaeAldrich, St Louis, MO, USA). 2.2. Derivation of NCSCs from CmESCs CmESCs were washed twice with phosphate-buffered saline (PBS) and then disaggregated into small clusters by incubating with 1 mg/ml dispase (Roche, Indianapolis, IN, USA) at 37 C for 5e10 min. For embryoid body formation, clusters were suspended in KSR medium consisting of 80% KnockOut DMEM, 20% KnockOut Serum Replacement (Life Technologies), 1 mM L-glutamine, 1% NEAA, and 0.1 mM 2mercaptoethanol. Embryoid bodies were cultured for 5 days in ultra-lowattachment culture dishes with daily changes of medium. Embryoid bodies were then replated on dishes coated with poly-L-ornithine (15 mg/ml; SigmaeAldrich) and laminin (10 mg/ml; Millipore, Temecula, CA, USA) and cultured in neural crest culture medium (NCM), consisting of DMEM/F-12 and Neurobasal medium (1:1 ratio) (both from Life Technologies), 1% N2, 2% B27, 1 mM L-glutamine, and 0.1 mM 2mercaptoethanol supplemented with 20 ng/ml EGF (epidermal growth factor) and 20 ng/ml bFGF. CmESC-derived NCSCs were labeled with antibodies against HNK1 (also known as CD57/B3GAT1) and p75 (also known as low affinity nerve growth factor receptor, NGFR) (both from BD-Pharmingen, Palo Alto, CA, USA) for fluorescence-activated cell sorting (FACS) using a BD Influx cell sorter (BD-Pharmingen). Sorted cells were replated on polyornithine/laminin-coated dishes for adherent culture (50,000e100,000 cells/cm2) or were cultured in suspension on ultra-low-attachment plates for the formation of neural crest spheres (1000e10,000 cells/cm2). 2.3. Differentiation of CmNCSCs For differentiation of CmNCSCs towards the peripheral nervous system, cells were cultured in NCM without FGF2 and EGF and supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml nerve growth factor (NGF) and 10 ng/ml neurotrophin-3 (NT3) (all from Peprotech, Rocky Hill, New Jersey, USA), as well as 200 mM ascorbic acid (AA) and 0.5 mM dibutyryl-cAMP (db-cAMP) (both from SigmaeAldrich) [23]. Cells were cultured for 2e4 weeks, and media were changed every 2e3 days. Differentiated cells were analyzed by assessing the expression of neural markers by immunocytochemistry. For Schwann cell differentiation, CmNCSCs were first cultured in NCM for at least 4 weeks. Differentiation was then induced by culturing in NCM without FGF2 and EGF and supplemented with 10 ng/ml ciliary neurotrophic factor (CNTF; Peprotech), 20 ng/ml neuregulin (SigmaeAldrich), and 0.5 mM db-cAMP for 3e4 weeks [23]. Media were changed every 2e3 days. Cells were then examined for the expression of Schwann cell protein markers by immunostaining. For mesenchymal differentiation, CmNCSCs were cultured for more than 2 weeks on uncoated tissue culture dishes in a-MEM containing 10% FBS. The differentiation potential of CmNCSC-derived MSCs (CmNCSC-MSCs) was assessed by testing their ability to differentiate into adipocytes, osteoblasts, chondrocytes and smooth muscle cells, as previously described [25]. The phenotype of CmNCSC-MSCs was assessed by FACS analysis and subsequent assay of differentiation into cells of the mesenchymal lineage (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). For chondrogenic differentiation, CmNCSC-MSCs attached in tissue culture plates were cultured in induction medium, consisting of DMEM containing 1% FBS, 10 ng/ml transforming growth factor-b3 (TGF-b3; Peprotech), 6.25 mg/ml insulin (SigmaeAldrich), 6.25 mg/ml transferrin (Life Technologies), 1.25 mg/ml bovine serum albumen (BSA; Life Technologies) and 1 mM pyruvate (SigmaeAldrich) for 3e4 weeks. Media were replaced every 3 days. Cells were then fixed with 4% paraformaldehyde (PFA) and examined by toluidine blue staining and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). For adipogenic differentiation, CmNCSC-MSCs were grown to confluence and exposed to differentiation medium containing 1 mM dexamethasone (SigmaeAldrich), 10 mg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; SigmaeAldrich) for 3 weeks. For osteogenic differentiation, CmNCSC-MSCs were plated at low density (5 103 cells/cm2) in DMEM containing 10 mM b-glycerol phosphate (SigmaeAldrich), 10 nM dexamethasone, and 200 mM AA for 21days. For smooth muscle cell differentiation, CmNCSC-MSCs (2 103 cells/cm2) were seeded onto laminin (10 ng/ml)-coated six-well plates and induced to differentiate by incubating in serum-free medium containing 2.5 ng/ml TGF-b1 (R&D Systems, Minneapolis, MN, USA) for 7 days. Media were changed every 2e3 days.
W. Li et al. / Biomaterials 39 (2015) 75e84 2.4. FACS analysis Expression of cell surface marker in CmNCSCs and CmNCSC-MSCs was determined by flow cytometry. Cells were trypsinized, harvested, and incubated with monoclonal antibodies against human antigens, including p75, HNK1, CD29, CD34, CD44, CD45, CD73, and CD166 (all from BD-Pharmingen). An irrelevant isotypeidentical antibody (BD Biosciences) served as a negative control. Samples were analyzed by collecting 10,000 events using FlowJo software (BD Biosciences). 2.5. Immunocytochemistry Undifferentiated and differentiated CmESCs, CmNCSCs, and CmNCSC-MSCs were fixed with 4% PFA at room temperature for 20 min and rinsed three times with PBS. Cells were then blocked by incubating in PBS containing 0.2% Triton X-100 and 2% BSA or goat serum at room temperature for 1 h. The following antibodies were used at the indicated dilutions (see Supplementary Table 1): anti-p75 (1:200; Promega, San Luis Obispo, CA, USA), anti-HNK1 (1:300; SigmaeAldrich), anti-Sox10 (1:50; Abcam), anti-Ap2a (1:200; DSHB, Iowa City, IA, USA), anti-Slug (1:20; Abcam, Cambridge, UK), anti-bIII Tubulin (Tubb3; 1:100; R&D Systems), anti-peripherin (1:200; SigmaeAldrich), anti-S100b (1:200; Millipore), anti-GFAP (glial fibrillary acidic protein; 1:200; Millipore), anti-Brn3a (1:200; Millipore), anti-Nestin (1:500; Abcam), anti-Pax6 (1:2; DSHB), anti-Sox2 (1:400; Millipore), anti-Vimentin (1:500; Abcam), anti- Smooth Muscle Actin alpha (aSMA). (1:1500; Abcam), anti-Neurofilament-Light (NF-L) (1:500; Millipore), anti-Ki67 (1:200; Abcam) and anti-CD31 (1:50; Abcam). After washing, cells were incubated with Alexa Fluor 488/Alexa Fluor 594-conjugated goat anti-mouse/anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h in the dark. Nuclei were counterstained with 40 ,6-diamidino-2phenylindole (DAPI) (SigmaeAldrich). Undifferentiated cells were used as negative controls. The results were analyzed by direct observation under a fluorescence microscope. Adipogenic-differentiated cells were fixed and incubated with Oil Red O (SigmaeAldrich) for 30 min to detect lipid droplets. Osteogenic-differentiated cells were fixed and incubated with Alizarin Red S (Sigma) for 20 min to detect calcium deposits. For assessment of chondrogenic differentiation, cells were fixed and then incubated with 0.1% toluidine blue for 10 min. The results were analyzed by direct observation under a light microscope. 2.6. qRT-PCR analyses Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. After digestion with DNase I (Fermentas, Glen Burnie, MD, USA), total RNA was reverse transcribed to cDNA using a Quantitect Reverse Transcription kit (Qiagen, Valencia, CA, USA), as described by the manufacturer. qPCR was performed using a DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific, Rutherford, NJ, USA) and the LightCycler 480 Detection System (Roche). DNA was amplified using the following thermocycling conditions: 40 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 10 s, and extension at 72 C for 30 s 18S rRNA served as an internal control. mRNA levels were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and changes in gene expression were calculated as fold changes using the DDCt method. Primer details are provided in Supplementary Table 2. 2.7. Time-lapse analysis and electrophysiology The migration of FACS-purified HNK1þ/p75þ CmNCSCs was monitored using a Zeiss Axio Observer Z1 microscope within 6e24 h after isolation. Cells were cultured at a constant 37 C in an enclosed chamber with CO2 control. Cell migration was quantified at 10-min intervals for 12 h using computer-assisted tracking. The migration distances of individual cells were determined and analyzed [23]. Electrophysiological recordings were performed using an EPC-10 patch-clamp amplifier; results were recorded and analyzed using Patch master V2X69 software (HEKA Electronics Gmbh, Lambrecht, Germany) [26]. The extracellular solution consisted of 128 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 15 mM HEPES (SigmaeAldrich) and15 mM glucose (SigmaeAldrich), adjusted to a pH of 7.2e7.4 and an osmolarity of 340 mOsm. The patch pipettes had resistances of 3e5 Mega Ohm after filling with a solution containing 135 mM KCl, 5 mM Na2-phosphocreatine, 2 mM EGTA, 2 mM MgATP, 0.3 mM Na2GTP and 5 mM HEPES (SigmaeAldrich), adjusted to a pH of 7.2e7.4 and an osmolarity of 315 mOsm. 2.8. In vivo transplantation Neural crest stem cells derived from CmESCs were confirmed to be functional by transplantation into the fetal brain of a non-human primate or chick embryos. CmNCSCs were dissociated using accutase (Life Technologies) and labeled with CellTracker CM-DiI (Life Technologies) according to manufacturer's protocol. For in utero transplantation, maternal cynomolgus monkeys at approximately 8e12 weeks of pregnancy were injected intramuscularly with ketamine and instilled with a 3% isoflurane mixture via an endotracheal tube [27]. Under monitoring with a B-ultrasonic diagnostic apparatus, DiI-labeled CmNCSCs were injected into the fetal brain of monkey embryos through the seam in the skull bone
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connection using laparoscopic techniques. Postnatal cynomolgus monkeys were fixed with 4% PFA by vascular perfusion. For chick embryo transplantation, fertile chicken eggs were incubated at 37 C in a humidified incubator until they reached Hamburger and Hamilton (HH) stage 10e11 [23]. A small window was made and embryos were visualized using India Ink (1:10). DiI-labeled CmNCSCs were then injected into the cranial neural tube and the intersomite space of chick embryos. Eggs were incubated for 3 days posttransplantation, and the embryos were fixed and cryosectioned. All experimental procedures involving animals were approved by the Animal Ethics Committee of Sun Yat-sen University. 2.9. Statistical analysis All results presented representative data collected from at least three independent experiments. Statistical analyses of data employed one-way analyses of variance (ANOVA). A P-value
