Isolation and propagation of neural crest stem cells from mouse embryonic stem cells via cranial neurospheres.

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Stem Cells and Development Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres (doi: 10.1089/scd.2014.0152) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1

Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres

Yuki Minamino,1,2 Yuichi Ohnishi, 1,2 Kenji Kakudo,1 and Masami Nozaki2

1

Second Department of Oral and Maxillofacial Surgery, Osaka Dental University,

1-5-17 Otemae, Chuo-ku, Osaka 540-0008, Japan. 2

Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita,

Osaka 565-0871, Japan.

Yuki Minamino, Tel; 81-6-6910-1510, Fax; 81-6-6910-1030, E-mail; [email protected] Yuichi Ohnishi, Tel; 81-6-6910-1510, Fax; 81-6-6910-1030, E-mail; [email protected] Kenji Kakudo, Tel; 81-6-6910-1510, Fax; 81-6-6910-1030, E-mail; [email protected] Masami Nozaki, Tel and Fax; 81-6-6879-8339, E-mail; [email protected]

Running title: Cranial neural crest cells from ES cells

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Corresponding author; Masami Nozaki, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel and Fax; 81-6-6879-8339, e-mail; [email protected]

Abbreviations NC; neural crest NIM; neural induction medium NIM+KG; knockout serum replacement and L-glutamine supplemented to NIM

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Abstract

The developmental fate of the multipotent neural crest (NC) is determined along with the neural axis in which NC cells are generated. Only the cranial NC can differentiate to mesectodermal derivatives such as osteoblasts, chondrocytes, and adipocytes in vivo. Here, we attempted to selectively differentiate mouse embryonic stem (ES) cells into cranial NC stem cells and propagate them to explore their developmental potential to differentiate into mesectodermal derivatives. Using aggregation cultures in feeder- and serum-free neural induction medium (NIM) without serum replacement and L-glutamine, we obtained NIM neurospheres composed of neuroepithelium. The NIM neurospheres expressed the rostral markers Otx1 and Otx2, but not non-rostral markers Hoxb4, Hoxb9, Lbx1, and TH, which characterize cranial neurospheres. Subsequently, AP2α, Sox9, p75, Snail, Slug, and Twist-positive NC cells were differentiated in 4-day adhesion cultures of cranial neurospheres. Additionally, sphere clusters in adhesion cultures were differentiated into osteoblast while migrating cells were not. By taking advantage of the sphere-formation capability, we isolated and propagated NC stem cells from the sphere clusters and confirmed their multipotency. NC stem cells expressed NC and stem cell markers, and maintained differentiation potency in the NC derivatives. These results show that cranial NC stem cells were obtained reproducibly and efficiently without special inducing factors, gene transfection or fluorescence-activated cell sorting (FACS) selection.

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Introduction

In the vertebrate embryo, the neural crest (NC) is composed of a transient multipotent cell population that arises at the border of the nonneural ectoderm and the neural plate. NC cells migrate extensively throughout the embryo and give rise to neurons and glia in the peripheral nervous system, skin melanocytes, smooth muscle cells, adipocytes, craniofacial bone, and cartilage [1,2]. The developmental fate of NC cells is determined based on their position within the anteroposterior neural axis, which can be divided into two groups, the cranial and trunk [3]. NC cells at a given axial level follow distinct migratory routes and differentiate into a predictable array of progeny at their final destination [4-6]. Studies investigating the mechanisms of lineage progression and disease pathogenesis have shown that the majority of cranial NC cells contribute to the morphogenesis of the craniofacial region including the peripheral nervous system and connective tissue of the cranial muscles, adipocytes, chondrocytes, osteoblasts, and odontoblasts [7,8]. A subpopulation of posterior cranial NC cells, called the cardiac NC cells, contributes to the cardiac outflow tract [9]. The NC cells in the trunk give rise to limited cell types including peripheral nerve cells, melanocytes, and chromaffin cells. Transplantation experiments in the amniotic vertebrate have suggested that the trunk NC could be scarcely differentiated into cranial NC derivatives [10], although avian trunk NC cells were differentiated into chondrocyte by long-term in vitro cultures [11,12]. Moreover, when mouse trunk NC cells were recombined with branchial arch 1 epithelium, they differentiated into dentin and bone [13]. Trunk NC cells were

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differentiated into mesenchymal derivatives in cultures with specific factors (14-16). These data suggest that differences in developmental potency between the cranial and trunk NC cells depend on the environmental signals. To clarify when and how the differences of developmental potency are established between two segments of the NC, analyzing their undifferentiated NC precursor or stem cells, respectively, is necessary. Several studies have reported that mammalian multipotent migratory trunk NC cells were isolated from the neural tube in the rat embryo [17]. Recently, a cranial NC cell line was isolated from the mouse embryo [18]. In mammals, some NC cells having characteristics that resemble those in embryonic NC cells are retained after a period of organogenesis and in the adult tissues [19]. Therefore, part of the stem cells isolated from dental pulp and the periodontal ligament may be derived from cranial NC stem cells [20,21]. However, whether these stem cells are necessarily equivalent to embryonic NC precursor or stem cells has not been confirmed. Therefore, the derivation of precursor/stem cells of the cranial and trunk NC from embryonic stem (ES) cells might be effective in elucidating the determination of NC fate. ES cells can be used to analyze early events in mammalian development because of their pluripotency and ability to recapitulate the developmental process in vitro. In addition, ES cells are capable of proliferating indefinitely in vitro, and that ability can facilitate biochemical analyses. Moreover, ES cells are useful in a variety of gene analyses because they can easily undergo gene transfection. To differentiate ES cells into NC cells, one must first differentiate the neuroepithelium. Because embryoid bodies (EBs) formed in culture medium with serum tend to differentiate into three germ

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layers consisting of nonneural cells, fluorescence-activated cell sorter (FACS) is necessary to obtain a small number of NC cells in EBs [22]. A single-step method for the generation of NC-like stem cells from human ES and iPS cells in chemically defined media was reported (23). This approach is efficient for NC differentiation via neuroepithelium, but region specificity of the NC cells was not elucidated. In addition, several methodologies for deriving neuroepithelium from ES cells with high efficiency have been developed.

These protocols are based on stromal-derived inducing activity

(SDIA) and serum-free suspension (SFEB) cultures, which can selectively differentiate into a nervous system that alters the rostral/caudal or dorsoventral axes by the addition of a variety of inducing factors [24-27].

In particular, neurosphere culture [28] is

suited for analyzing the process of NC differentiation from the neuroepithelium of a specific region because of the serum-free culture withdrawal of stromal feeder cells. In this study, we showed that NC cells were differentiated from mouse ES cells via neurospheres that had rostral characteristics under feeder- and serum-free conditions, without serum replacement and L-glutamine supplement. In addition, we isolated and propagated cranial NC-like stem cells using sphere formation as a functional approach in stem cell isolation. These NC cells tended to differentiate into mesenchymal derivatives including osteoblasts and adipocytes.

Materials and Methods

Cell culture

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Mouse ES (mES) cells (E14; ATCC, Manassas, VA) were maintained without feeders in ES cell medium consisting of Grasgow’s modified Eagle’s medium (GMEM) (Sigma-Aldrich) supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 1× nonessential amino acids, 0.1 mM 2-mercaptoethanol, 100 U/ml penicilin, 100 μg/ml streptomycin (all from Invitrogen), and 1000 U of leukemia inhibitory factor (LIF, ESGRO; Millipore) on 0.1% gelatin-coated 25-cm2 plastic flasks (Corning) at 37℃ in a humidified atmosphere with 5% CO2. The medium was changed every day, and the cells were passaged every 2 days. For sphere formation to neuroepithelial induction, mES cells were dissociated using 0.25% trypsin-EDTA to single cells and seeded in 100-μl of serum-free neural induction medium [NIM; 1:1 ratio of Dulbecco’s MEM (DMEM)-F12 and neurobasal medium supplemented with 0.5× N2, 0.5× B27, 20 ng/ml insulin, 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), and 100 U/ml penicilin and 100 μg/ml streptomycin; all from Invitrogen] in low cell-adhesion wells of 96-well plates (Sumilon Spheroid plates; Sumitomo). For NC differentiation, NIM neurospheres were transferred to a 24-well plate coated with 50 μg/ml fibronectin in serum-free medium consisting of αMEM supplemented with 15% Serum Replacement 3 (Sigma), 2 mM L-glutamine, 1× nonessential amino acids, 100 U/ml penicilin, 100 μg/ml streptomycin, and 0.1 mM 2-mercaptoethanol with or without 100 ng/ml bone morphogenetic protein 4 (BMP4; PROSPEC). For NC cell expansion, NC cells were cultured on fibronectin-coated plates in the expansion medium consisting of DMEM/F12

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supplemented with Serum Replacement 3, 10 µg/ml bFGF, 50 µg/ml EGF, 1 mg/ml Fibronectin and 100 ng/ml Heparin. For differentiation to NC derivatives, NC cells were cultured on fibronectin-coated plates in the respective differentiation media. For osteogenic differentiation, NC cells were cultured in osteogenic medium consisting of αMEM with 15% FBS, 2 mM L-glutamine, 1× nonessential amino acids, 100 U/ml penicilin, 100 μg/ml streptomycin, 0.1 mM 2-mercaptoethanol, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10-7 M dexamethasone for 3-4 weeks. For adipocyte differentiation, NC cells were cultured in adipogenic medium consisting of αMEM with 10% FBS, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 10 μg/ml insulin for 2 weeks.

For chondrocyte differentiation, NC cells were densely cultured in

chondrogenic medium consisting of 200 μM ascorbic acid, 10 ng/ml TGFβ3 in αMEM containing 10% FBS for 4 weeks. For myocyte differentiation, NC cells were cultured in myogenic medium consisting of a 1:1 ratio of DMEM/F12 and neurobasal medium supplemented with 1 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 2.5× B27, 10% knockout serum replacement (GIBCO) and 1 ng/ml TGFβ3. For neural differentiation, NC cells were cultured in neural differentiation medium no.1 consisting of 10% chicken embryo extract, 20 ng/ml bFGF, 1× N2, 1× B27 (-vitamine A), 50 μM 2-mercaptomethanol, 35 ng/ml retinoic acid, 25 U/ml penicillin and 25 µg/ml streptomycin in DMEM-low glucose. After 4 days culture, the medium was changed to the neural differentiation medium no.2, consisting of 1% chicken embryo extract and 10 ng/ml bFGF in neural differentiation medium no.1.

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Immunostainning

Cells were fixed with 3.5% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized using 0.2% TritonX-100 for 5 min and blocked with Image-iTTM FX Signal Enhancer (Invitrogen) for 30 min. Spheres were fixed with 3.5% paraformaldehyde and embedded in paraffin. The 6-μm sections were deparaffinized in xylene, and rehydrated in alcohol and deionized water. Primary antibodies (Supplementary Table 1) were applied and allowed to react for overnight at 4℃. Cells and sections were then washed three times with PBS, and incubated for 1 h at room temperature using Alexa Fluor 488 conjugated secondary antibodies. Cells and sections were washed three times and incubated with DAPI to label the cell nuclei. Images of immunostaining were captured using a fluorescence microscope (DMRXE; Leica) or confocal microscope (FV10i; Olympus).

Histochemistry

To assess the osteogenic, chondrogenic, and adipogenic differentiation, we stained the cells with Alizarin Red S, Alcian Blue, and Oil Red O, respectively. For Alizarin Red S staining, cells were fixed in 3.5% paraformaldehyde for 10 min at room temperature, washed three times with PBS and H2O, and incubated for 15 min with a 2% Alizarin Red S (Sigma-Aldrich). For Alcian Blue staining, specimens were fixed in 3.5% paraformaldehyde for 10 min at room temperature, washed three times with PBS

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and H2O, and subsequently incubated with an Alcian Blue stain solution (pH 2.5; Nacalai Tesque) for 24 h and washed with dH2O several times. For Oil Red O staining, cells were fixed with 3.5% paraformaldehyde for 10 min at room temperature, equilibrated with 60% isopropyl alcohol (IPA), incubated with 0.18% Oil Red O solution in 60% IPA and then washed in 60% IPA to eliminate excess staining.

Reverse transcription-polymerase chain reaction (RT-PCR) assay

Total RNA was extracted from the cultured cells using TRIzoL (Invitrogen), and 500 ng of total RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) following the manufacturer’s instructions. PCR analyses were performed using a standard protocol. Primer sequences, product length, and annealing temperatures are

provided

(Supplementary Table

2).

The

amplification

fragments

were

electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The expression level of each sample was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression.

Results

Neuroepithelium differentiation from undifferentiated mouse ES cells

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The NC arises on the dorsal neural tube during the early stage of vertebrate embryonic development [29]. Thus, we first differentiated mouse ES cells into neuroepithelial cells prior to carrying out neural crest differentiation. ES cell aggregates in serum- and feeder-free cultures have been found to be susceptible to differentiation into neuroepithelium; however, large-sized aggregates tend to develop cavities resulting in increased cell death and generation of nonneural lineages [30]. We examined the appropriate aggregate size for efficient neural differentiation; however, controlling the sphere size in suspension culture on low cell-adhesion culture dishes coated with polyhydroxyl-ethyl-methacrylate (Poly-Hema) or EZ-BindShut dishes (Asahi Glass Co. Ltd.) was difficult. We attempted to control the size of cell aggregates depending upon the number of seeding cells using low cell-adhesion U shape-bottomed 96-well plates following the method of Eiraku et al. [31]; 1 × 103, 2 × 103, and 5 × 103 of dissociated ES cells were seeded and cultured in NIM on the plates. Aggregate size could be reproductively controlled by the number of cells used (Fig. 1A).

These aggregates were collected, and the RNA was extracted and

examined for neuroepithelial differentiation using RT-PCR (Fig. 1B). On day 6 of culture, expression levels of Nestin, neural cell adhesion molecule (Ncam), Evi3/Zfp521, and Sox1 in aggregates generated from 1 × 103 of ES cells were higher than those from 2 × 103 and 5 × 103 of ES cells. In addition, the expression of the mesoderm marker Brachyury was detected in larger aggregates from 2 × 103 and 5 × 103 cells, but not in smaller aggregates from 1 × 103 cells. The localization of marker proteins in 1 × 103 and 5 × 103 aggregates was examined by immunohistochemical staining (Fig. 1C). The

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neuroepithelial marker Nestin was detected in most cells in both the 1 × 103 and 5 × 103 aggregates. Colonization of the undifferentiated cell marker E-cadherin was detected in 5 × 103 cell aggregates but not in 1 × 103 aggregates. Rosette-like structures were observed in culture cells in which the apical side was densely stained by N-cadherin in 1 × 103 aggregates. However, such rosette-like structures were not observed in 5 × 103 aggregates. A small number of cells in which Brachyury was detected was observed in 5 × 103 aggregates but not in 1 × 103 aggregates. These results (summarized in Table 1) suggest that ES cells were induced to differentiate into neuroepithelium by cell aggregation using a low cell-adhesion U-shaped 96-well plates. Furthermore, it is critical to make small aggregates of 1000 cells for efficient neuralization, because larger aggregates tended to be induced to multiple cell types and undifferentiated cells were remained. During the growth of 1 × 103 aggregates in NIM, we analyzed the expression of undifferentiated ES cells, neuroepithelium, mesoderm, and endoderm markers (NIM in Fig. 2A). The undifferentiated ES cell markers Nanog and Oct3/4 were expressed in day 3 aggregates, but not in day 6 aggregates. The neuroectodermal markers Evi3/Zfp521, Nestin, Sox1 and Ncam, were detected at low levels in day 3 aggregates, which were upregulated in day 6 aggregates. Furthermore, the dorsal marker Pax3 was clearly detected in day 6 aggregates, whereas the ventral marker Nkx2.2 was not. These data indicate that 1 × 103 ES cell aggregates cultured in NIM converted neurospheres containing dominant dorsal neuroepithelium. In this report, the aggregates cultured in NIM are referred to as NIM neurospheres.

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Several studies have demonstrated differentiation of ES cells to neuroepithelium using feeder- and serum-free suspension culture methods, in which the media were supplemented with L-glutamine and serum replacement [28,31] or not [30, present report]. Thus, we investigated the effects of adding knockout serum replacement and L-glutamine supplemented to NIM (NIM+KG) on the differentiation into neurospheres (NIM+KG in Fig. 2A).

The expression levels of Evi3/zfp521, Nestin, Sox1, and Ncam

were detected in day 3 and day 6 culture with NIM+KG and in NIM cultured neurospheres. However, the levels of neural markers were not upregulated in day 6 cultures with NIM+KG, and NIM+KG-cultured spheres expressed Nkx2.2 and Pax3, indicating that the spheres in both culture media differentiated into neuroepithelium, but NIM+KG-cultured spheres were not restricted to the dorsal neuroepithelium. In addition, the undifferentiated markers Nanog and Oct3/4, mesoderm markers Brachyury and Mox1, and endoderm marker GATA6 were detected in day 6 NIM+KG-cultured spheres. These results suggest that aggregates under the NIM supplemented with knockout serum replacement and L-glutamine maintain undifferentiated cells; they differentiate not only to neuroepithelium regardless of dorsoventral side, but also to the endodermal and mesodermal cells. Therefore, immunostaining was performed to investigate the localization of the differentiation marker proteins (Fig. 2B). The undifferentiated markers Oct3/4 and E-cadherin were detected in small clusters of cells.

Most cells

expressed N-cadherin and Nestin. Brachyury was detected in a small number of cells in NIM+KG-cultured spheres. We next examined the rostral/caudal identity of neuroepithelium in NIM-cultured

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neurospheres using RT-PCR. The rostral markers Otx1 and Otx2 were detected, but the caudal markers Hoxb4, Hoxb9, and Lbx1 and the midbrain and hindbrain marker tyrosine hydroxylase (TH) were not detected (Fig.2C), suggesting that the rostral neuroepithelium was selectively differentiated in NIM-cultured neurospheres. The NIM+KG spheres were positive not only for Otx2 but also for Hoxb4 and Hoxb9 (Fig.2C), which suggested that they differentiated into neuroepithelium without the deflection of the rostral/caudal axis. However, the NIM+KG-cultured spheres did not express Otx1 and Lbx1, which were detected in caudalized spheres treated by the caudalizing factor retinoic acid (RA) (25) (Fig.2C), suggesting that the rostralization and caudalization in the NIM+KG-cultured spheres was insufficient. These data indicate that the cranial neural cell spheres can be differentiated in serum-free defined medium without serum replacement and L-glutamine.

Neural crest induction from NIM neurospheres

The mRNA expression of neural crest markers in the NIM neurospheres was analyzed by RT-PCR. AP2α, Sox9, p75, and Snail were expressed at low levels in day 6 NIM-cultured neurospheres. High-level expression of Slug and Twist was detected in day 6 NIM-cultured neurospheres (Fig. 3A). These results suggest that day 6 NIM-cultured neurospheres from ES cells largely contained neuroepithelium with a small number of NC-like cells. Several growth factors including BMPs have been shown their potential roles in NC

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development [32]. To generate larger quantities of NC cells from NIM-cultured neurospheres, we transferred day 6 NIM-cultured neurospheres to fibronectin-coated plates and cultured them in the BMP4 containing serum-free medium for 4-5 days. Neurospheres were attached in a single day; the tegmental cells emigrated, and the spheres remained as clusters in central parts. To confirm NC differentiation of plated spheres, we examined the expression of NC markers using RT-PCR and immunostaining. RT-PCR analysis showed the upregulation of NC markers in the NIM-cultured neurospheres after the 4-day culture with BMP4 (day 10 neurospheres; Fig. 3A). The NC marker AP2α-positive cells increased in the cultured cells after BMP4 treatment based on immunostaining (Fig 3B, C). These data indicate that the NC cells from the NIM-cultured neurospheres consisting of cranial neuroepithelium were induced in day 10 neurospheres. The craniofacial skeleton originates mainly from the cranial NC. The possibility exists that NC cells from the cranial neurospheres are cranial NC cells that tend to be osteoblasts. To examine this possibility, we tested whether day 10 neurospheres could differentiate into osteoblasts. After 4 weeks culture of the day 10 neurospheres in osteogenic medium, only sphere clusters were positive for Alizarin Red S staining, and emigrating cells were not (Fig.4A). Moreover, induced NC cells from the NIM-cultured neurospheres in medium without BMP4 also differentiated into Alizarin Red S-positive clusters (Fig.4B). During osteogenesis, molecular markers, collagen I and Runx II were upregulated (Fig. 4C). Next, we mechanically separated the adherent spheres from the migratory cells after BMP4 treatment. The adherent spheres and migratory cells were

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dissociated by trypsinization, and each group of cells was analyzed. NC markers Sox9 and AP2α were expressed in both cell populations. Cells from adherent spheres expressed Pax7 and FoxD3. Migratory cells expressed Pax7, but not FoxD3 (Suppl Fig. 1). Furthermore, both groups of cells were cultured in osteogenic medium under high-density monolayer culture conditions. The cells derived from spheres formed nodules, which were stained with Alizarin Red S (Fig.4D), whereas migratory cells were not (Fig.4E). In these experiments, almost sphere clusters and nodule-like cell aggregates were differentiated to Alizarine Red S positive, but spreading mono-layer cells were not. These results suggested that the cells within day 10 neurospheres differentiated into NC cells and cranial NC cells mainly existed in adherent spheres.

Isolation and expansion of NC stem cells from ES derived neurospheres

We attempted to isolate NC stem cells in day 10 neurospheres, and subsequently propagate and differentiate them to various NC derivatives. Day 10 neurospheres were trypsinized and the dissociated cells were expanded in expansion medium. We next determined whether these cells were able to give rise to a variety of NC derivatives. We confirmed that the expanded NC cells differentiated into adipocytes, smooth muscle cells, chondrocytes, and osteoblasts (Suppl Fig. 2). These data indicate that day 10 neurospheres contain NC stem cells, which possess multipotentialities. The isolation of stem cells from various tissues and tumors has been achieved using flow cytometry [33-35] or sphere formation assays based on the properties of the stem

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cells [36]. In these experiments, we isolated NC stem cells from expanded NC cells using their sphere-formation ability under the cultivation of defined serum-free medium with growth factors. Spheres from single cells of expanded NC cells were transferred to low adhesion U-shaped-bottomed 96-well plates and cultured for 2 weeks. Dissociated cells from 14 spheres were cultured on fibronectin-coated 96-well plate; nine clones were expanded successfully and the ability to differentiate to osteoblasts was examined. Two of the nine expanded clones (clones #11 and #14) that differentiated into Alizarin Red S-positive osteoblasts were analyzed for NC stem cell characteristics. The expression of NC markers AP2α, Sox9, p75, Snail, Slug, and Twist and those of stem cell markers Nanog, Oct3/4, and Sox2 were detected in clone #11 (Fig. 5A), showing that clone #11 possessed characteristics of NC stem cells. Furthermore, clone #11 could be clonally amplified and maintained for >30 passages, and was differentiated into peripherin-positive peripheral neurons, Pparγ-, and Oil Red O-positive adipocytes, smooth muscle actin-positive myocytes, Alcian Blue-positive chondrocytes, and Alizarin Red S-positive osteoblasts (Fig. 5B-J).

These data indicate that we

successfully established NC stem cell lines from ES cell differentiation.

Discussion

In this study, we demonstrated that cranial NC cells were efficiently differentiated from mouse ES cells under culture conditions, in which ES cell aggregates were preferentially induced to cranial neuroepithelium in serum-free medium. In this

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procedure, the aggregate size was reproducibly dependent upon the number of cells seeded into each well of the low-adhesive U-shaped 96-well plates. Spheres formed from 1 × 103 ES cells selectively promoted neuralization, in which Nestin, N-CAM, Evi3, and Sox1 were expressed. In larger-size aggregates, neuralization of ES cells was suppressed and mesoderm differentiation was promoted. These results are consistent with a previous study [30] reporting that the formation of small aggregates consisting of a small number of cells is associated with the efficient neuralization of ES cells. In this report, trypsin-dissociated undifferentiated ES cells were reaggregated in each well of the U-shaped low cell-adhesion 96-well plates; a previous report selectively used aggregates composed of 50-100 cells in floating culture. Although, these reports differ markedly in the initial seeding number of cells, ES cell aggregates consisting of less than 1 × 103 cells may be prone to differentiate to neuroepithelium. Another study reported that the majority of neurospheres composed of Bf1+ telencephalic progenitors were formed using 3 × 103 cells under the same culture system using low cell-adhesion 96-well plates [31]. Based on our results, these neurospheres might tend to differentiate into nonneural cells because their neurospheres are composed of a comparatively large cell count and cultured in medium with serum replacement and L-glutamine. However, the authors added Dkk-1 and Lefty-1 to the culture medium to suppress differentiation of nonneuronal cells in aggregate for suppressing Wnt and TGFβ signaling, which impedes neural differentiation. Therefore, the primary culture conditions in our study were conductive to growth of neurospheres rich in neural cells in medium without drug additives.

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In this study, the ES cell-derived NIM neurospheres expressed the neuroepithelial markers Nestin, Sox1, and N-CAM and the rostral markers Otx1 and Otx2, but not the caudal markers Hoxb4, Hoxb9, and Lbx; this profile suggests that the cranial neuroepithelium consisted of NIM-cultured neurospheres. In addition, the dorsal neuroepithelial marker Pax3 [37] was detected in NIM neurospheres, but the ventral neuroepithelial marker Nkx2.2 [38] was not. Taken together, these findings indicated that NIM-cultured neurospheres differentiated into the dorsal side of the cranial neuroepithelium. Developmentally, the cranial NC arises from the dorsal side of the cranial neuroepithelium and migrates extensively throughout the head; then, they differentiate into mesectoderm derivatives [39] including osteoblasts and chondrocytes to contribute to the morphogenesis of the maxillofacial region, which are not derived from the trunk NC in vivo. In this study, NC cells derived from NIM-cultured neurospheres consisted of the dorsal side of the cranial neuroepithelium and differentiated to osteoblasts. These results indicate that the cranial NC cells were selectively obtained. As shown in Fig. 2, RA treatment suppressed the rostral markers Otx1 and Otx2, whereas it induced caudal markers Hoxb4, Hoxb9, Lbx1 and TH in neurospheres. In addition, NC cells were generated from RA-treated caudalized neurospheres. NC markers expressed in NC cells from RA-treated caudalized spheres were distinct from those in NC cells from NIM-cultured neurospheres, indicating that region-specific NC cells may be obtained under appropriate culture conditions. NIM-cultured neurospheres expressed low levels of NC markers Sox9 and p75.

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Histological and immunohistochemical analyses of the neurospheres showed that the neural tube like structures with apical/basal polarity formed in the spheres, in which most cells were Nestin- and N-CAM-positive (Fig. 1C). In addition, the neurospheres showed highly expression of Slug and Twist (Fig.3A), which contribute to the epithelial-mesenchymal transition. Thus, part of the NC cells may have arisen from already epithelialized neural cells. Moreover, the NC markers AP2α, Sox9, and p75 were upregulated markedly after 4 days adhesion culture of NIM-cultured neurospheres on plates, showing that NC cells were differentiated. The neural tube like structures in the NIM-cultured neurospheres are considered to be disorganized in adhesion cultures, resulting in the promotion of the neuroepithelium to the epithelial-mesenchymal transition; thus, NC cells are induced from neural cells. Some emigrating cells from neurospheres that attached on the fibronectin-coated plates differentiated into Sox10-positive emigrating NC cells [28]. Replated sphere clusters without migrating cells gave rise to Sox10-positive emigrating NC cells again [28]. Here, we showed that osteogenesis that occurred within the sphere cluster of the NIM-cultured neurospheres formed nodules and that osteogenesis took place under osteogenic-dense culture conditions; however, emigrating cells from the NIM-cultured neurospheres expressed NC markers AP2α and Sox9, but did not differentiate to osteoblast. These results indicate that the premigratory NC stem cells present in the clusters derived from the NIM-cultured neurospheres tended to differentiate into osteoblasts, whereas migrating NC cells from the NIM-cultured neurospheres tended to differentiate into various NC derivatives other than osteoblasts.

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In this study, we attempted to propagate NC stem cells in clusters from the NIM-cultured neurospheres, which likely consist of premigratory NC stem cells. Alhough previous studies reported propagation of NC stem cells derived from ES cells via the neuroepithelium [28,40,41], most studies isolated NC stem cells from emigrating cells using FACS for the putative stem cell markers p75 and HNK1. In contrast, we succeeded in propagating cranial NC stem cells from premigratory NC cells in cranial neuroepithelium-rich clusters by taking advantage of their sphere-formation capability to assess the stem cell potential in combination with the osteogenic potential for assessment of NC differentiation. In this study, we developed a system for reproducible differentiation and propagation of cranial NC stem cells. Because cranial NC stem cells tend to differentiate into mesenchymal derivatives, this system could be a useful tool for elucidating the pathogenesis of NC disorders and developing ways to treat diseases with poorly characterized differentiation processes in osteogenesis and adipogenesis.

Acknowlegements Fundings for this study was from the Osaka University and Osaka Dental University.

Disclosure statement The authors declare no potential conflicts of interest to the authorship and/or publication of this article.

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20. Coura GS, RC Garcez, CB de Aguiar, M Alvarez-Silva, RS Magini and AG Trentin. (2008). Human periodontal ligament: a niche of neural crest stem cell. J Periodont Res 43:531-536.

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31. Eiraku M, K Watanabe, M Matsuo-Takasaki, M Kawada, S Yonemura, M Matsumura, T Wataya, A Nishiyama, K Muguruma and Y Sasai. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3:519-532.

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Stem Cells and Development Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres (doi: 10.1089/scd.2014.0152) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 29 of 47

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Figure legends


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Figure 1. Differentiation of aggregates from various numbers of cells (1 × 10 3, 2 × 103, and 5 × 103) of mouse embryonic stem (ES) cells. (A) Phase-contrast photomicrographs of each aggregate cultured in neural induction medium (NIM) using low cell-adhesion 96-well plates. (B) Expression of neural (Nestin, Ncam, Evi3, and Sox1) and mesoderm (Brachyury)

differentiation

markers

in

day

6

aggregates

by

reverse

transcription-polymerase chain reaction (RT-PCR). (C) Localization of the marker proteins E-cadherin, N-cadherin, Nestin, and Brachyury in 1 × 103 and 5 × 103 aggregates cultured for 6 days. Left panels represent immunofluorescence. Right panels represent diamidino-2-phenylindole (DAPI) staining.


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Figure 2. Neural differentiation of spheres cultured in neural induction medium (NIM). (A) Expression patterns of differentiation markers in spheres cultured in NIM or NIM with knockout serum replacement and L-glutamine (NIM+KG) at culture day 0 (0), day 3 (3), or day 6 (6). Expression of markers of undifferentiated embryonic stem (ES) cells (Nanog and Oct3/4), neural differentiation (Evi3, Nestin, Sox1, and, Ncam), dorsoventral differentiation (Nkx2.2 and Pax3), mesoderm differentiation (Brachyury and Mox1), and endoderm differentiation (GATA6) were analyzed by reverse transcription-polymerase chain reaction(RT-PCR). (B) Immunohistochemical analysis of marker proteins, E-cadherin and Oct3/4 (undifferentiated ES cells), N-cadherin and Nestin (neuroepithelium), and Brachyury (mesenchymal) in NIM+KG-cultured spheres. (C) Expression of rostral/caudal positional markers, Otx1 and Otx2 (rostral), Hoxb4, Hoxb9 and Lbx1 (caudal), and TH (mid- and hindbrain) in spheres by RT-PCR. Spheres were cultured in NIM (NIM), NIM with retinoic acid (RA) treatment during days 2-5 (NIM+RA) and NIM+KG at day 6, respectively.


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Figure 3. Neural crest (NC) cell differentiation of neural induction medium (NIM)-cultured neurospheres. NIM-cultured neurospheres were cultured for 6 days in NIM, transferred to fibronectin-coated dishes, and cultured for 4-5 days in medium containing bone morphogenic protein 4 (BMP4). (A) Expression of NC markers in NIM-cultured neurospheres (0, 3, and 6) and NC culture for 4 days (10) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). (B, C) Immunostaining for the NC marker AP2α. AP2α protein expression of cells in sphere on day 6 (B) and day 11 (C).


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Figure 4. Osteogenic differentiation from neural induction medium (NIM)-cultured neurospheres. NIM neurospheres cultured in NIM supplemented with (A) or without (B) bone morphogenic protein 4 (BMP4) for 4 days were subsequently cultured in osteogenic medium for 4 weeks and stained with Alizarin Red S. (C) Expression of osteogenic markers collagen I and Runx II in NIM-cultured neurospheres (0, 3, and 6), NC (11), and osteogenic cells (22) were analyzed by RT-PCR. Dissociated cells from adherent spheres (D) and migrating cells (E) treated with BMP4 for 4 days were subsequently cultured in osteogenic medium for 19 days using high-density monolayer culture conditions and stained with Alizarin Red S. All sphere clusters and nodule-like cell aggregates were differentiated to Alizarine Red S positive, but spreading monolayer cells were not.


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Figure 5. Characterization of neural crest (NC) stem cells in CKB-EK clone #11. (A) Expression of NC and stem cell markers in CKB-EK clone #11 was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). (B-J) Immunofluorescence staining of peripherin (B), Pparγ (C), and smooth muscle actin (D), in NC derivatives from CKB-EK clone #11 and diamidino-2-phenylindole (DAPI), respectively. (E) Alizarin Red S staining of cultured cells in osteogenic medium. (F) Oil Red O staining of cultured cells in adipogenic medium. (G) Alcian Blue staining of cultured cells in chondrogenic medium.

Supplementary Figure 1. Characterization of neural crest (NC) cells derived from neurospheres. Day 6 NIM neurospheres and RA-treated neurospheres were cultured for 4 days in fibronectin-coated plates. Expression of NC markers AP2α, Sox9, Pax7, Sox10 and FoxD3 in NC cells within adherent clusters (Sphere) and emigrating cells (Emigrate) derived from NIM neurospheres, and clusters from RA-treated neurospheres (RA) was examined by RT-PCR.

Supplementary Figure 2. Multiple differentiation potential of the expanded NC cells. Immunofluorescence staining of Pparγ (A) and collagen II (C) in NC derivatives. (B) Oil Red O staining of cells cultured in adipogenic medium. (D) Alcian Blue staining of


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cultured cells in chondrogenic medium. (E) Alizarin Red S staining of cultured cells in

Osteogenic medium. About half of cells in adipogenic culture were Pparγ positive.

Almost all cells expressed collagen II; dense cell aggregates differentiated into alcian

blue-positive chondrocytes.

Stem Cells and Development Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres (doi: 10.1089/scd.2014.0152) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f

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TABLE 1. Characteristics of Aggregates with Different Amount of Cells in NIM Gene expression No. of cells

Size of aggregates

Nestin

Ncam

Evi3

Sox1

Brachyury

1x103

small

＋

＋＋

＋＋＋

＋

－

2x103

midlle

－

＋

＋＋

＋

±

5x103

large

－

＋

＋

＋

＋

Immunostaining No. of cells

E-cad

N-cad

Nestin

Brachyury

1x103

－

＋＋**

＋＋

－

2x103

ND

ND

ND

ND

5x103

＋＋*

＋

＋

＋

－; no expression, ±; weak expression, ＋; moderate expression, ＋＋; strong expression, ＋＋＋; very strong expression, ND; Not done, ＋＋*; colonize positive cells, ＋＋**; strong expression was localized at the apical side of rosette-like structure.


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Stem Cells and Development Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres (doi: 10.1089/scd.2014.0152) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 43 of 47

43


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Supplementary Table 1 Antibody purchasing and dilution

Antibody

Host

Dilution

AP2α

Mouse

1:50 Santa Cruz

p75(NGFR)

Goat

1:50 Santa Cruz

Peripherin

Rabbit

Pparγ

Mouse

1:50 Santa Cruz

Smooth muscle actin

Mouse

1:50 Santa Cruz

E-cadherin

Rat

20μg/ml

Takara

N-cadherin

Rabbit

20μg/ml

Takara

Nestin

Rabbit

Brachyury

Goat

1:50

Santa Cruz

CollagenII

Mouse

1:50

Abcam

1:100

Supplier

Millipore

1:50 Santa Cruz

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Supplementary Table 2 Primers used for RT-PCR

Gene name Nanog

Oct3/4

GATA6

Brachyury

Mox1

Nestin

Evi3

Sox1

Ncam Pax3

Nkx2.2

AP2α

Anealing(°C) sense

5’-AGGCCCAGCTGTGTGCACTC-3’

anti-sense

5’-GGACTGAGCGGTCCAGGCCT-3’

sense

5’-TGCCCGAAGCCCTCCCTACA-3’

anti-sense

5’-TGTCTACCTCCCTTGCCTTGGCTC-3’

sense

5’-CAGGAGCGCAGGCTGCAGTT-3’

anti-sense

5’-CCCGGGAGCAGGAGGAGGAC-3’

sense

5’-GTGGGCCTGGAGGAGAGCGA-3’

anti-sense

5’-CCCCAGGTACCCACTCCCCG-3’

sense

5’-TCGCCAATGAGACGGAGAAG-3’

anti-sense

5’-GGTCCAGGTTGACTGCAATC-3’

sense

5’-CAGGTGAACAAGACTCCGTA-3’

anti-sense

5’-GGGCATCTGTCAAAATCGGGA-3’

sense

5’-TGGGGAGGCAGTAGACTG-3’

anti-sense

5’-CTCCATCCTGGAGCCAGA-3’

sense

5’-GCACACAGCGTTTTCTCGG-3’

anti-sense

5’-ACATCCGACTCCTCTTCCC-3’

sense

5’-TGAGGGTACTTACCGCTGTG-3’

anti-sense

5’-GTTGCTGGCAGTGCAGATGT-3’

sense

5’-CCAGGATGATGCGGCCCGGCCCGGG-3’

anti-sense

5’-AGGATGCGGCTGATAGAACTCACTG-3’

sense

5’-GAAGCGCCGAGTGCTCTTCTCC-3’

anti-sense

5’-GCCGAGCTGTACTGGGCGTTGT-3’

sense

5’-GCCGTCCACCTAGCCAGGGA-3’

anti-sense Sox9

Page 47 of 47

P75

Slug

60

60

62

58

60

60

60

55

60

58

55

60

5’-ATGGCCTTGAGGGCCTCGGT-3’

sense

5’-CCCTTCGTGGAGGAGGCGGA-3’

anti-sense

5’-CGTCGCGGAAGTCGATGGGG-3’

sense

5’-CCTGCCTGGACAGTGTTACG-3’

anti-sense

5’-GCCAAGATGGAGCAATAGACA-3’

sense

5’-CGCCGCCTGGACCGTTATCC-3’

anti-sense

5’-AAGGCGGGGGACTTACACGC-3’

60

60

60


47

Twist

Otx1

Otx2

Hoxb4

Hoxb9

Lbx1

TH

Pax7

Sox10

FoxD3

Gapdh

sense

5’-GGAGGATGGAGGGGGCCTGG-3’

anti-sense

5’-TCCACGGGCCTGTCTCGCTT-3’

sense

5’-GCTGTTGGCAAAGACTCGCTAC-3’

anti-sense

5’-ATGGCTCTGGCACTGATACGGATG-3’

sense

5’-CCATGACCTATACTCAGGCTTCAGG-3’

anti-sense

5’-GAAGCTCCATATCCCTGGGTGGAAAG-3’

sense

5’-CCTGGATGCGCAAAGTTC-3’

anti-sense

5’-GTGTTGGGCAACTTGTGGTC-3’

sense

5’-GGAAGCGAGGACAAAGAGAG-3’

anti-sense

5’-TTGAGGAGTCTGGCCACTTC-3’

sense

5’-AAGACCTTTAAGGGGCTGGT-3’

anti-sense

5’-AGGTGATGACCTGTGCATTG-3’

sense

5’-TCCTGCACTCCCTGTCAGAG-3’

anti-sense

5’-CCAAGAGCAGCCCATCAAAGG-3’

sense

5’-TGCCGATATCAGGAGACTG-3’

anti-sense

5’-CCAGGATGCCATCGAT-3’

sense

5’-CTAGGCAAGCTTGGAGGTT-3’

anti-sense

5’-AGGTATTGGTCCAGCTCAGT-3’

sense

5’-TGACCCGAACAAGCCCAAGAACA-3’

anti-sense

5’-AGGCTCCGAAGCTCTGCATCATCA-3’

sense

5’-AGAGACGGCCCCATCTTCTTGT-3’

anti-sense

5’-ATGTTCTGGGCAGCCCCACG-3’

64

60

60

52

60

60

58

60

50

60

60

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells.

MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

Fndc5 overexpression facilitated neural differentiation of mouse embryonic stem cells.

Generating trunk neural crest from human pluripotent stem cells.

Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and lead to generation of neural crest cells from human embryonic stem cells.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

Time-sensitive effects of hypoxia on differentiation of neural stem cells derived from mouse embryonic stem cells in vitro.

Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

Embryonic Origin of Postnatal Neural Stem Cells.

Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

A porous membrane-mediated isolation of mesenchymal stem cells from human embryonic stem cells.

Enteric neurospheres are not specific to neural crest cultures: implications for neural stem cell therapies.

Sequential Differentiation of Embryonic Stem Cells into Neural Epithelial-Like Stem Cells and Oligodendrocyte Progenitor Cells.

The use of human pluripotent stem cells for the in vitro derivation of cranial placodes and neural crest cells.

Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells.

Isolation and propagation of neural stem cells in caprine (Capra hircus).

Generation of neurospheres from human adipose-derived stem cells.

Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells.

Differentiation of Adipocytes in Monolayer from Mouse Embryonic Stem Cells.

Generation of v2a interneurons from mouse embryonic stem cells.

Correction: MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

Chromosome instability in mouse embryonic stem cells.

LIF signal in mouse embryonic stem cells.

Electrospun fibrous scaffolds of Poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells.