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OPEN ACCESS ARTICLE
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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS |
aDepartment of Physiology, Keio University, School of Medicine, Shinjuku-ku, Tokyo, Japan;
bDepartment of Neurology, Nagoya University, Graduate School of Medicine, Showa-ku, Nagoya, Japan;
cDepartment of Neurology, Tohoku University, Graduate School of Medicine, Aoba-ku, Sendai, Japan
Key Words. Neural stem/progenitor cells • Embryonic stem cells • Neurosphere • Temporal identity • Spatial identity • Regenerative medicine
Correspondence: Correspondence: Hideyuki Okano, M.D., Ph.D., Department of Physiology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Telephone: +81-3-5363-3747; Fax: +81-3-3357-5445; e-mail: hidokano{at}sc.itc.keio.ac.jp
Received on March 25, 2008;
accepted for publication on August 12, 2008.
First published online in STEM CELLS EXPRESS August 28, 2008.
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ABSTRACT |
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 TopFootnotes Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Neurospheres and other methods for culturing NS/PCs in vitro have been reported and are widely used [3–5]. In these culture systems, NS/PCs are usually derived from the brain at mid to late gestation or from the adult brain, both of which are easy to manipulate and yield NS/PCs in large quantities, but these NS/PCs have limited plasticity. On the other hand, the culture of early embryonic brain requires special dissection techniques, and only a limited number of NS/PCs can be obtained from each brain. Furthermore, it is impossible to expand these early NS/PCs maintaining their early identities. Thus, it would be valuable to establish a culture system for generating NS/PCs with early temporal identities and high cell-fate plasticity from ES cells and to be able to control the spatiotemporal identities of the NS/PCs in vitro.
Several methods have been reported for deriving neural cells from mouse embryonic stem (ES) cells [6–14]. However, each method directs the induction of only certain types of neural cells with specific temporal and spatial identities, such as forebrain progenitors, dopaminergic neurons, motor neurons, cerebellar neurons, and neural crest cells. In addition, the differentiation protocols are quite varied, and therefore the methods used must be changed according to the desired cell type. Moreover, the requirement for different conditions means that neither the characteristics of NS/PCs with different spatial or temporal identities nor the possible associations between temporal and spatial identities can be evaluated within the same culture system. Furthermore, most of these culture protocols have a risk of contamination by undifferentiated cells, non-neural cells, and feeder cells or rely on a long and complicated protocol to obtain particular cells.
To solve these problems, we examined whether we could recapitulate in vivo CNS development in vitro and established a simple ES cell culture system in which purified early NS/PCs are derived as neurospheres and their spatial (rostrocaudal and dorsoventral) and temporal identities are regulated in a single culture system. This system provides a powerful in vitro model for investigating the mechanisms underlying early CNS development and the pathogenesis of neurological disorders and will be applicable to regenerative therapy for neurodegenerative disorders.
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MATERIALS AND METHODS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Lentivirus Transduction and Clonal Neurosphere Formation
For clonal neurosphere analysis, primary neurospheres were initiated from dissociated EBs transduced with lentivirus expressing either Venus or monomeric red fluorescent protein (mRFP) under the EF1
promoter (pCSII-EF-Venus or pCSII-EF-mRFP) [17]. Venus- and mRFP-labeled primary neurospheres were dissociated, plated at a 1:1 ratio at a cell density of 0.5–1 x 104 cells/ml, and cultured for 10–13 days in the MHM with 0.8% methylcellulose (22223-52; Nacalai Tesque, Kyoto, Japan, http://nacalai.co.jp) and 20 ng/ml FGF2 to form secondary neurospheres as described previously [18, 19].
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
RNA isolation and reverse transcription (RT)-polymerase chain reaction (PCR) were performed as described previously [16]. The amount of cDNA was normalized to β-actin mRNA. Total RNA from embryonic day (E) 11.5 whole embryos was used as a positive control. Real-time RT-PCR was performed using MX3000P (Stratagene, La Jolla, CA, http://www.stratagene.com), with SYBR Premix ExTaq (Takara, Otsu, Japan, http://www.takara.co.jp). Data are expressed as the amount of mRNA relative to that of neurospheres derived from E14.5 striatum. Primer sequences and PCR cycling conditions are listed in supplemental online Table 1.
Immunohistochemical Analysis
Immunohistochemical analyses for cultured cells (immunocytochemistry [ICC]) and embryonic tissues (immunohistochemistry [IHC]) were performed as described previously [16]. Detailed conditions for the ICC and IHC are in the supplemental online Materials and Methods. For statistical analysis of the ICC results, at least 60 colonies/cover glasses were examined, and the number of colonies that immunoreacted with each antibody was counted and expressed as the percentage of the total number of colonies.
Bisulfite Sequencing
Sodium bisulfite treatment of genomic DNA was performed as described previously [20] with slight modifications, as described in supplemental online Materials and Methods. The DNA fragment containing the Stat3 recognition sequence was amplified by PCR, the products were cloned into the pGEM-T easy vector (Promega, Madison, WI, http://www.promega.com), and 10–14 clones randomly picked from each of four independent PCRs were sequenced.
Patch-Clamp Recording Procedure
ES cell-derived neurospheres were allowed to differentiate for 10–14 days on poly-L-ornithine/fibronectin-coated cover glasses, on an astrocyte feeder layer, and processed for patch-clamp analysis as described in the supplemental online Materials and Methods.
Transplantation of ES Cell-Derived Neurospheres and Immunohistochemistry
Low-RA neurospheres were transplanted into wild-type Sprague-Dawley rats (CLEA Japan, Inc., Tokyo, http://www.clea-japan.com) or amyotrophic lateral sclerosis (ALS) model rats harboring a mutant human SOD1G93A gene [21, 22] at approximately 90 days of age. The procedure for the transplantation is described in the supplemental online Materials and Methods. All of the animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals of the Keio University, School of Medicine.
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RESULTS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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To confirm whether these neurospheres were derived from NS/PCs, we examined their self-renewal and multipotency. Most of the single primary noggin or low-RA neurospheres that were cultured at low density (2.5 x 104 cells/ml) and deposited into 96-well plates (single neurosphere/well) generated secondary neurospheres (103/133 [77%] and 115/122 [94%], respectively, from more than two independent experiments). Moreover, both types of ES cell-derived neurospheres could be passaged repeatedly to form tertiary neurospheres (Fig. 1D), indicating that they could self-renew.
We also examined the differentiation potentials of noggin and low-RA neurospheres grown at low density (<2.5 x 104 cells/ml at plating) by subjecting 5-day differentiated neurospheres to immunocytochemical analysis with markers for neurons (N) (βIII-tubulin), astrocytes (A) (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (O) (O4). Most of the colonies from primary neurospheres contained neurons only (93.4% and 74.5% in noggin and low-RA neurospheres, respectively), whereas only a small number contained glial cells. When single primary neurosphere-derived secondary neurospheres and tertiary neurospheres cultured at low density (2.5–5 x 104 cells/ml) were differentiated using the same method, they generated more colonies containing glial cells, and the proportions of multipotent colonies (NAO colonies) increased gradually in the secondary and tertiary neurospheres (Fig. 2A–2C; supplemental online Fig. S1A–S1C). This sequential generation of neurons and glial cells also corresponds well with in vivo CNS development, in which neurons are generated first and glial cells later [1, 29]. Notably, colonies that only generated neurons (N colonies) were observed in up to 44.9% of the secondary noggin neurospheres, whereas all of the colonies from secondary low-RA neurospheres contained glial cells, suggesting that the low-RA neurospheres preceded noggin neurospheres in their temporal differentiation properties (Fig. 2; supplemental online Fig. S1).
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We also examined the expression of cell type-specific markers in ES cell-derived neurospheres by RT-PCR. Both noggin-treated and low-RA-treated EBs expressed markers for neural progenitors (Sox1), along with markers for undifferentiated ES cells, such as Nanog, Eras, and Oct3/4 (Fig. 1E). In contrast, primary and secondary neurospheres derived from EBs treated with either agent expressed much lower levels of markers for undifferentiated ES cells or other lineages, including markers for endoderm (Pdx1), mesoderm (Nkx2.5), and epiderm (Ck-17). These results indicate that the repeated formation of neurospheres from single-cell suspensions in serum-free medium facilitates the selection of NS/PCs and the elimination of unwanted undifferentiated and non-neural cells.
Primary Neurospheres and Secondary/Tertiary Neurospheres Have the Properties of Early and Late NS/PCs, Respectively
Because the long-term expansion of ES cell-derived neurospheres through repeated passages resulted in a sequential generation of neurons and glial cells similar to that seen in vivo (Fig. 2A–2C; supplemental online Fig. S1A–S1C), we next focused on the temporal specification of ES cell-derived NS/PCs, characterizing them by several other temporally restricted properties of NS/PCs seen in vivo.
In vivo, NS/PCs initially proliferate only in response to FGF, and then acquire responsiveness to EGF when the EGF receptor is expressed at mid gestation [30, 31]. We therefore first studied the expression of the involved receptors by NS/PCs and their responsiveness to these growth factors during neurosphere formation. Real-time RT-PCR analysis showed that Fgfr1 and Fgfr2 were constantly expressed by EBs, primary neurospheres, and secondary neurospheres. In contrast, Egfr expression was low in EBs and primary neurospheres but was dramatically upregulated in the secondary neurospheres (Fig. 2D; supplemental online Fig. S1D). Consistent with these results, EBs and primary neurospheres efficiently generated primary and secondary neurospheres only in the presence of FGF2 and not EGF, but tertiary neurospheres could be efficiently formed in the presence of either FGF2 or EGF (Fig. 2E; supplemental online Fig. S1E). These results suggest that this in vitro system also recapitulates the temporal change in growth factor responsiveness that occurs during NS/PC development in vivo.
Our results so far showed that the temporal changes in differentiation potentials and proliferation in ES cell-derived NS/PCs were similar to those of embryonic NS/PCs. These changes are thought to be regulated in vivo by epigenetic mechanisms such as DNA methylation and chromatin modification, at least in part. To examine these mechanisms in our system, we focused on the methylation status of CpGs around the Stat3 recognition sequence in the GFAP promoter. This region is methylated during neurogenesis, at around E11.5–12.5 in mice, and gradually demethylated during gliogenesis, after E14.5, to regulate the transcription of the GFAP gene and astrocyte differentiation [20, 32, 33]. By bisulfite sequencing, we found that the proportion of unmethylated CpGs around the Stat3 recognition sequence (–1568 to –1460) (Fig. 2F) gradually increased in the primary and secondary noggin and low-RA neurospheres (Fig. 2G). Thus, temporal epigenetic changes in the ES cell-derived neurospheres also recapitulated those seen in vivo.
Regulation of Rostrocaudal Identities by Noggin and Various Concentrations of RA in ES Cell-Derived NS/PCs
Because noggin and RA are involved in the formation of the rostral and caudal neural tube, respectively, we next examined whether exposure to noggin or various concentrations of RA during EB formation could alter the rostrocaudal identities of the neurospheres that were subsequently derived. We found that noggin neurospheres expressed forebrain to midbrain markers (Foxg1, Otx1, Otx2, and En1), whereas low-RA neurospheres were caudalized to some extent, expressing hindbrain markers (Pax2, Gbx2, Hoxb1, Hoxa2, and Hoxb4) as well as forebrain to midbrain markers (Fig. 3A). However, neither type of neurosphere expressed spinal cord markers such as Hoxc4 and Hoxc6.
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This regulation of marker expression correlating with the rostrocaudal axis was confirmed by subjecting 5-day differentiated neurospheres to immunocytochemical analysis for Otx1 (forebrain to midbrain), Pax2 (midbrain to spinal cord), and Hoxb4 (hindbrain and spinal cord) (supplemental online Fig. S5A). Approximately 70% of the colonies from noggin neurospheres contained Otx1+ cells, whereas few or none contained Pax2+ or Hoxb4+ cells (Fig. 3B, 3C), suggesting a forebrain-to-midbrain identity. In contrast, many colonies from low-RA neurospheres contained Pax2+, Hoxb4+, and Otx1+ cells (forebrain-to-hindbrain identity), and high-RA neurospheres generated Pax2+ and Hoxb4+ but not Otx1+ colonies (hindbrain-to-spinal cord identity). These data indicate that the caudalization of NS/PCs in neurospheres could be driven by RA added during EB formation in a concentration-dependent manner. These rostrocaudal marker-positive cells were also positive for neural progenitor markers, such as Sox1/2/3 and hSox (supplemental online Fig. S3) ([34, 35]), or the neuronal marker NeuN, confirming that they were neural cells (Fig. 3B). Moreover, noggin and low-RA neurospheres differentiated into many neurons that were glutamic acid decarboxylase (GAD67)-positive (GABAergic) or choline acetyltransferase (ChAT)-positive (cholinergic), and small numbers of neurons that were tyrosine hydroxylase (TH)-positive (catecholaminergic) or serotonin (5-HT)-positive (serotonergic). In contrast, the high-RA neurospheres differentiated into GAD67+ and ChAT+ neurons but not into TH+ or 5-HT+ neurons (Fig. 3D, 3E). There was no significant difference in the frequency of differentiation into GAD67+ or ChAT+ neurons between the noggin and low-RA neurospheres. The generation of these neuronal subtypes shows overall consistency with the identity of neurons generated in the corresponding rostrocaudal region in vivo. Thus, the rostrocaudal identity of ES cell-derived NS/PCs was controlled by administering noggin or various concentrations of RA during EB formation.
Dorsoventral Regulation of ES Cell-Derived NS/PCs
In addition to the rostrocaudal axis, we next attempted to control the dorsoventral identities of ES cell-derived NS/PCs by adding several secreted factors during neurosphere formation, including Sonic hedgehog (Shh), wingless (Wnt) 3a, and BMP4, which act as morphogens in dorsoventral specification in the developing neural tube [2, 36].
Because the neuronal subtypes and the dorsoventral axis are determined during the neural fold and neural tube stages in vivo by Shh from the ventral notochord and floor plate and by Wnts and BMPs from the dorsal roof plate [2, 36] and because this follows the determination of the rostrocaudal axis (during gastrulation in vivo or EB formation in vitro) [2, 27, 37], we added Shh, Wnt3a, and BMP4 during neurosphere formation.
As with BMP4, only small-cell clusters (ragged "spheres") were formed, and very few spheres were formed from noggin-treated EBs (supplemental online Fig. S4). Therefore, we examined the effects of sonic hedgehog N-terminal peptide (Shh-N) and Wnt3a on the expression of dorsoventral markers of the forebrain in noggin neurospheres, and the effects of Shh-N, Wnt3a, and BMP4 on the expression of caudal dorsoventral markers in low- and high-RA neurospheres.
Noggin neurospheres expressed Nkx2.1, a marker for the basal forebrain (supplemental online Fig. S5B), in an Shh-N concentration-dependent manner, generating 69.2 ± 8.2% Nkx2.1+ ventral colonies with 300 nM Shh-N (Fig. 4A, 4D, 4E). On the other hand, these neurospheres expressed Pax6 and Emx1, markers for dorsal telencephalon (supplemental online Fig. S5B), in a Wnt3a concentration-dependent manner, generating 84.0 ± 3.1% Pax6+ dorsal colonies with 50 ng/ml Wnt3a (Fig. 4A, 4D, 4E). The expression of Gsh2 and Dlx2, which are found in the mid forebrain (supplemental online Fig. S5B), was not significantly altered by any of the treatments, and Gsh2+ colonies were generated in the presence or absence of Shh-N or Wnt3a (Fig. 4A, 4D, 4E).
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Finally, we examined whether Wnt3a and BMP4 directed ES cell-derived neurospheres into the neural crest lineages. Although Wnt3a increased the expression of dorsal markers, including Pax3 and Pax7, it only slightly upregulated markers for neural crest lineages, Slug and Snail, in contrast to BMP4, which strongly upregulated Slug and Snail (Fig. 4B, 4C). Consistent with these results, only a small number of cells in Wnt3a-treated neurospheres appeared to be differentiated into peripherin+ peripheral neurons and SMA+ smooth muscle progenitors, as in control cultures, whereas virtually all of the cells that differentiated from BMP4-treated spheres expressed peripherin or SMA (Fig. 4G). Moreover, Wnt3a-treated neurospheres generated Brn3a+/peripherin+ sensory neurons and a small number of Phox2b+/peripherin+ autonomic neurons, whereas most of the peripherin+ neurons derived from BMP4-treated low- and high-RA neurospheres were Phox2b+ autonomic neurons (85.8 ± 5.0 and 89.2 ± 0.6%, respectively) and none at all were Brn3a+ sensory neurons (Fig. 4H). These results were consistent with the in vivo effects of Wnt3a and BMP4 on neural crest stem cells [38]. Taken together, these results indicate that our culture system provides a variety of neural progenitors with a wide range of temporal and spatial identities, including those of the neural crest lineages (summarized in Fig. 5).
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-bungarotoxin, suggesting that these neurons could form synaptic contacts in vitro (supplemental online Fig. S6).
ES Cell-Derived NS/PCs Survived and Differentiated into a Variety of Neuronal Subtypes That Formed Synaptic Connections in Rat Spinal Cord
Finally, we transplanted low-RA neurospheres derived from CAG-EGFP ES cells into the ventral lumbar spinal cord of approximately 90-day-old Sprague-Dawley rats to assess the in vivo differentiation potentials of the ES cell-derived NS/PCs. Two weeks later, the rats were sacrificed and processed for immunohistochemical analysis. The transplanted cells survived in the ventral spinal cord as cell clusters (Fig. 7A). In the treated rats, the ES cell-derived NS/PCs differentiated into NeuN+ neurons and GFAP+ astrocytes in some animals, but never into 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-positive oligodendrocytes (Fig. 7B; supplemental online Table 2). The neurons were mostly GAD+ (GABAergic), and there were relatively few ChAT+ (cholinergic), TH+ (catecholaminergic), or 5-HT+ (serotonergic) neurons (Fig. 7B–7F; supplemental online Table 2). Moreover, the EGFP+ neurites of the transplanted neurons were extensively labeled for and surrounded by the presynaptic marker synaptophysin, indicating the formation of synaptic contacts with host neurons (Fig. 7I–7K). Similar results were obtained when low-RA neurospheres were transplanted into the lumbar spinal cord of ALS model rats harboring the mutant human SOD1G93A gene [21, 22], although their differentiation properties in vivo varied somewhat from animal to animal (supplemental online Fig. S7; supplemental online Table 2). Thus, our ES cell-derived neurosphere culture system could be applicable to regenerative therapy for neurological disorders.
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DISCUSSION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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We used noggin and RA for neural induction, which also respectively determined the rostral and caudal identities of the NS/PCs. Several lines of evidence have suggested a model in which the default positional fate of the neural plate is the rostral brain, with factors such as RA, FGFs, Wnts, and growth differentiation factors (GDFs) inducing the caudalization of neural cells in early vertebrate development [39]. One report demonstrated the effective induction of rostral neural progenitors with a combination of Dickkopf-1 and LeftyA [10]; here, we chose to use noggin instead, because BMP antagonism is essential for mammalian forebrain development [25, 26]. In our present study, noggin treatment of EBs dose dependently increased the number of neurospheres by inhibiting BMP signals in the serum-containing EB medium. Moreover, consistent with the above-mentioned model, caudalizing signal-independent noggin neurospheres adopted rostral identities. On the other hand, RA promotes the neural differentiation of ES cells concurrently with the caudalization of neural progenitors in EBs in a concentration-dependent manner in vitro: low RA produces more neural progenitors with slightly caudalized identities around the midbrain to hindbrain, and high RA induces postmitotic neurons with caudal neural tube identities rather than proliferative neural progenitors [16]. RA also causes cell cycle arrest in neuroblastoma cells by increasing the level of cyclin-dependent kinase inhibitors, such as p27kip1, through downregulation of the ubiquitin-proteasome-dependent degradation pathway [40]. Thus, the efficient generation of neurospheres from low-RA-treated rather than high-RA-treated EBs in the present study was consistent with this concentration-dependent effect of RA on neural induction from ES cells. Moreover, we obtained neurospheres with caudal identities in an RA concentration-dependent manner by using the low-RA neurosphere protocol (forebrain, midbrain, and hindbrain) and the high-RA/short-exposure protocol (hindbrain and spinal cord). These results indicated that the rostrocaudal identity in primary neurospheres could be preserved from that acquired in the EB stage, except that the low-RA neurospheres exhibited not only midbrain-to-hindbrain but also forebrain identity. Taking into consideration the effect of RA on cell cycle arrest, these findings suggest that the rostral NS/PCs, a relatively minor population in the low-RA-treated EBs, were selectively expanded in the neurosphere condition.
In the developing CNS, distinct NS/PCs with different temporal identities are generated, depending on the developmental stage. The earliest are leukemia inhibitory factor-dependent primitive NS/PCs (E5.5–E7.5) and FGF-responsive (but not EGF-responsive) NS/PCs (E8.5–E11.5), which have the potential to generate early-born neurons; the latest are EGF-responsive NS/PCs with gliogenic potentials that cannot generate early-born neurons [1, 29–31, 41]. In addition, although ES cell-derived NS/PCs that are identical to primitive NS/PCs have been reported, these cells do not show a transition into EGF-responsive NS/PCs unless stimulated by an exogenous Notch signal [9, 41]. In contrast, our neurosphere culture system successfully enables the sequential generation of NS/PCs with early and late temporal identities just as in vivo, and it could clearly recapitulate the temporal transition of neurogenic early NS/PCs into gliogenic late NS/PCs and the acquisition of EGF responsiveness.
The gradual increase in the number of unmethylated CpGs in the GFAP promoter region, which regulates the timing of GFAP expression [20, 32, 33], from undifferentiated ES cells to secondary neurospheres, suggests that in vivo developmental changes in the epigenetic status of this region are also recapitulated to some extent in our system. Despite the remarkable augmentation of differentiation into GFAP+ astrocytes from secondary neurospheres, the increase in unmethylated CpGs was not as dramatic. It is possible that cells of the neuronal lineage within the neurospheres masked this change in stem cells.
It is also noteworthy that the acquisition of gliogenic potential in the noggin neurosphere cultures was delayed compared with that in the low-RA neurosphere cultures (Fig. 2A–2C; supplemental online Fig. S1A–S1C). This is consistent with in vivo development, in which the acquisition of later identities in the caudal neural tube precedes that in the forebrain [29–31, 42, 43], indicating another advantage of our culture system, the simultaneous recapitulation of temporal and spatial specification.
We also showed that dorsoventral identity can be controlled by the administration of Shh-N, Wnt3a, and BMP4 during the neurosphere formation. The dorsoventral identity in noggin neurospheres was sharply regulated by Shh-N and Wnt3a, but the neurosphere-initiating progenitors from low- and high-RA-treated EBs seemed relatively less competent to respond to these factors. We previously showed that EBs treated with low-RA express more of the active form of Shh-N and are ventralized and those treated with high-RA express less Shh-N and acquire a dorsal identity [16]. Thus, low- and high-RA neurospheres exhibited the default identities of ventral and dorsal neural tubes, respectively. On the other hand, BMP4 had a drastic effect. BMP4 induced neurospheres that largely adopted neural crest lineages, generating peripherin+ peripheral neurons and -SMA+ smooth muscle cells. Given that BMP2 and BMP4 promote cell death and inhibit the proliferation of rat early cortical progenitors in vitro [44], this difference between BMP4 and other factors in the magnitude of their effects on NS/PCs, including negative effects on neurosphere formation and the potential to differentiate into neural crest lineage cells, may be explained by a mechanism of selective survival. Focusing on neural crest development, because BMP2/4 and Wnt1/3a play important, concerted roles in the formation of the dorsal neural tube, including the neural crest, and are respectively involved in the generation of Phox2b+ autonomic and Brn3a+ sensory neurons in vivo [36, 38], the differentiation of Phox2b+ autonomic and Brn3a+ sensory neurons from neurospheres treated with BMP4 and Wnt3a, respectively, was consistent with the in vivo development of the peripheral nervous system (PNS), demonstrating the possible application of our culture system to the generation of neural crest lineages, including the PNS.
Another important finding of this study is that low-RA neurospheres differentiated into GAD+, ChAT+, TH+, and 5-HT+ neurons both in vitro and when transplanted into the lumbar spinal cord of adult rats (Figs. 3 and 7; supplemental online Fig. 7; supplemental Table 2), indicating that these ES cell-derived NS/PCs may have maintained their in vitro-acquired identities of mainly midbrain to hindbrain even after transplantation into the more caudal in vivo environment. Interestingly, the low-RA neurospheres generated some GAD+ GABAergic neurons (20%–40%) but relatively few ChAT+ cholinergic neurons (up to 5%) in vivo (Fig. 7E, 7H; supplemental online Fig. S7G, S7J, S7K, S7N; supplemental online Table 2), even though they differentiated into many GAD+ GABAergic and ChAT+ cholinergic neurons in vitro (Fig. 3D, 3E). It is well known that neurons that make contact with their target cells and form the appropriate synaptic circuitry selectively survive better than those that do not form such connections in the nervous system [45–47]. Thus, it is reasonable to speculate that GABAergic interneurons derived from the grafted low-RA neurospheres might have been able to form synaptic contact with their target cells, which are abundant within the host spinal cord [48] and thereby survived well even in vivo. On the other hand, grafted neurosphere-derived cholinergic neurons, including motor neurons, might not have been able to connect with their target cells, resulting in poor survival after transplantation. For example, graft-derived cholinergic motor neurons might be prevented from extending their axons out of the spinal cord by myelin-derived axonal inhibitors in the spinal cord white matter [49] and thus might have had difficulty contacting their target muscles. Alternatively, the adult spinal cord simply might not be an appropriate environment for the cholinergic differentiation of the precursor cells. Moreover, it is noteworthy that neurons derived from the transplanted NS/PCs that survived formed many synaptic contacts with the host tissues, both in wild-type and disease model animals, indicating that the ES cell-derived NS/PCs can generate functional neurons and may have the potential to regenerate functional networks in vivo, even in diseased neural tissues.
Because the complicated structure and function of the mammalian CNS develops through appropriate spatiotemporally regulated patterning, our in vitro model, which recapitulates the in vivo CNS development, may provide a simple and powerful tool for investigating the mechanisms underlying mammalian CNS development and be applicable to regenerative medicine for neurological disorders. Further studies should involve the application of a variety of factors to our culture system to mimic in vivo development, to obtain more types of neurons or neural progenitors, and to produce NS/PCs from a variety of stem cells, such as human ES cells, nuclear transfer ES cells, and induced pluripotent stem cells, which may lead to effective regenerative therapy for the damaged CNS.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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FOOTNOTES |
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REFERENCES |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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), whereas those containing fewer than 10 are indicated as weakly positive (+, 
). Scale bar = 50 µm. (F): Low-RA neurospheres were differentiated for 5 days and immunostained for Isl-1/2 and HB9, markers for ventral somatic motor neurons and with the neuronal marker Dcx. Scale bar = 20 µm. (G): Immunocytochemical analysis of differentiated neurospheres treated with dorsalizing factor, Wnt3a or BMP4. Differentiated neurospheres were immunostained for 
