Mol. Cells 2018; 41(2): 110-118
Published online January 31, 2018
https://doi.org/10.14348/molcells.2018.2210
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: dwkim2@yuhs.ac (DWK); sonnet10@korea.ac.kr (DSK)
The objective of this study was to induce the production of isthmic organizer (IsO)-like cells capable of secreting fibroblast growth factor (FGF) 8 and WNT1 from human embryonic stem cells (ESCs). The precise modulation of canonical Wnt signaling was achieved in the presence of the small molecule CHIR99021 (0.6 μM) during the neural induction of human ESCs, resulting in the differentiation of these cells into IsO-like cells having a midbrain-hindbrain border (MHB) fate in a manner that recapitulated their developmental course
Keywords FGF, human pluripotent stem cells, isthmic organizer, neural differentiation, Wnt
The secondary organizer, a specific group of cells that emerges during early embryonic development, can influence the identity of surrounding tissues (Kiecker and Lumsden, 2012). These cells have the ability to secret morphogens, thus specifying the fate of adjacent cells in a space- and time-dependent manner, and elaborating the development of a given tissue. The isthmic organizer (IsO) in the vertebrate central nervous system (CNS) has been comprehensively characterized. Located at the midbrain-hindbrain border (MHB) of the developing neural tube, the IsO influences the induction, proliferation, and differentiation of neural cells between the midbrain and hindbrain by secreting Wnt1 and fibroblast growth factor (FGF) 8 (Rhinn and Brand, 2001; Wurst and Bally-Cuif, 2001). The development of the IsO has been intensively studied in lower vertebrates, such as chick embryos. IsO development begins with anteroposterior (AP) patterning in the neural plate (Partanen, 2007), during which the canonical Wnt signal plays a crucial role in the establishment of the MHB (Ciani and Salinas, 2005; Kiecker and Lumsden, 2005; Wurst and Bally-Cuif, 2001). It is known that the position of the IsO is determined by the juxtaposition of two homeobox domain-containing transcription factors (Otx2 and Gbx2) (Simeone, 2000), followed by the expression of key transcription factors, such as En1, En2, Pax2, and Pax5, as well as Wnt1 and FGF8 in the MHB in a temporally and spatially controlled manner (Wurst and Bally-Cuif, 2001). Previous studies have shown that either the targeted ablation of Wnt1 or the aberrant expression of FGF8 will disturb the normal morphogenesis of the midbrain and hindbrain, and may cause cerebellar malformations (Crossley et al., 1996; McMahon et al., 1992), thus substantiating their pivotal roles in MHB formation. It has been shown that convergent Wnt and FGF signals precisely induce the formation of the IsO (Olander et al., 2006) and that the expression of both Wnt1 and FGF8 in the IsO can maintain the integrity of the MHB (Canning et al., 2007; Ciani and Salinas, 2005; Kiecker and Lumsden, 2005). On the other hand, the development of the IsO in higher mammals, like humans, remains unexplored, primarily due to the lack of an appropriate model system. Considering several congenital developmental defects that are relevant to the IsO (Barkovich et al., 2009; Basson and Wingate, 2013), there is a demand for a faithful model system in which to study the cellular and molecular mechanisms underlying MHB and IsO development.
In this study, we induced the differentiation of human ESCs into cells with characteristics of the IsO. Taking advantage of a neural differentiation technology, we attempted to regionalize human ESC-derived neural precursors (NPs) to the MHB by the precise control of canonical Wnt signaling. We also tested whether exogenous FGF8 was required for the generation of IsO-like cells. Finally, we characterized IsO-like cells by microarray analysis and assessed their functionality using lysates and conditioned media generated from these cells.
Human ESCs (H9, WiCell Inc., USA) were cultured in human ESC medium composed of DMEM/F12 medium (Invitrogen, USA) supplemented with 20% Knockout-Serum Replacement (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM beta-mercaptoethanol (Sigma, USA), and 4 ng/mL basic FGF (Peprotech, USA). For differentiation, embryoid bodies (EBs) were formed by mechanically detaching ESC colonies and culturing them in DMEM/F12:Neurobasal media (Invitrogen) (1:1), 1% N2 supplement (Invitrogen), and 2% B27 supplement without vitamin A (Invitrogen). On day 4 of differentiation, these EBs were plated onto Matrigel (BD Biosciences, USA)-coated dishes and cultured in the same medium except that the concentrations of N2 and B27 supplements were reduced by half (0.5%) for five days. During the first four days, 5 μM dorsomorphin (Calbiochem, USA) and 10 μM SB431542 (SB) (Sigma) were added to the medium to facilitate neural induction. To induce IsO-like cells, CHIR99021 (CHIR) at various concentrations (0–1.2 μM) (Calbiochem) and 100 ng/ml FGF8 (Peprotech) were added to the medium as described in
Total RNA was isolated using the Easy-Spin Total RNA Extraction kit (iNtRON Biotechnology, Korea). cDNA was synthesized from 1 μg total RNA using the PrimeScript RT Master Mix (Takara Bio, Japan). Transcript levels of each marker gene were quantified by real-time PCR using SYBR Premix Ex
IsO-like cells and control cells (DMSO-treated and/or dorsomorphin + SB treated cells) were differentiated from human ESCs until day 7. After a thorough rinse with Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen) to avoid potential contamination with any exogenous factor added to the culture, cells were detached from the culture dish using a curved Pasteur pipette to spontaneously form spherical masses that were then cultured in media devoid of CHIR and FGF8. One day later, the same number of spheres were obtained from each experimental group and sonicated completely in lysis buffer. Supernatants were isolated from the cellular debris by centrifugation, and the concentration of total protein was measured using the Bradford protein assay. Human WNT1 and FGF8 kits (Cat. # CSB-EL026128HU and CSB-E15861h, CUSABIO Life-Science, Baltimore, MD, USA) were used to detect WNT1 and FGF8 protein levels in the cell lysates, following the manufacturer’s protocol. Protein levels of WNT1 and FGF8 were quantified relative to total protein levels.
Ten micrograms of total RNA from each sample were collected and analyzed using a Human HT-12 Expression v.4.0 bead array (Macrogen, Korea). For clustering analysis, normalized data were narrowed down to 14,548 using a cutoff value that was based on a fail count of less ≤ 3. Gene Ontology (GO) analysis was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery). Significantly upregulated and downregulated genes were compared against DAVID’s GO FAT database to clarify their biological significance.
Cells were fixed with 4% of paraformaldehyde at day 7, and permeablized with 0.1% of triton X-100 DPBS solution for 10 min and then blocked with 2% bovine serum albumin solution for at least 1 h. Then, cells were incubated with primary antibodies (mouse anti-WNT1 antibody, Abcam (ab91191), Cambridge, UK, 1:200; mouse anti-FGF8 antibody, Novus Biologicals (47109), Littleton, CO, USA, 1:50) for overnight at 4°C. After washing with DPBS, cells were exposed to a fluorescence-tagged secondary antibody (anti-mouse Alexa Fluor 488, Invitrogen, 1:500) for 1 h and mounted in DAPI-containing medium (Vector Laboratories, USA). IX71 microscope equipped with a DP71 digital camera (Olympus, Japan) was used to obtain images.
Cell lysates were obtained using the same method described above. Cell lysates obtained from IsO-like cells and control cells were added to the culture after EB attachment at two different concentrations (1×, 96 μg/ml; 0.5×, 48 μg/ml) for four days. To collect conditioned media from IsO-like cells, human ESCs were differentiated for 6.5 days using the protocol described in
All data are presented as the mean ± SEM from at least three independent experiments. Statistical significance was evaluated using a two-tailed Student’s
We induced neuroectoderm formation from human ESCs through the simultaneous inhibition of BMP and activ-in/nodal signals with the small molecule inhibitors dorsomorphin and SB (Kim et al., 2010). Human ESCs were cultured as EBs in chemically defined conditions and supplemented with dorsomorphin and SB for four days. EBs were allowed to attach onto Matrigel-coated dishes for induction of primitive NPs.
The early developmental program of the vertebrate CNS favors an anterior fate unless caudalizing cues are present (Stern, 2001). Mounting evidence has shown that NPs that are differentiated from human ESCs retain forebrain-like characteristics, supporting the notion of ‘default differentiation’ (Lupo et al., 2014). Therefore, induction of the IsO characteristics requires caudalization of NPs into the caudal midbrain/anterior hindbrain. It is known that AP patterning in the early vertebrate neural tube is established by graded Wnt signaling and that caudal neural cells are specified by high levels of Wnt ligand derived from paraxial mesoderm (Nordström et al., 2002). Therefore, we decided to modulate canonical Wnt signaling. Instead of using a natural Wnt ligand, a well-known GSK3β inhibitor (CHIR) was used to treat NPs as a Wnt signaling agonist, because it has been shown to be less toxic and a more potent activator of the Wnt/β-catenin pathway than other GSK3β inhibitors (Naujok et al., 2014).
Since graded Wnt signaling gives rise to neural cells with a distinctive regional identity along the AP axis (Nordström et al., 2002), we first determined the optimal concentration of CHIR for IsO generation. After treatment with CHIR at various concentrations (0–1.2 μM) for nine days, relative gene expression analysis revealed that neural cells tended to have a posterior fate with increased expression of
To further test whether this culture condition generates IsO-like cells, we examined the expression of two IsO markers: The
Next, we examined the time-course expression of various marker genes during the induction of IsO-like cells. Once differentiation was initiated, pluripotent markers, such as
To characterize IsO-like cells in greater detail, we analyzed global gene expression profiles. Microarray data were subjected to GO enrichment analysis using DAVID (Fig. 4A and
The key feature of the IsO is its ability to influence the fate of adjacent cells by producing functional WNT1 and FGF8 (Wurst and Bally-Cuif, 2001). To examine whether our differentiated IsO-like cells had such an activity, we first measured the levels of WNT1 and FGF8 in cell lysates on differentiation day 7 by ELISA. Protein levels of WNT1 were found to be 2.6 times higher in IsO-like cells than in DMSO-treated control cells (185.4 pg/μg total protein vs. 69.8 pg/μg total protein, respectively). There was a slight increase in WNT1 protein levels in IsO-like cells compared to those in dorsomorphin + SB treatment control cells (169.5 pg/μg total protein). However, this increase was not statistically significant (Fig. 4B). In contrast, protein levels of FGF8 in IsO-like cells were significantly higher than those in both DMSO-treated and dorsomorphin + SB treated cells (114.4 pg/μg total protein vs. 6.1 and 15.3 pg/μg total protein, respectively), consistent with its transcriptional levels (Figs. 2 and 4B and 4C). Immunocytochemical staining also revealed that many IsO-like cells were positively labeled by specific antibodies for WNT1 and FGF8 (Figs. 4D and 4E).
We hypothesized that if both WNT1 and FGF8 in cell lysates were functionally active, MHB gene expression might be induced in differentiating human ESCs. To test this hypothesis, we treated EBs with cell lysates at two concentrations (0.5× and 1×) after attachment on a Matrigel-coated dish. After four days of treatment, gene expression analysis showed that expression levels of
To further validate the IsO-like activity of these cells, we treated EBs with conditioned media. To our surprise, expression levels of all genes including
In this study, we describe a method for generating cells of the MHB with IsO activity from human ESCs. We found that a graded activation of canonical Wnt signaling during the neural induction of human ESCs transformed neural cells from a rostral to a caudal fate with antagonistic expression of
Our comprehensive analysis of the transcriptome determined that induced IsO-like cells exhibited gene expression profiles with features of the MHB. Most of the highly upregulated genes in IsO-inducing conditions were involved in midbrain and hindbrain formation and early neurogenesis (e.g.,
The ability to produce and secrete Wnt1 and FGF8 is a hallmark of the activity of the IsO as a local signaling center. Even though our differentiation conditions generated cells with such an activity, it is intriguing that the increase in the production of FGF8 was more prominent than that of Wnt1 (Fig. 4B–4C). Previously, FGF8 was shown to exhibit a partial organizing activity of the IsO in chick embryos, in which the implantation of FGF8-soaked microbeads into the hindbrain modulated gene expression similar to that of ectopically transplanted IsO tissue (Irving and Mason, 2000). Therefore, one may doubt that induction of MHB genes in differentiating human ESCs by IsO-like cell-conditioned medium might be solely attributed to the secretion of FGF8, which is reminiscent of the activity of the anterior neural ridge (ANR), another secondary organizer responsible for the maintenance of forebrain identity (Shimamura and Rubenstein, 1997). In this previous study (Irving and Mason, 2000), however, the molecular machinery for caudal regionalization was already present in the host cells. Therefore, FGF8 beads might be able to mimic the IsO. In contrast, differentiating ESCs acquire a rostral fate by default unless a caudalizing signal (i.e., Wnt) is present. If FGF8 is the primary organizing factor in the conditioned medium, it would foster a rostral fate as an ‘ANR’-like activity. However, we found that the upregulation of MHB genes by the conditioned media of IsO-like cells is consistent with the activity of WNT1 secreted from IsO-like cells.
One important finding obtained from the ectopic transplantation experiment with IsO tissue in avian embryos was that the inductive effect of the IsO graft is always asymmetrical (Wurst and Bally-Cuif, 2001). In other words, new midbrain and hindbrain structures were induced in a polarized manner, depending on the rostrocaudal orientation of the IsO graft. Although the molecular basis underlying such an asymmetrical induction has not been clearly demonstrated, numerous genetic studies have provided evidence that genetic interactions between Otx2 and Gbx2 in IsO grafts could orchestrate the induction and maintenance of newly formed midbrain and hindbrain in the host, and may involve the locally restricted expression of Wnt1 and FGF8 in the rostral (Otx2-positive) and caudal (Gbx2-positive) regions, respectively (Wurst and Bally-Cuif, 2001). An asymmetric inductive effect is a key feature of the IsO. Unfortunately, our system was unable to recapitulate such activity, because
In conclusion, our differentiation conditions, which involve the precise modulation of canonical Wnt signaling along with co-stimulation of FGF8 signaling during neural induction, give rise to IsO-like cells with gene expression profiles of the MHB and the ability to secrete functional Wnt1 and FGF8. We believe that our new model system can be used to study CNS development in humans and the etiology of congenital malformations related to the midbrain and hindbrain.
Mol. Cells 2018; 41(2): 110-118
Published online February 28, 2018 https://doi.org/10.14348/molcells.2018.2210
Copyright © The Korean Society for Molecular and Cellular Biology.
Junwon Lee1,2,3,5, Sang-Hwi Choi1,3,5, Dongjin R Lee1,3, Dae-Sung Kim4,*, and Dong-Wook Kim1,3,*
1Department of Physiology, Yonsei University College of Medicine, Seoul 03722, Korea, 2Department of Ophthalmology, Yonsei University College of Medicine, Seoul 03722, Korea, 3Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Korea, 4Department of Biotechnology, Brain Korea 21 PLUS Project for Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea
Correspondence to:*Correspondence: dwkim2@yuhs.ac (DWK); sonnet10@korea.ac.kr (DSK)
The objective of this study was to induce the production of isthmic organizer (IsO)-like cells capable of secreting fibroblast growth factor (FGF) 8 and WNT1 from human embryonic stem cells (ESCs). The precise modulation of canonical Wnt signaling was achieved in the presence of the small molecule CHIR99021 (0.6 μM) during the neural induction of human ESCs, resulting in the differentiation of these cells into IsO-like cells having a midbrain-hindbrain border (MHB) fate in a manner that recapitulated their developmental course
Keywords: FGF, human pluripotent stem cells, isthmic organizer, neural differentiation, Wnt
The secondary organizer, a specific group of cells that emerges during early embryonic development, can influence the identity of surrounding tissues (Kiecker and Lumsden, 2012). These cells have the ability to secret morphogens, thus specifying the fate of adjacent cells in a space- and time-dependent manner, and elaborating the development of a given tissue. The isthmic organizer (IsO) in the vertebrate central nervous system (CNS) has been comprehensively characterized. Located at the midbrain-hindbrain border (MHB) of the developing neural tube, the IsO influences the induction, proliferation, and differentiation of neural cells between the midbrain and hindbrain by secreting Wnt1 and fibroblast growth factor (FGF) 8 (Rhinn and Brand, 2001; Wurst and Bally-Cuif, 2001). The development of the IsO has been intensively studied in lower vertebrates, such as chick embryos. IsO development begins with anteroposterior (AP) patterning in the neural plate (Partanen, 2007), during which the canonical Wnt signal plays a crucial role in the establishment of the MHB (Ciani and Salinas, 2005; Kiecker and Lumsden, 2005; Wurst and Bally-Cuif, 2001). It is known that the position of the IsO is determined by the juxtaposition of two homeobox domain-containing transcription factors (Otx2 and Gbx2) (Simeone, 2000), followed by the expression of key transcription factors, such as En1, En2, Pax2, and Pax5, as well as Wnt1 and FGF8 in the MHB in a temporally and spatially controlled manner (Wurst and Bally-Cuif, 2001). Previous studies have shown that either the targeted ablation of Wnt1 or the aberrant expression of FGF8 will disturb the normal morphogenesis of the midbrain and hindbrain, and may cause cerebellar malformations (Crossley et al., 1996; McMahon et al., 1992), thus substantiating their pivotal roles in MHB formation. It has been shown that convergent Wnt and FGF signals precisely induce the formation of the IsO (Olander et al., 2006) and that the expression of both Wnt1 and FGF8 in the IsO can maintain the integrity of the MHB (Canning et al., 2007; Ciani and Salinas, 2005; Kiecker and Lumsden, 2005). On the other hand, the development of the IsO in higher mammals, like humans, remains unexplored, primarily due to the lack of an appropriate model system. Considering several congenital developmental defects that are relevant to the IsO (Barkovich et al., 2009; Basson and Wingate, 2013), there is a demand for a faithful model system in which to study the cellular and molecular mechanisms underlying MHB and IsO development.
In this study, we induced the differentiation of human ESCs into cells with characteristics of the IsO. Taking advantage of a neural differentiation technology, we attempted to regionalize human ESC-derived neural precursors (NPs) to the MHB by the precise control of canonical Wnt signaling. We also tested whether exogenous FGF8 was required for the generation of IsO-like cells. Finally, we characterized IsO-like cells by microarray analysis and assessed their functionality using lysates and conditioned media generated from these cells.
Human ESCs (H9, WiCell Inc., USA) were cultured in human ESC medium composed of DMEM/F12 medium (Invitrogen, USA) supplemented with 20% Knockout-Serum Replacement (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM beta-mercaptoethanol (Sigma, USA), and 4 ng/mL basic FGF (Peprotech, USA). For differentiation, embryoid bodies (EBs) were formed by mechanically detaching ESC colonies and culturing them in DMEM/F12:Neurobasal media (Invitrogen) (1:1), 1% N2 supplement (Invitrogen), and 2% B27 supplement without vitamin A (Invitrogen). On day 4 of differentiation, these EBs were plated onto Matrigel (BD Biosciences, USA)-coated dishes and cultured in the same medium except that the concentrations of N2 and B27 supplements were reduced by half (0.5%) for five days. During the first four days, 5 μM dorsomorphin (Calbiochem, USA) and 10 μM SB431542 (SB) (Sigma) were added to the medium to facilitate neural induction. To induce IsO-like cells, CHIR99021 (CHIR) at various concentrations (0–1.2 μM) (Calbiochem) and 100 ng/ml FGF8 (Peprotech) were added to the medium as described in
Total RNA was isolated using the Easy-Spin Total RNA Extraction kit (iNtRON Biotechnology, Korea). cDNA was synthesized from 1 μg total RNA using the PrimeScript RT Master Mix (Takara Bio, Japan). Transcript levels of each marker gene were quantified by real-time PCR using SYBR Premix Ex
IsO-like cells and control cells (DMSO-treated and/or dorsomorphin + SB treated cells) were differentiated from human ESCs until day 7. After a thorough rinse with Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen) to avoid potential contamination with any exogenous factor added to the culture, cells were detached from the culture dish using a curved Pasteur pipette to spontaneously form spherical masses that were then cultured in media devoid of CHIR and FGF8. One day later, the same number of spheres were obtained from each experimental group and sonicated completely in lysis buffer. Supernatants were isolated from the cellular debris by centrifugation, and the concentration of total protein was measured using the Bradford protein assay. Human WNT1 and FGF8 kits (Cat. # CSB-EL026128HU and CSB-E15861h, CUSABIO Life-Science, Baltimore, MD, USA) were used to detect WNT1 and FGF8 protein levels in the cell lysates, following the manufacturer’s protocol. Protein levels of WNT1 and FGF8 were quantified relative to total protein levels.
Ten micrograms of total RNA from each sample were collected and analyzed using a Human HT-12 Expression v.4.0 bead array (Macrogen, Korea). For clustering analysis, normalized data were narrowed down to 14,548 using a cutoff value that was based on a fail count of less ≤ 3. Gene Ontology (GO) analysis was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery). Significantly upregulated and downregulated genes were compared against DAVID’s GO FAT database to clarify their biological significance.
Cells were fixed with 4% of paraformaldehyde at day 7, and permeablized with 0.1% of triton X-100 DPBS solution for 10 min and then blocked with 2% bovine serum albumin solution for at least 1 h. Then, cells were incubated with primary antibodies (mouse anti-WNT1 antibody, Abcam (ab91191), Cambridge, UK, 1:200; mouse anti-FGF8 antibody, Novus Biologicals (47109), Littleton, CO, USA, 1:50) for overnight at 4°C. After washing with DPBS, cells were exposed to a fluorescence-tagged secondary antibody (anti-mouse Alexa Fluor 488, Invitrogen, 1:500) for 1 h and mounted in DAPI-containing medium (Vector Laboratories, USA). IX71 microscope equipped with a DP71 digital camera (Olympus, Japan) was used to obtain images.
Cell lysates were obtained using the same method described above. Cell lysates obtained from IsO-like cells and control cells were added to the culture after EB attachment at two different concentrations (1×, 96 μg/ml; 0.5×, 48 μg/ml) for four days. To collect conditioned media from IsO-like cells, human ESCs were differentiated for 6.5 days using the protocol described in
All data are presented as the mean ± SEM from at least three independent experiments. Statistical significance was evaluated using a two-tailed Student’s
We induced neuroectoderm formation from human ESCs through the simultaneous inhibition of BMP and activ-in/nodal signals with the small molecule inhibitors dorsomorphin and SB (Kim et al., 2010). Human ESCs were cultured as EBs in chemically defined conditions and supplemented with dorsomorphin and SB for four days. EBs were allowed to attach onto Matrigel-coated dishes for induction of primitive NPs.
The early developmental program of the vertebrate CNS favors an anterior fate unless caudalizing cues are present (Stern, 2001). Mounting evidence has shown that NPs that are differentiated from human ESCs retain forebrain-like characteristics, supporting the notion of ‘default differentiation’ (Lupo et al., 2014). Therefore, induction of the IsO characteristics requires caudalization of NPs into the caudal midbrain/anterior hindbrain. It is known that AP patterning in the early vertebrate neural tube is established by graded Wnt signaling and that caudal neural cells are specified by high levels of Wnt ligand derived from paraxial mesoderm (Nordström et al., 2002). Therefore, we decided to modulate canonical Wnt signaling. Instead of using a natural Wnt ligand, a well-known GSK3β inhibitor (CHIR) was used to treat NPs as a Wnt signaling agonist, because it has been shown to be less toxic and a more potent activator of the Wnt/β-catenin pathway than other GSK3β inhibitors (Naujok et al., 2014).
Since graded Wnt signaling gives rise to neural cells with a distinctive regional identity along the AP axis (Nordström et al., 2002), we first determined the optimal concentration of CHIR for IsO generation. After treatment with CHIR at various concentrations (0–1.2 μM) for nine days, relative gene expression analysis revealed that neural cells tended to have a posterior fate with increased expression of
To further test whether this culture condition generates IsO-like cells, we examined the expression of two IsO markers: The
Next, we examined the time-course expression of various marker genes during the induction of IsO-like cells. Once differentiation was initiated, pluripotent markers, such as
To characterize IsO-like cells in greater detail, we analyzed global gene expression profiles. Microarray data were subjected to GO enrichment analysis using DAVID (Fig. 4A and
The key feature of the IsO is its ability to influence the fate of adjacent cells by producing functional WNT1 and FGF8 (Wurst and Bally-Cuif, 2001). To examine whether our differentiated IsO-like cells had such an activity, we first measured the levels of WNT1 and FGF8 in cell lysates on differentiation day 7 by ELISA. Protein levels of WNT1 were found to be 2.6 times higher in IsO-like cells than in DMSO-treated control cells (185.4 pg/μg total protein vs. 69.8 pg/μg total protein, respectively). There was a slight increase in WNT1 protein levels in IsO-like cells compared to those in dorsomorphin + SB treatment control cells (169.5 pg/μg total protein). However, this increase was not statistically significant (Fig. 4B). In contrast, protein levels of FGF8 in IsO-like cells were significantly higher than those in both DMSO-treated and dorsomorphin + SB treated cells (114.4 pg/μg total protein vs. 6.1 and 15.3 pg/μg total protein, respectively), consistent with its transcriptional levels (Figs. 2 and 4B and 4C). Immunocytochemical staining also revealed that many IsO-like cells were positively labeled by specific antibodies for WNT1 and FGF8 (Figs. 4D and 4E).
We hypothesized that if both WNT1 and FGF8 in cell lysates were functionally active, MHB gene expression might be induced in differentiating human ESCs. To test this hypothesis, we treated EBs with cell lysates at two concentrations (0.5× and 1×) after attachment on a Matrigel-coated dish. After four days of treatment, gene expression analysis showed that expression levels of
To further validate the IsO-like activity of these cells, we treated EBs with conditioned media. To our surprise, expression levels of all genes including
In this study, we describe a method for generating cells of the MHB with IsO activity from human ESCs. We found that a graded activation of canonical Wnt signaling during the neural induction of human ESCs transformed neural cells from a rostral to a caudal fate with antagonistic expression of
Our comprehensive analysis of the transcriptome determined that induced IsO-like cells exhibited gene expression profiles with features of the MHB. Most of the highly upregulated genes in IsO-inducing conditions were involved in midbrain and hindbrain formation and early neurogenesis (e.g.,
The ability to produce and secrete Wnt1 and FGF8 is a hallmark of the activity of the IsO as a local signaling center. Even though our differentiation conditions generated cells with such an activity, it is intriguing that the increase in the production of FGF8 was more prominent than that of Wnt1 (Fig. 4B–4C). Previously, FGF8 was shown to exhibit a partial organizing activity of the IsO in chick embryos, in which the implantation of FGF8-soaked microbeads into the hindbrain modulated gene expression similar to that of ectopically transplanted IsO tissue (Irving and Mason, 2000). Therefore, one may doubt that induction of MHB genes in differentiating human ESCs by IsO-like cell-conditioned medium might be solely attributed to the secretion of FGF8, which is reminiscent of the activity of the anterior neural ridge (ANR), another secondary organizer responsible for the maintenance of forebrain identity (Shimamura and Rubenstein, 1997). In this previous study (Irving and Mason, 2000), however, the molecular machinery for caudal regionalization was already present in the host cells. Therefore, FGF8 beads might be able to mimic the IsO. In contrast, differentiating ESCs acquire a rostral fate by default unless a caudalizing signal (i.e., Wnt) is present. If FGF8 is the primary organizing factor in the conditioned medium, it would foster a rostral fate as an ‘ANR’-like activity. However, we found that the upregulation of MHB genes by the conditioned media of IsO-like cells is consistent with the activity of WNT1 secreted from IsO-like cells.
One important finding obtained from the ectopic transplantation experiment with IsO tissue in avian embryos was that the inductive effect of the IsO graft is always asymmetrical (Wurst and Bally-Cuif, 2001). In other words, new midbrain and hindbrain structures were induced in a polarized manner, depending on the rostrocaudal orientation of the IsO graft. Although the molecular basis underlying such an asymmetrical induction has not been clearly demonstrated, numerous genetic studies have provided evidence that genetic interactions between Otx2 and Gbx2 in IsO grafts could orchestrate the induction and maintenance of newly formed midbrain and hindbrain in the host, and may involve the locally restricted expression of Wnt1 and FGF8 in the rostral (Otx2-positive) and caudal (Gbx2-positive) regions, respectively (Wurst and Bally-Cuif, 2001). An asymmetric inductive effect is a key feature of the IsO. Unfortunately, our system was unable to recapitulate such activity, because
In conclusion, our differentiation conditions, which involve the precise modulation of canonical Wnt signaling along with co-stimulation of FGF8 signaling during neural induction, give rise to IsO-like cells with gene expression profiles of the MHB and the ability to secrete functional Wnt1 and FGF8. We believe that our new model system can be used to study CNS development in humans and the etiology of congenital malformations related to the midbrain and hindbrain.
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