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Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue.

Sakaguchi H, Kadoshima T, Soen M, Narii N, Ishida Y, Ohgushi M, Takahashi J, Eiraku M, Sasai Y - Nat Commun (2015)

Bottom Line: Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases.Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs).Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Organogenesis and Neurogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20(+)/Prox1(+) granule neurons and Zbtb20(+)/KA1(+) pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

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Transient exposure of dorsalizing factors can induce patterned dorsomedial telencephalic tissues.(a) Schematic of mouse medial pallium and neighbouring tissues at E12.5–13.5. (b) Schematic of condition to induce medial pallium tissues. (c) Bright-field view of aggregates cultured in condition 3 on day 36. (d,e) By shortening the period (day 18–21), the aggregates expressed Foxg1::Venus with patterning into Foxg1::Venus+ (arrow) and Venus− (arrowhead) NE domains. (f) Histogram of percentage of Foxg1::Venus− protrusion and Foxg1::Venus+ main body induction. Patterning of Foxg1::Venus+/Venus− NE domains were induced in around 70–80% of aggregates (bars in graph, s.e.m.). (g–l) The Venus− NE domains of aggregates (g) showed TTR+/Lmx1a+ in distal parts (h,i) and Lmx1a+/TTR− in proximal parts (j–l, box in h). (m–o) In hESC-derived NE, Foxg1::Venus+ main bodies expressed Lef1 (m) and Lhx2 (n). (o) On the basal side, the thick cell layer of the aggregate expressed Tbr1 and Lhx5. (p) Foxg1::Venus and Lef1 co-expressed in continuous NE (p,q, box in h), suggesting medial pallium-like NE generation. (r) Schematic of hESC-derived dorsomedial telencephalic tissues. Choroid plexus-, hem- and medial pallium-like regions were continuously generated as seen in vivo. (s) Schematic of method to examine Wnts/BMPs expression in Venus− protrusions. The aggregates were cut into Venus− protrusions and Venus+ main bodies at day 35–40, and wnts/bmps expression were examined using RT–qPCR. (t) qPCR for genes expressed in Venus− protrusion versus Venus+ main body (**P<0.01; ***P<0.001, n=3, unpaired t-test). foxg1 was significantly attenuated in Venus− protrusions, and a significant increase in lmx1a/ttr mRNA expression in Venus− protrusions was confirmed. wnt 2b/3a/5a and bmp 4/6 significantly increased in Venus− protrusions. (u) Schematic summary of conditions examined in Figs 1 and 2. Scale bars, 500 μm (c–e); 200 μm (g–p). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
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f2: Transient exposure of dorsalizing factors can induce patterned dorsomedial telencephalic tissues.(a) Schematic of mouse medial pallium and neighbouring tissues at E12.5–13.5. (b) Schematic of condition to induce medial pallium tissues. (c) Bright-field view of aggregates cultured in condition 3 on day 36. (d,e) By shortening the period (day 18–21), the aggregates expressed Foxg1::Venus with patterning into Foxg1::Venus+ (arrow) and Venus− (arrowhead) NE domains. (f) Histogram of percentage of Foxg1::Venus− protrusion and Foxg1::Venus+ main body induction. Patterning of Foxg1::Venus+/Venus− NE domains were induced in around 70–80% of aggregates (bars in graph, s.e.m.). (g–l) The Venus− NE domains of aggregates (g) showed TTR+/Lmx1a+ in distal parts (h,i) and Lmx1a+/TTR− in proximal parts (j–l, box in h). (m–o) In hESC-derived NE, Foxg1::Venus+ main bodies expressed Lef1 (m) and Lhx2 (n). (o) On the basal side, the thick cell layer of the aggregate expressed Tbr1 and Lhx5. (p) Foxg1::Venus and Lef1 co-expressed in continuous NE (p,q, box in h), suggesting medial pallium-like NE generation. (r) Schematic of hESC-derived dorsomedial telencephalic tissues. Choroid plexus-, hem- and medial pallium-like regions were continuously generated as seen in vivo. (s) Schematic of method to examine Wnts/BMPs expression in Venus− protrusions. The aggregates were cut into Venus− protrusions and Venus+ main bodies at day 35–40, and wnts/bmps expression were examined using RT–qPCR. (t) qPCR for genes expressed in Venus− protrusion versus Venus+ main body (**P<0.01; ***P<0.001, n=3, unpaired t-test). foxg1 was significantly attenuated in Venus− protrusions, and a significant increase in lmx1a/ttr mRNA expression in Venus− protrusions was confirmed. wnt 2b/3a/5a and bmp 4/6 significantly increased in Venus− protrusions. (u) Schematic summary of conditions examined in Figs 1 and 2. Scale bars, 500 μm (c–e); 200 μm (g–p). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).

Mentions: Since the medial pallium is the precursor to the hippocampus3, and the hem is known to function as a hippocampal organizer16, we next investigated whether hem and medial pallium tissues, which are located adjacent to choroid plexus (Fig. 2a), could be induced by modifying culture conditions. Since Foxg1 was expressed from medial pallium to neocortex, but not in choroid plexus and hem (Fig. 2a; Supplementary Fig. 1a)17, we tried to define the condition that produces aggregates with patterning into Foxg1::Venus+ (estimated as the medial pallium region) and Venus− (estimated as the choroid plexus or hem region) NE domains. We adjusted the timing of the dorsalizing treatment, the concentration of CHIR or BMP4, and the treatment period. These experiments revealed that CHIR+BMP4 exposure during a specific time window was critical for induction of the aggregates with patterning into Foxg1::Venus+ and Venus− NE domains (Fig. 2b–e; Supplementary Fig. 2a). As the exposure period was shortened, Foxg1::Venus expression increased (Supplementary Fig. 2d). We also noticed that the domain of Venus− NE generally formed protrusions in the aggregates (Fig. 2d,e, arrowhead), and the main body of the aggregates expressed Foxg1::Venus (Fig. 2e, arrow). Since the 3-day treatment (days 18–21) was sufficient to induce the patterned expression of Foxg1::Venus in 70–80% of aggregates (Fig. 2b–f, condition 3), we focused on this condition for more in-depth analysis.


Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue.

Sakaguchi H, Kadoshima T, Soen M, Narii N, Ishida Y, Ohgushi M, Takahashi J, Eiraku M, Sasai Y - Nat Commun (2015)

Transient exposure of dorsalizing factors can induce patterned dorsomedial telencephalic tissues.(a) Schematic of mouse medial pallium and neighbouring tissues at E12.5–13.5. (b) Schematic of condition to induce medial pallium tissues. (c) Bright-field view of aggregates cultured in condition 3 on day 36. (d,e) By shortening the period (day 18–21), the aggregates expressed Foxg1::Venus with patterning into Foxg1::Venus+ (arrow) and Venus− (arrowhead) NE domains. (f) Histogram of percentage of Foxg1::Venus− protrusion and Foxg1::Venus+ main body induction. Patterning of Foxg1::Venus+/Venus− NE domains were induced in around 70–80% of aggregates (bars in graph, s.e.m.). (g–l) The Venus− NE domains of aggregates (g) showed TTR+/Lmx1a+ in distal parts (h,i) and Lmx1a+/TTR− in proximal parts (j–l, box in h). (m–o) In hESC-derived NE, Foxg1::Venus+ main bodies expressed Lef1 (m) and Lhx2 (n). (o) On the basal side, the thick cell layer of the aggregate expressed Tbr1 and Lhx5. (p) Foxg1::Venus and Lef1 co-expressed in continuous NE (p,q, box in h), suggesting medial pallium-like NE generation. (r) Schematic of hESC-derived dorsomedial telencephalic tissues. Choroid plexus-, hem- and medial pallium-like regions were continuously generated as seen in vivo. (s) Schematic of method to examine Wnts/BMPs expression in Venus− protrusions. The aggregates were cut into Venus− protrusions and Venus+ main bodies at day 35–40, and wnts/bmps expression were examined using RT–qPCR. (t) qPCR for genes expressed in Venus− protrusion versus Venus+ main body (**P<0.01; ***P<0.001, n=3, unpaired t-test). foxg1 was significantly attenuated in Venus− protrusions, and a significant increase in lmx1a/ttr mRNA expression in Venus− protrusions was confirmed. wnt 2b/3a/5a and bmp 4/6 significantly increased in Venus− protrusions. (u) Schematic summary of conditions examined in Figs 1 and 2. Scale bars, 500 μm (c–e); 200 μm (g–p). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Transient exposure of dorsalizing factors can induce patterned dorsomedial telencephalic tissues.(a) Schematic of mouse medial pallium and neighbouring tissues at E12.5–13.5. (b) Schematic of condition to induce medial pallium tissues. (c) Bright-field view of aggregates cultured in condition 3 on day 36. (d,e) By shortening the period (day 18–21), the aggregates expressed Foxg1::Venus with patterning into Foxg1::Venus+ (arrow) and Venus− (arrowhead) NE domains. (f) Histogram of percentage of Foxg1::Venus− protrusion and Foxg1::Venus+ main body induction. Patterning of Foxg1::Venus+/Venus− NE domains were induced in around 70–80% of aggregates (bars in graph, s.e.m.). (g–l) The Venus− NE domains of aggregates (g) showed TTR+/Lmx1a+ in distal parts (h,i) and Lmx1a+/TTR− in proximal parts (j–l, box in h). (m–o) In hESC-derived NE, Foxg1::Venus+ main bodies expressed Lef1 (m) and Lhx2 (n). (o) On the basal side, the thick cell layer of the aggregate expressed Tbr1 and Lhx5. (p) Foxg1::Venus and Lef1 co-expressed in continuous NE (p,q, box in h), suggesting medial pallium-like NE generation. (r) Schematic of hESC-derived dorsomedial telencephalic tissues. Choroid plexus-, hem- and medial pallium-like regions were continuously generated as seen in vivo. (s) Schematic of method to examine Wnts/BMPs expression in Venus− protrusions. The aggregates were cut into Venus− protrusions and Venus+ main bodies at day 35–40, and wnts/bmps expression were examined using RT–qPCR. (t) qPCR for genes expressed in Venus− protrusion versus Venus+ main body (**P<0.01; ***P<0.001, n=3, unpaired t-test). foxg1 was significantly attenuated in Venus− protrusions, and a significant increase in lmx1a/ttr mRNA expression in Venus− protrusions was confirmed. wnt 2b/3a/5a and bmp 4/6 significantly increased in Venus− protrusions. (u) Schematic summary of conditions examined in Figs 1 and 2. Scale bars, 500 μm (c–e); 200 μm (g–p). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
Mentions: Since the medial pallium is the precursor to the hippocampus3, and the hem is known to function as a hippocampal organizer16, we next investigated whether hem and medial pallium tissues, which are located adjacent to choroid plexus (Fig. 2a), could be induced by modifying culture conditions. Since Foxg1 was expressed from medial pallium to neocortex, but not in choroid plexus and hem (Fig. 2a; Supplementary Fig. 1a)17, we tried to define the condition that produces aggregates with patterning into Foxg1::Venus+ (estimated as the medial pallium region) and Venus− (estimated as the choroid plexus or hem region) NE domains. We adjusted the timing of the dorsalizing treatment, the concentration of CHIR or BMP4, and the treatment period. These experiments revealed that CHIR+BMP4 exposure during a specific time window was critical for induction of the aggregates with patterning into Foxg1::Venus+ and Venus− NE domains (Fig. 2b–e; Supplementary Fig. 2a). As the exposure period was shortened, Foxg1::Venus expression increased (Supplementary Fig. 2d). We also noticed that the domain of Venus− NE generally formed protrusions in the aggregates (Fig. 2d,e, arrowhead), and the main body of the aggregates expressed Foxg1::Venus (Fig. 2e, arrow). Since the 3-day treatment (days 18–21) was sufficient to induce the patterned expression of Foxg1::Venus in 70–80% of aggregates (Fig. 2b–f, condition 3), we focused on this condition for more in-depth analysis.

Bottom Line: Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases.Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs).Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Organogenesis and Neurogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20(+)/Prox1(+) granule neurons and Zbtb20(+)/KA1(+) pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

Show MeSH
Related in: MedlinePlus