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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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Expression of hippocampus marker Zbtb20 in medial pallium-like tissue.Under the optimized conditions, the aggregate was cultured with less formation of rosette-like NE (a,b). (e–h is box in c) Immunostaining on day 61 showed that the medial pallium-like continuous NE, positive for Foxg1::Venus (e) and Lef1 (f) were formed adjacent to the Lmx1a+ choroid plexus-like domain (c,d). (g) The more basal layer of the Lef1+ NE was positive for Nrp2. (h) Zbtb20 was expressed in the Lef1+ NE. (i) Zbtb20 mRNA expression at day 73 showed a significant increase compared with day 18. *P<0.05, n=3, Unpaired t-test with Welch's correction. (j) The medial pallium-like continuous NE, positive for Foxg1::Venus and Lef1 were clearly formed adjacent to the Lmx1a+ choroid plexus-like domain even at day 75. (k,l) Zbtb20 expression in cells beneath the Lef1+ NE was clearly seen at day 75 (l is enlarged box in k). Scale bars, 300 μm (a,b); 200 μm (c–h); 100 μm (j,k); 50 μm (l). Bar in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
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f3: Expression of hippocampus marker Zbtb20 in medial pallium-like tissue.Under the optimized conditions, the aggregate was cultured with less formation of rosette-like NE (a,b). (e–h is box in c) Immunostaining on day 61 showed that the medial pallium-like continuous NE, positive for Foxg1::Venus (e) and Lef1 (f) were formed adjacent to the Lmx1a+ choroid plexus-like domain (c,d). (g) The more basal layer of the Lef1+ NE was positive for Nrp2. (h) Zbtb20 was expressed in the Lef1+ NE. (i) Zbtb20 mRNA expression at day 73 showed a significant increase compared with day 18. *P<0.05, n=3, Unpaired t-test with Welch's correction. (j) The medial pallium-like continuous NE, positive for Foxg1::Venus and Lef1 were clearly formed adjacent to the Lmx1a+ choroid plexus-like domain even at day 75. (k,l) Zbtb20 expression in cells beneath the Lef1+ NE was clearly seen at day 75 (l is enlarged box in k). Scale bars, 300 μm (a,b); 200 μm (c–h); 100 μm (j,k); 50 μm (l). Bar in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).

Mentions: Under these conditions, the aggregates had less rosette-like NE (Fig. 3a,b), and on day 61, Lef1+/Foxg1::Venus+ medial pallium-like continuous NE portions clearly formed adjacent to the Lmx1a+ choroid plexus- and hem-like domain (Fig. 3c–f). Importantly, a more basal layer of the Lef1+ NE was positive for Nrp2 (neuropillin 2) (Fig. 3g), as seen in the embryonic hippocampus21. In addition, the Lef1+ NE expressed Zbtb20 (BTB/POZ zinc-finger family; Fig. 3h), a marker for hippocampal neurons and their precursors (Supplementary Fig. 3c)22. This was also confirmed using RT–qPCR (Fig. 3i). Even in culture for over 70 days, Lef1+/Foxg1::Venus+ continuous NE clearly formed adjacent to the Lmx1a+ /Foxg1::Venus− domain (Fig. 3j,k). IHC showed clear Zbtb20 expression in cells beneath the Lef1+ NE (Fig. 3l), and weak Zbtb20 expression in the NE (Fig. 3l). Collectively, these results demonstrate the successful in vitro generation of hippocampal primordium-like tissue.


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)

Expression of hippocampus marker Zbtb20 in medial pallium-like tissue.Under the optimized conditions, the aggregate was cultured with less formation of rosette-like NE (a,b). (e–h is box in c) Immunostaining on day 61 showed that the medial pallium-like continuous NE, positive for Foxg1::Venus (e) and Lef1 (f) were formed adjacent to the Lmx1a+ choroid plexus-like domain (c,d). (g) The more basal layer of the Lef1+ NE was positive for Nrp2. (h) Zbtb20 was expressed in the Lef1+ NE. (i) Zbtb20 mRNA expression at day 73 showed a significant increase compared with day 18. *P<0.05, n=3, Unpaired t-test with Welch's correction. (j) The medial pallium-like continuous NE, positive for Foxg1::Venus and Lef1 were clearly formed adjacent to the Lmx1a+ choroid plexus-like domain even at day 75. (k,l) Zbtb20 expression in cells beneath the Lef1+ NE was clearly seen at day 75 (l is enlarged box in k). Scale bars, 300 μm (a,b); 200 μm (c–h); 100 μm (j,k); 50 μm (l). Bar 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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f3: Expression of hippocampus marker Zbtb20 in medial pallium-like tissue.Under the optimized conditions, the aggregate was cultured with less formation of rosette-like NE (a,b). (e–h is box in c) Immunostaining on day 61 showed that the medial pallium-like continuous NE, positive for Foxg1::Venus (e) and Lef1 (f) were formed adjacent to the Lmx1a+ choroid plexus-like domain (c,d). (g) The more basal layer of the Lef1+ NE was positive for Nrp2. (h) Zbtb20 was expressed in the Lef1+ NE. (i) Zbtb20 mRNA expression at day 73 showed a significant increase compared with day 18. *P<0.05, n=3, Unpaired t-test with Welch's correction. (j) The medial pallium-like continuous NE, positive for Foxg1::Venus and Lef1 were clearly formed adjacent to the Lmx1a+ choroid plexus-like domain even at day 75. (k,l) Zbtb20 expression in cells beneath the Lef1+ NE was clearly seen at day 75 (l is enlarged box in k). Scale bars, 300 μm (a,b); 200 μm (c–h); 100 μm (j,k); 50 μm (l). Bar in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
Mentions: Under these conditions, the aggregates had less rosette-like NE (Fig. 3a,b), and on day 61, Lef1+/Foxg1::Venus+ medial pallium-like continuous NE portions clearly formed adjacent to the Lmx1a+ choroid plexus- and hem-like domain (Fig. 3c–f). Importantly, a more basal layer of the Lef1+ NE was positive for Nrp2 (neuropillin 2) (Fig. 3g), as seen in the embryonic hippocampus21. In addition, the Lef1+ NE expressed Zbtb20 (BTB/POZ zinc-finger family; Fig. 3h), a marker for hippocampal neurons and their precursors (Supplementary Fig. 3c)22. This was also confirmed using RT–qPCR (Fig. 3i). Even in culture for over 70 days, Lef1+/Foxg1::Venus+ continuous NE clearly formed adjacent to the Lmx1a+ /Foxg1::Venus− domain (Fig. 3j,k). IHC showed clear Zbtb20 expression in cells beneath the Lef1+ NE (Fig. 3l), and weak Zbtb20 expression in the NE (Fig. 3l). Collectively, these results demonstrate the successful in vitro generation of hippocampal primordium-like tissue.

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