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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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Hippocampal marker-positive pyramidal and granule-like neurons were generated by long-term dissociation culture.(a–c) Immunostaining of dissociated cells at day 197. Foxg1::Venus+ neurons (a) were connected to one another by MAP2+ neurites (b,c). GFAP+ glial-like cells were also detected (d). (e,f) KA1+/Foxg1::Venus+ neurons (CA type) tended to have large cell bodies; in contrast, Prox1+/Foxg1::Venus+ neurons (DG type) mostly had small somata (f is enlarged box in e). Zbtb20 was co-expressed with both KA1+ and Prox1+ neurons (g,h). (i) The percentage of Zbtb20+ cells was about 75% (means: 76.5%, s.e.m.: 1.65) and the percentages of KA1+ (means: 34.4%, s.e.m.: 1.83) and Prox1+ (means: 33.8%, s.e.m.: 2.33) were both about 34%. Countered neurons: Zbtb20+ (n=2764), KA1+ (n=147), Prox1+ (n=188). (j) Cell body size was significantly larger in KA1+ cells. ***P<0.001, paired t-test. Countered neurons: KA1+ (n=56), Prox1+ (n=55). (k,l) CaMKII was expressed in KA1+ (k) and Prox1+ (l) neurons. Scale bars, 200 μm (a–c); 50 μm (d); 20 μm (e,g–h); 10 μm (f,k,l). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
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f4: Hippocampal marker-positive pyramidal and granule-like neurons were generated by long-term dissociation culture.(a–c) Immunostaining of dissociated cells at day 197. Foxg1::Venus+ neurons (a) were connected to one another by MAP2+ neurites (b,c). GFAP+ glial-like cells were also detected (d). (e,f) KA1+/Foxg1::Venus+ neurons (CA type) tended to have large cell bodies; in contrast, Prox1+/Foxg1::Venus+ neurons (DG type) mostly had small somata (f is enlarged box in e). Zbtb20 was co-expressed with both KA1+ and Prox1+ neurons (g,h). (i) The percentage of Zbtb20+ cells was about 75% (means: 76.5%, s.e.m.: 1.65) and the percentages of KA1+ (means: 34.4%, s.e.m.: 1.83) and Prox1+ (means: 33.8%, s.e.m.: 2.33) were both about 34%. Countered neurons: Zbtb20+ (n=2764), KA1+ (n=147), Prox1+ (n=188). (j) Cell body size was significantly larger in KA1+ cells. ***P<0.001, paired t-test. Countered neurons: KA1+ (n=56), Prox1+ (n=55). (k,l) CaMKII was expressed in KA1+ (k) and Prox1+ (l) neurons. Scale bars, 200 μm (a–c); 50 μm (d); 20 μm (e,g–h); 10 μm (f,k,l). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).

Mentions: We first tried further long-term culture to assess CA and DG fields, but the organoids could not be maintained beyond day 100 in a healthy condition. Thus, to evaluate the neuronal population and function of the hESC-derived dorsomedial telencephalic tissues, we utilized a dissociation culture that enables long-term culture and facilitates neuronal maturation. Cells were dissociated at days 73–84 and plated on poly-D-lysine-laminin-coated plates (Supplementary Fig. 4c). At day 197 (17 weeks after dispersion), these cells made multiple small aggregations (Fig. 4a), which were connected to one another by numerous MAP2+ neurites (Fig. 4b). The majority of the cells expressed Zbtb20 (Fig. 4b,c). Unexpectedly, we also detected Foxg1::Venus+/GFAP+ glial-like cells (Fig. 4d; Supplementary Fig. 4d–g). Other markers for astrocytes such as S100 beta23 and vimentin24 were also detected in the glial-like cells (Supplementray Fig. 4h–k,l–o). These data demonstrate the generation of hippocampal-like neurons and astrocyte-like cells from hESC-derived dorsomedial telencephalic tissues.


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)

Hippocampal marker-positive pyramidal and granule-like neurons were generated by long-term dissociation culture.(a–c) Immunostaining of dissociated cells at day 197. Foxg1::Venus+ neurons (a) were connected to one another by MAP2+ neurites (b,c). GFAP+ glial-like cells were also detected (d). (e,f) KA1+/Foxg1::Venus+ neurons (CA type) tended to have large cell bodies; in contrast, Prox1+/Foxg1::Venus+ neurons (DG type) mostly had small somata (f is enlarged box in e). Zbtb20 was co-expressed with both KA1+ and Prox1+ neurons (g,h). (i) The percentage of Zbtb20+ cells was about 75% (means: 76.5%, s.e.m.: 1.65) and the percentages of KA1+ (means: 34.4%, s.e.m.: 1.83) and Prox1+ (means: 33.8%, s.e.m.: 2.33) were both about 34%. Countered neurons: Zbtb20+ (n=2764), KA1+ (n=147), Prox1+ (n=188). (j) Cell body size was significantly larger in KA1+ cells. ***P<0.001, paired t-test. Countered neurons: KA1+ (n=56), Prox1+ (n=55). (k,l) CaMKII was expressed in KA1+ (k) and Prox1+ (l) neurons. Scale bars, 200 μm (a–c); 50 μm (d); 20 μm (e,g–h); 10 μm (f,k,l). 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
getmorefigures.php?uid=PMC4660208&req=5

f4: Hippocampal marker-positive pyramidal and granule-like neurons were generated by long-term dissociation culture.(a–c) Immunostaining of dissociated cells at day 197. Foxg1::Venus+ neurons (a) were connected to one another by MAP2+ neurites (b,c). GFAP+ glial-like cells were also detected (d). (e,f) KA1+/Foxg1::Venus+ neurons (CA type) tended to have large cell bodies; in contrast, Prox1+/Foxg1::Venus+ neurons (DG type) mostly had small somata (f is enlarged box in e). Zbtb20 was co-expressed with both KA1+ and Prox1+ neurons (g,h). (i) The percentage of Zbtb20+ cells was about 75% (means: 76.5%, s.e.m.: 1.65) and the percentages of KA1+ (means: 34.4%, s.e.m.: 1.83) and Prox1+ (means: 33.8%, s.e.m.: 2.33) were both about 34%. Countered neurons: Zbtb20+ (n=2764), KA1+ (n=147), Prox1+ (n=188). (j) Cell body size was significantly larger in KA1+ cells. ***P<0.001, paired t-test. Countered neurons: KA1+ (n=56), Prox1+ (n=55). (k,l) CaMKII was expressed in KA1+ (k) and Prox1+ (l) neurons. Scale bars, 200 μm (a–c); 50 μm (d); 20 μm (e,g–h); 10 μm (f,k,l). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
Mentions: We first tried further long-term culture to assess CA and DG fields, but the organoids could not be maintained beyond day 100 in a healthy condition. Thus, to evaluate the neuronal population and function of the hESC-derived dorsomedial telencephalic tissues, we utilized a dissociation culture that enables long-term culture and facilitates neuronal maturation. Cells were dissociated at days 73–84 and plated on poly-D-lysine-laminin-coated plates (Supplementary Fig. 4c). At day 197 (17 weeks after dispersion), these cells made multiple small aggregations (Fig. 4a), which were connected to one another by numerous MAP2+ neurites (Fig. 4b). The majority of the cells expressed Zbtb20 (Fig. 4b,c). Unexpectedly, we also detected Foxg1::Venus+/GFAP+ glial-like cells (Fig. 4d; Supplementary Fig. 4d–g). Other markers for astrocytes such as S100 beta23 and vimentin24 were also detected in the glial-like cells (Supplementray Fig. 4h–k,l–o). These data demonstrate the generation of hippocampal-like neurons and astrocyte-like cells from hESC-derived dorsomedial telencephalic tissues.

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