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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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Generation of choroid plexus-like tissues with pleated structure from hESCs.(a) Schematic of examined conditions to induce choroid plexus tissues. (b) Immunostaining of neocortical structure induced from hESCs on day 35. (c) Comparison of aggregate formation from Foxg1::Venus knock-in hESCs on day 35. CHIR (GSK3 inhibitor) plus BMP4 treatment (condition 2) induced thin epithelia with many folds with significant attenuation of Foxg1::Venus expression. (d,e) qPCR for genes expressed in dorsomedial telencephalon (***P<0.001). (d) foxg1 significantly attenuated in condition 2 (n=3, unpaired t-test). (e) ttr significantly increased in condition 2 (n=3, unpaired t-test with Welch's correction). (f) Bright-field view of one aggregate cultured in condition 2 on day 42. (g–k) The induction of choroid plexus tissues with pleated structure from hESCs. The pleated epithelia are Lmx1a+ (g,h), Otx2+ (i) and TTR+ (h). TTR mainly stained apically (h), and Aquaporin-1 stained mainly in apical parts, with less staining in basal parts (j). ZO-1 stained the surface of epithelia (k). Scale bars, 50 μm (b; 500 μm (c, f); 200 μm (g); 20 μm (h–k). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
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f1: Generation of choroid plexus-like tissues with pleated structure from hESCs.(a) Schematic of examined conditions to induce choroid plexus tissues. (b) Immunostaining of neocortical structure induced from hESCs on day 35. (c) Comparison of aggregate formation from Foxg1::Venus knock-in hESCs on day 35. CHIR (GSK3 inhibitor) plus BMP4 treatment (condition 2) induced thin epithelia with many folds with significant attenuation of Foxg1::Venus expression. (d,e) qPCR for genes expressed in dorsomedial telencephalon (***P<0.001). (d) foxg1 significantly attenuated in condition 2 (n=3, unpaired t-test). (e) ttr significantly increased in condition 2 (n=3, unpaired t-test with Welch's correction). (f) Bright-field view of one aggregate cultured in condition 2 on day 42. (g–k) The induction of choroid plexus tissues with pleated structure from hESCs. The pleated epithelia are Lmx1a+ (g,h), Otx2+ (i) and TTR+ (h). TTR mainly stained apically (h), and Aquaporin-1 stained mainly in apical parts, with less staining in basal parts (j). ZO-1 stained the surface of epithelia (k). Scale bars, 50 μm (b; 500 μm (c, f); 200 μm (g); 20 μm (h–k). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).

Mentions: Over the past several years, substantial progress in human pluripotent stem cell (hPSC) technology has enabled the generation of neuroectodermal tissues in vitro78910111213. For example, we previously described an efficient three-dimensional (3D) culture method for generating stratified neocortical structure from human embryonic stem cells (hESCs)1112. In this method, called serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq), several thousand dissociated hESCs are quickly reaggregated by plating into each well of a 96-well plate with a special low-adhesion coating. When the floating cell aggregates are cultured in medium suitable for cortical differentiation (Fig. 1a), the majority of aggregates express the telencephalic marker Foxg1 (ref. 12). However, generation of medial pallium from PSCs of any species has not yet been achieved.


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)

Generation of choroid plexus-like tissues with pleated structure from hESCs.(a) Schematic of examined conditions to induce choroid plexus tissues. (b) Immunostaining of neocortical structure induced from hESCs on day 35. (c) Comparison of aggregate formation from Foxg1::Venus knock-in hESCs on day 35. CHIR (GSK3 inhibitor) plus BMP4 treatment (condition 2) induced thin epithelia with many folds with significant attenuation of Foxg1::Venus expression. (d,e) qPCR for genes expressed in dorsomedial telencephalon (***P<0.001). (d) foxg1 significantly attenuated in condition 2 (n=3, unpaired t-test). (e) ttr significantly increased in condition 2 (n=3, unpaired t-test with Welch's correction). (f) Bright-field view of one aggregate cultured in condition 2 on day 42. (g–k) The induction of choroid plexus tissues with pleated structure from hESCs. The pleated epithelia are Lmx1a+ (g,h), Otx2+ (i) and TTR+ (h). TTR mainly stained apically (h), and Aquaporin-1 stained mainly in apical parts, with less staining in basal parts (j). ZO-1 stained the surface of epithelia (k). Scale bars, 50 μm (b; 500 μm (c, f); 200 μm (g); 20 μm (h–k). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660208&req=5

f1: Generation of choroid plexus-like tissues with pleated structure from hESCs.(a) Schematic of examined conditions to induce choroid plexus tissues. (b) Immunostaining of neocortical structure induced from hESCs on day 35. (c) Comparison of aggregate formation from Foxg1::Venus knock-in hESCs on day 35. CHIR (GSK3 inhibitor) plus BMP4 treatment (condition 2) induced thin epithelia with many folds with significant attenuation of Foxg1::Venus expression. (d,e) qPCR for genes expressed in dorsomedial telencephalon (***P<0.001). (d) foxg1 significantly attenuated in condition 2 (n=3, unpaired t-test). (e) ttr significantly increased in condition 2 (n=3, unpaired t-test with Welch's correction). (f) Bright-field view of one aggregate cultured in condition 2 on day 42. (g–k) The induction of choroid plexus tissues with pleated structure from hESCs. The pleated epithelia are Lmx1a+ (g,h), Otx2+ (i) and TTR+ (h). TTR mainly stained apically (h), and Aquaporin-1 stained mainly in apical parts, with less staining in basal parts (j). ZO-1 stained the surface of epithelia (k). Scale bars, 50 μm (b; 500 μm (c, f); 200 μm (g); 20 μm (h–k). Bars in graph, s.e.m. Nuclear counter staining (blue), 4,6-diamidino-2-phenylindole (DAPI).
Mentions: Over the past several years, substantial progress in human pluripotent stem cell (hPSC) technology has enabled the generation of neuroectodermal tissues in vitro78910111213. For example, we previously described an efficient three-dimensional (3D) culture method for generating stratified neocortical structure from human embryonic stem cells (hESCs)1112. In this method, called serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq), several thousand dissociated hESCs are quickly reaggregated by plating into each well of a 96-well plate with a special low-adhesion coating. When the floating cell aggregates are cultured in medium suitable for cortical differentiation (Fig. 1a), the majority of aggregates express the telencephalic marker Foxg1 (ref. 12). However, generation of medial pallium from PSCs of any species has not yet been achieved.

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