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Self-Organization of Axial Polarity, Inside-Out Layer Pattern, and Species-Specific Progenitor Dynamics in Human ES Cell–Derived Neocortex

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Self-Organization of Axial Polarity, Inside-Out Layer Pattern, and Species-Specific Progenitor Dynamics in Human ES Cell–Derived Neocortex Correction NEUROSCIENCE Correction for “Self-organization of axial polarity, inside-out December 10, 2013, of Proc Natl Acad Sci USA (110:20284– layer pattern, and species-specific progenitor dynamics in human 20289; first published November 25, 2013; 10.1073/pnas. ES cell–derived neocortex,” by Taisuke Kadoshima, Hideya 1315710110). Sakaguchi, Tokushige Nakano, Mika Soen, Satoshi Ando, The authors note that Fig. 2 appeared incorrectly. The corrected Mototsugu Eiraku, and Yoshiki Sasai, which appeared in issue 50, figure and its legend appear below. A Foxg1::Venus BCDFoxg1::Venus phospho-MLC2 AcTubulin Day21 Sox2 aPKC Day24 EFGHt=0hr (Day24.5) t=20hr t=40hr t=60hr (Day27) I apical JKFoxg1::Venus Foxg1::Venus L AcTubulin AcTubulin w/o cont +Y-27632 rolling with rolling Day30 cont +Y-27632 MNFoxg1::Venus Foxg1::Venus OP Otx2 Coup-TF1 Gsh2 Venus Nkx2.1 Sox2 GAD65 Day26 Q R t=1.50 t=2.30 t=3.10 t=3.50 t=4.30 t=5.10 t=5.50 (basal) Pax6 (apical) Day24.5- ( pax6::venus ) Fig. 2. Asymmetric rounding morphogenesis in self-organized cortical NE. (A–I) Asymmetric progression of rounding morphogenesis of hESC-derived cortical NE. Arrows, boundary of a cortical NE domain in A and rolling epithelium in (B–D). Red arrowheads, rolling epithelium in E. Red arrows, rounding movements of the NE (F–I). (J–L) Effect of the ROCK inhibitor Y-27632 on the rolling of cortical NE. (L) Attenuation of rolling morphogenesis with ROCK inhibitor. ***P < 0.001 in contingency table analysis (2 × 2) with Fisher’s exact test. Treatment group, n = 187 NE domains; control group, n = 130. (M and N) The rolling shape was preferentially observed on the side with strong expression of Otx2 and Coup-TF1 (dorsal and caudal markers). (O–Q) Adjacent formation of NE structures of cortex (Pax6+) and LGE (Gsh2+; with GAD65+ GABAergic neurons underneath) on day 35. The cortical side contacting the LGE domain was opposite to the side with strong rolling (arrow). (R) Interkinetic nuclear migration in the hESC-derived cortical NE on day 24 (two-photon imaging). Visualized with partial mixing of pax6::venus reporter hESCs with nonlabeled hESCs. Two daughter cells with both apical and basal processes were generated from an apically dividing progenitor (red dots). (Scale bars, 200 μminA;100μminB–H, J, K, M, and N; 200 μminO–Q.) Nuclear counter staining (blue), DAPI. www.pnas.org/cgi/doi/10.1073/pnas.1407159111 7498 | PNAS | May 20, 2014 | vol. 111 | no. 20 www.pnas.org Downloaded by guest on October 1, 2021 Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell–derived neocortex Taisuke Kadoshimaa,b,1, Hideya Sakaguchia,b, Tokushige Nakanoa,2, Mika Soena, Satoshi Andoa,2, Mototsugu Eirakuc, and Yoshiki Sasaia,b,3 aLaboratory of Organogenesis and Neurogenesis and cFour-Dimensional Tissue Analysis Unit, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; and bDepartment of Medical Embryology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan Edited by Chen-Ming Fan, Carnegie Institution of Washington, Baltimore, MD, and accepted by the Editorial Board October 17, 2013 (received for review August 21, 2013) Here, using further optimized 3D culture that allows highly selective optimized culture generates species-specific progenitors in the induction and long-term growth of human ES cell (hESC)-derived outer subventricular zone (oSVZ), called outer radial glia cortical neuroepithelium, we demonstrate unique aspects of self- (oRG), which are abundantly present in the human neocortex organization in human neocorticogenesis. Self-organized cortical (11, 12) but rare in the mouse cortex (13, 14). Thus, an un- tissue spontaneously forms a polarity along the dorsocaudal- expectedly wide range of self-organizing events is internally ventrorostral axis and undergoes region-specificrollingmorpho- programmed within the cortical NE. genesis that generates a semispherical structure. The neuroepithe- Results lium self-forms a multilayered structure including three neuronal Intracortical Polarity in Self-Organized Cortical NE. zones (subplate, cortical plate, and Cajal-Retzius cell zones) and For the improved SFEBq culture (Fig. S2 A and A′), we formed aggregates by three progenitor zones (ventricular, subventricular, and inter- × 3 mediate zones) in the same apical-basal order as seen in the hu- plating 9 10 dissociated hESCs into each well of V-bottomed man fetal cortex in the early second trimester. In the cortical plate, 96-well plates (15) and culture them in medium supplemented with the Rho kinase inhibitor (16) (Fig. S2A). Then, the aggre- NEUROSCIENCE late-born neurons tend to localize more basally to early-born neu- gates were transferred to petri dishes and cultured under 40% O2 rons, consistent with the inside-out pattern seen in vivo. Further- conditions. The addition of chemically defined lipid concentrate, more, the outer subventricular zone contains basal progenitors FBS, heparin, and a low concentration of Matrigel improved that share characteristics with outer radial glia abundantly found long-term maintenance of VZ progenitors, whereas the addition in the human, but not mouse, fetal brain. Thus, human neocortico- of a TGFβ inhibitor and a Wnt inhibitor for the first 18 d mod- genesis involves intrinsic programs that enable the emergence of erately promoted telencephalic differentiation. complex neocortical features. Under these conditions, all hESC aggregates were positive for foxg1::Venus (marking telencephalic tissue) (2) on days 26 (Fig. corticogenesis | stratification 1A and Fig. S2B), and >75% of total cells (day 34) expressed foxg1::Venus, in contrast to the previous culture (Fig. 1B and Fig. + he mammalian neocortex has a multilayered structure (layers S2C). The foxg1::Venus NE contained epithelial domes with a I–VI) (1). The neocortex arises from the neuroepithelium ventricle-like cavity inside (Fig. 1C; day 42). These thick epi- T thelia had a cell-dense VZ positive for Pax6 and Sox2 on the (NE) of the dorsal telencephalon, which evaginates to form + a semispherical brain vesicle (Fig. S1A) (2). The dorsocaudal luminal side (Fig. 1 D and E), whereas pH3 progenitors under mitosis were found exclusively in its innermost part (Fig. 1F), as side of the neocortex is flanked by the cortical hem, whereas its + seen in vivo. TuJ1 neurons occupied the zone outside of the VZ ventrorostral side is neighbored by the lateral ganglionic emi- (CP; Fig. 1G), and expressed markers of early CP neurons such nence (LGE; striatum anlage) and septum. Layer I [its fetal primordium is called the marginal zone (MZ); Fig. S1B]is fi qualitatively different from other layers, as this superficial-most Signi cance + layer is mainly composed of Reelin Cajal-Retzius (CR) cells, which are largely derived from neighboring tissues such as the Using 3D culture of human ES cells, we show new self-organizing cortical hem and septum (3) (in the case of human cortex, some aspects of human corticogenesis: spontaneous development of + Reelin cells also arise directly from neocortical NE) (4). The intracortical polarity, curving morphology, and complex zone rest of the cortical layers are generated with the “inside-out” separations. Moreover, this culture generates species-specific pattern: the deeper the layer, the earlier the neurons are born progenitors, outer radial glia, which are abundantly present in from cortical progenitors (Fig. S1B) (5, 6). the human, but not mouse, neocortex. Our study suggests an A detailed understanding of early human corticogenesis unexpectedly wide range of self-organizing events that are remains elusive because of the limited access to fetal brain tis- driven by internal programs in human neocortex development. sues. We previously established a 3D culture of mouse and human ES cell (hESC) aggregates that recapitulates early steps of Author contributions: T.K. and Y.S. designed research; T.K., H.S., T.N., M.S., S.A., and M.E. corticogenesis [or serum-free floating culture of embryoid body- performed research; T.K. and Y.S. analyzed data; and T.K. and Y.S. wrote the paper. like aggregates with quick reaggregation (SFEBq)] (7–9). This The authors declare no conflict of interest. method has been also applied to human induced pluripotent stem This article is a PNAS Direct Submission. C.-M.F. is a guest editor invited by the (iPS) cell culture (10). In this self-organization culture, large Editorial Board. domains of cortical NE self-form within a floating hESC aggre- 1Present address: Faculty of Exploratory Pharmacology, Asubio Pharma Co., Ltd., Kobe gate and spontaneously develop ventricular zone (VZ), cortical 650-0047, Japan. plate (CP) (mostly deep-layer neurons), and MZ by culture day 2Present address: Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 40–45. This cortical NE was still immature, mimicking human Osaka 554-8558, Japan. corticogenesis during the first trimester (Fig. S1C)(7). 3To whom correspondence should be addressed. E-mail: [email protected]. Here, using an optimized culture, we revealed unique self- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. organizing aspects of human corticogenesis. Moreover, the 1073/pnas.1315710110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315710110 PNAS Early Edition | 1of6 as Ctip2 and Tbr1 (1, 2) (Fig. 1H and Fig. S2D). The neuronal surface of the aggregate (Fig. 2A, Lower). On day 21, the NE + zone also contained Reelin cells (Fig. 1I), and a Laminin-rich structure started to break into several large domains (Fig. 2A), zone near the surface (Fig. 1J) (4). Thus, self-organizing lami- and subsequently became apically concave (Fig. 2 B–D and Fig. nation occurs in this hESC-derived cortical NE. S3A, Upper). Interestingly, the cortical NE frequently had an axial polarity. Each cortical NE domain had an asymmetrically curved struc- Expression of Coup-TF1 in the VZ (Fig. 1K, red), which forms ture.
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