Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells

Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine

自己組織化によって構築されたヒトES細胞由来大脳皮質組織における軸極性の獲得、インサイド-アウトの層形成、および種特異的な神経幹細胞の再現

京都大学0048新制・課程博士博士(医学)甲第18183号医博第3903号新制||医||1004(附属図書館)31041京都大学大学院医学研究科医学専攻(主査)教授 渡邉 大, 教授 髙橋 良輔, 教授 髙橋 淳, 教授 江藤 浩之学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

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

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

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

<div><p>In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.</p> </div

Systemic treatment with a novel basic fibroblast growth factor mimic small-molecule compound boosts functional recovery after spinal cord injury.

Neurotrophic factors have been regarded having promising potentials for neuronal protection and regeneration, and thus promoting beneficial effects of kinesiological functions. They can be suspected to play important roles in cell/tissue grafting for various neural diseases. The clinical applications of such trophic factors to the central nervous system (CNS), however, have caused problematic side effects on account of the distinctive bioactive properties. In the course of developing synthetic compounds reflecting beneficial properties of basic fibroblast growth factor (bFGF), we conducted screening candidates that stimulate to trigger the intracellular tyrosine phosphorylation of FGF receptor and lead to the subsequent intracellular signaling in neurons. A small synthetic molecule SUN13837 was characterized by mimicking the beneficial properties of bFGF, which have been known as its specific activities when applied to CNS. What is more remarkable is that SUN13837 is eliminated the bioactivity to induce cell proliferation of non-neuronal somatic cells. On the bases of studies of pharmacology, behavior, physiology and histology, the present study reports that SUN13837 is characterized as a promising synthetic compound for treatment of devastating damages onto the rat spinal cord

Multi-layered cortical NE on day 12.

<p>(A–B) Day-12 aggregates carrying continuous Foxg1::venus⁺ NE epithelia. (A) Fluorescent image of a Foxg1::venus⁺ aggregate. (B) Immunostaining for Foxg1. (C–H) Layered formation in continuous Foxg1::venus⁺ NE epithelia. (C–E) Cortical progenitors (Pax6⁺ and Emx1⁺) form a zone on the apical side of Foxg1::venus⁺ NE. (F) Immunostaining for Ngn2⁺ cells located in the basal part of the progenitor zone. A majority of basal progenitors (Tbr2⁺) were found outside of the Ngn2⁺ cell zone. (G–H) Early cortical plate neurons (Tbr1⁺, Ctip2⁺) were found on the basal side of Foxg1::venus⁺ NE epithelia. (I–M) Differential location of layer-specific cortical neurons in Foxg1::venus⁺ NE epithelia. (I–K) Immunostaining for Ctip2 (I–J) and Tbr1 (K). (L–M) Immunostaining for Brn2 (L) and Cux1 (M). Dashed lines indicate the apical and basal borders of NE. Scale bars, 200 µm (A–B); 100 µm (C–M).</p

Formation and spatial arrangement of non-cortical telencephalic tissues.

<p>(A) Expression pattern of markers for cortex, cortical hem, and choroid plexus on embryonic day 12 (E12). (B) Hem-derived Cajal-Retzius cells expressing p73 on E12. (C–D) The hem-like tissues (Lmx1a⁺, Otx2⁺) formed adjacent to cortical tissues (Foxg1::venus⁺) in culture. The hem-like tissues also contained p73⁺ cells. (E) Schematic for SFEBq/gfCDM+IwL culture with Shh treatment. (F–H) <i>In vivo</i> expression of pallial and subpallial markers on E12. Cortical markers (Pax6, Ngn2, Tbr1), LGE markers (Gsh2, Dlx2, Mash1, Gad65), and MGE markers (Nkx2.1, Dlx2, Mash1, Gad65). (I) Schematic of the marker expression pattern. (J–M) Shh treatment induced the formation of subpallial tissues. LGE tissues (Gsh2⁺, Dlx2⁺, Mash1⁺) were located between cortical tissues (Pax6⁺, Ngn2⁺, Tbr1⁺) and MGE tissues (Nkx2.1⁺, Dlx2⁺, Mash1⁺). Arrowheads indicate a transition between pallial and subpallial tissues. (N) Expression of Gsh2::venus in thickened NE tissue on day 14. (O–Q) Immunostaing of Gsh2::venus⁺ NE on day 14. Immunostaining for Gsh2 (O), Mash1 (P) and Gad67/Dlx2 (Q). chp, choroid plexus; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; PSB, pallial-subpallial boundary; Th, thalamus. Scale bars, 100 µm (A–D,J–M,O–Q); 200 µm (F–H,N).</p

Multi-layered cortical NE on day 15.

<p>(A) Day-15 aggregate carrying continuous Foxg1::venus⁺ NE epithelia. Immunostaining for GFP. (B–G) Layer formation without the inside-out pattern. (B–C) Cortical progenitors (Foxg1::venus⁺/Pax6⁺) on the apical side. (D) Ngn2⁺ cells were located in a slightly deeper zone. Basal progenitors (Tbr2⁺) were outside of the Ngn2⁺ zone (E–G) Early cortical plate neurons (Tbr1⁺, Ctip2⁺) occupied the basal zone of continuous Foxg1::venus⁺ NE epithelia (E–F), while late cortical plate neurons (Brn2⁺, Cux1⁺) stayed on the apical side. (H) A schematic of cortical layer formation <i>in vitro</i> on day 15. Dashed lines indicate the apical or basal borders of NE. CR, Cajal-Retzius cell. Scale bars, 100 µm.</p

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis