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Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both in vitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7 months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues. One of two papers describing a reprogramming phenomenon called stimulus-triggered acquisition of pluripotency (STAP) -- in STAP, lineage-committed adult somatic cells are reprogrammed to pluripotency by transient exposure to low-pH treatment, and extensive analysis of the molecular features and developmental potential of STAP cells indicates that they represent a unique state of pluripotency. A new way to induce pluripotency The fates of the somatic cells that form the bulk of the mammalian body are thought to be largely determined by the time the cellular differentiation processes of development have been completed. Reprogramming in response to environmental stress has been observed in plants but not so far in mammalian cells. Now two manuscripts by Haruko Obokata and colleagues describe an unexpected reprogramming phenomenon, which the authors call stimulus-triggered acquisition of pluripotency (STAP). In STAP, mouse somatic cells such as CD45.sup.+ haematopoietic cells are reprogrammed to pluripotency by transient exposure to low pH. Extensive analysis of the molecular features and developmental potential of STAP cells suggests that they represent a unique state of pluripotency -- and provide an alternative source of pluripotent cells to the use of transcription factors, as has become routine for induced pluripotent stem cell production., Author(s): Haruko Obokata [sup.1] [sup.2] [sup.3], Teruhiko Wakayama [sup.3] [sup.8], Yoshiki Sasai [sup.4], Koji Kojima [sup.1], Martin P. Vacanti [sup.1] [sup.5], Hitoshi Niwa [sup.6], Masayuki Yamato [sup.7], Charles A. Vacanti [...]

One of two papers describing a reprogramming phenomenon called stimulus-triggered acquisition of pluripotency (STAP) -- in STAP, lineage-committed adult somatic cells are reprogrammed to pluripotency by transient exposure to low-pH treatment, and extensive analysis of the molecular features and developmental potential of STAP cells indicates that they represent a unique state of pluripotency. A new way to induce pluripotency The fates of the somatic cells that form the bulk of the mammalian body are thought to be largely determined by the time the cellular differentiation processes of development have been completed. Reprogramming in response to environmental stress has been observed in plants but not so far in mammalian cells. Now two manuscripts by Haruko Obokata and colleagues describe an unexpected reprogramming phenomenon, which the authors call stimulus-triggered acquisition of pluripotency (STAP). In STAP, mouse somatic cells such as CD45.sup.+ haematopoietic cells are reprogrammed to pluripotency by transient exposure to low pH. Extensive analysis of the molecular features and developmental potential of STAP cells suggests that they represent a unique state of pluripotency -- and provide an alternative source of pluripotent cells to the use of transcription factors, as has become routine for induced pluripotent stem cell production. We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP).sup.1. This reprogramming does not require nuclear transfer.sup.2,3 or genetic manipulation.sup.4. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency., Author(s): Haruko Obokata [sup.1] [sup.2] [sup.3], Yoshiki Sasai [sup.4], Hitoshi Niwa [sup.5], Mitsutaka Kadota [sup.6], Munazah Andrabi [sup.6], Nozomu Takata [sup.4], Mikiko Tokoro [sup.2], Yukari Terashita [sup.1] [sup.2], Shigenobu Yonemura [...]

Here we report a unique cellular reprogramming phenomenon, called stimulus- triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external [...]

We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP) (1). This reprogramming does not require nuclear transfer (2,3) or genetic manipulation (4). Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency., We recently discovered an intriguing phenomenon of cellular fate conversion: somatic cells regain pluripotency after experiencing sublethal stimuli such as a low-pH exposure (1). When splenic [CD45.sup.+] lymphocytes are exposed [...]

Our knowledge of the principles by which organ architecture develops through complex collective cell behaviours is still limited. Recent work has shown that the shape of such complex tissues as [...]

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo.We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties., Eye formation has attracted the attention of both classical and modern-day developmental biologists (1-12). The retinal anlage, which is demarcated by Rx (also called Rax) expression (13,14), first appears as [...]

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521., Previous studies, particularly in amphibians, have demonstrated that the initial step of vertebrate neural differentiation from uncommitted embryonic ectodermal cells occurs via a cell-intrinsic mechanism and requires only that inhibitory [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.11.032 Byline: Eri Mizuhara (a), Yasuko Minaki (a), Tomoya Nakatani (a), Minoru Kumai (b), Takeshi Inoue (a), Keiko Muguruma (c), Yoshiki Sasai (c), Yuichi Ono (a)(b) Keywords: Neph3; E-cadherin; Ptf1a; Purkinje cell; Origin; Neuronal subtype; Cell sorting; Cell surface marker; Fate mapping Abstract: GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3.sup.+ GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3.sup.+ populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3.sup.+ cells were progenitors for Pax2.sup.+ interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3.sup.+ cells. Sorting experiments revealed that the Neph3.sup.+ E-cadherin.sup.high population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2.sup.+ interneurons were enriched in the Neph3.sup.+ E-cadherin.sup.low population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium. Author Affiliation: (a) Group for Neuronal Differentiation, KAN Research Institute Inc., Kobe, Hyogo 650-0047, Japan (b) Group for Transgenic Technology, KAN Research Institute Inc., Kobe, Hyogo 650-0047, Japan (c) Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, Hyogo 650-0047, Japan Article History: Received 30 July 2009; Revised 12 November 2009; Accepted 30 November 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.07.008 Byline: Hidehiko Inomata (1), Tomoko Haraguchi (1), Yoshiki Sasai (1) Keywords: DEVBIO Abstract: Dorsal axial formation during vertebrate embryogenesis exhibits robust resistance to perturbations in patterning signals. However, how such stability is supported at the molecular level remains largely elusive. Here we show that Xenopus ONT1, an Olfactomedin-class secreted protein, stabilizes axial formation by restricting Chordin activity on the dorsal side. When ONT1 function is attenuated, the embryo becomes hyperdorsalized by a normally subeffective dose of Chordin. ONT1 binds Chordin and BMP1/Tolloid-class proteinases (B1TP) via distinct domains and acts as a secreted scaffold that enhances B1TP-mediated Chordin degradation by facilitating enzyme-substrate association. ONT1 is indispensable for fine-tuning BMP signaling in the axial tissue, and a similar role has been suggested for dorsally expressed BMPs such as ADMP. Simultaneous inhibition of ONT1 and dorsally expressed BMPs (ADMP and BMP2) synergistically caused drastic dorsalization. These results indicate that stable axial formation depends on two compensatory regulatory pathways involving ONT1/B1TP and dorsally expressed BMPs. Author Affiliation: (1) Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan Article History: Received 17 December 2007; Revised 30 April 2008; Accepted 2 July 2008 Article Note: (miscellaneous) Published: September 4, 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.03.035 Byline: Noriaki Sasai (1), Rieko Yakura (1), Daisuke Kamiya (1), Yoko Nakazawa (1), Yoshiki Sasai (1) Keywords: DEVBIO; SIGNALING; CELLBIO Abstract: During gastrulation of the amphibian embryo, specification of the three germ layers, endo-, ecto-, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-[beta] signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. Here, we report that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, we find that XFDL156 and its mammalian homologs interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53. Author Affiliation: (1) Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan Article History: Received 16 October 2007; Revised 31 January 2008; Accepted 24 March 2008 Article Note: (miscellaneous) Published: May 29, 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.010 Byline: Hong-Lin Su (a), Keiko Muguruma (a), Mami Matsuo-Takasaki (a), Mineko Kengaku (b), Kiichi Watanabe (a), Yoshiki Sasai (a) Keywords: Math1; Embryonic stem cells; Neural development; Purkinje cells; Rhombic lip; Cerebellum; Granule cells Abstract: Here, we report in vitro generation of Math1.sup.+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1.sup.+. The induced Math1.sup.+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1.sup.+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7.sup.+/Calbindin-D28K.sup.+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system. Author Affiliation: (a) Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan (b) Laboratory for Neural Cell Polarity, Brain Science Institute, RIKEN, Wako 351-0198, Japan Article History: Received 26 July 2005; Revised 24 October 2005; Accepted 4 November 2005

We report directed differentiaion of retinal precursors in vitro from mouse ES cells. [Six3.sup.+] rostral brain progenitors are generated by culturing ES cells under serum-free suspension conditions (SFEB culture) in the presence of Wnt and Nodal antagonists (Dkk1 and LeftyA), and subsequently steered to differentiate into [Rx.sup.+] cells (16%) by treatment with activin and serum. Consistent with the characteristics of early neural retinal precursors, the induced [Rx.sup.+] cells coexpress Pax6 and the mitotic marker Ki67, but not Nestin. The ES cell-derived precursors efficiently generate cells with the photoreceptor phenotype ([rhodopsin.sup.+], [recoverin.sup.+]) when cocultured with embryonic retinal cells. Furthermore, organotypic culture studies demonstrate the selective integration and survival of ES cell-derived cells with the photoreceptor phenotype (marker expression and morphology) in the outer nuclear layer of the retina. Taken together, ES cells treated with SFEB/Dkk1/LeftyA/ serum/activin generate neural retinal precursors, which have the competence of photoreceptor differentiation. regenerative medicine | differentiation | photoreceptor | induction | Six3

Formation of Spemann organizer is a very significant stage in dorsoventral axis determination of vertebrate development. A novel zebrafish homeobox gene, dharma, has been isolated. It can induce the organizer ectopically. For the first time a zygotic gene implicated in formation of an organizer-inducing center has been identified.

The molecular conservation of dorsoventral patterning mechanisms during evolution was used to investigate the function of the noggin gene. Noggin was found to promote ventral development in Drosophila, with ventral ectoderm and the central nervous system being specified in the absence of all endogenous ventral-specific zygotic gene expression. Constitutively active forms of dpp receptors were used to show that noggin blocks dpp signaling upstream of dpp receptor activation. Results suggest a mechanistic basis for the action of Spemann's organizer during Xenopus development and provide additional support for the conservation of dorsal-ventral patterning mechanisms between chordates and arthropods.

The Xenopus protein was expressed in the baculovirus system and a peptide antibody was raised against its NH2-terminus to study the biochemical function of chordin (Chd). A myc epitope was also introduced at the COOH-terminus. Radio-receptor binding assay results showed that Chd protein is capable of inhibiting the binding of BMP-4 protein to its cognate receptor at subnanomolar concentrations. The execution of noncell-autonomous effects of Spemann's organizer on dorsoventral patterning is partly caused by diffusible signals that directly bind to and neutralize ventral bone morphogenetic proteins during gastrulation. This creates pattern in the ectodermal and mesodermal germ layers.

An abundant cDNA enriched in Spemann's organizer, cerberus, and isolated by differential screening encodes a secreted protein that is expressed in a broad anterior domain of the gastrula. This domain includes the leading edge of the involuting endoderm and gives rise to foregut, liver and anterior midgut. Microinjection of cerberus mRNA into Xenopus embryos creates ectopic heads, and duplicated hearts and livers. These results support the role of a molecule expressed in the anterior endoderm in the creation of head structures in the vertebrate embryo.

Investigations on dorsoventral patterning reported that the structural formations of the germ layers ectoderm and mesoderm were directed by molecules associated with decapentaplegic genes/bone morphogenetic protein 4 (dpp/Bmp-4), and short gastrulation/Xenopus chordin (sog/chd). This mechanism was especially evidenced in Drosophila and also believed to be feasible with vertebrates. In the latter case, the organizer will release the signals that induce dorsal differentiation which in turn will be opposed by ventral positioning information released by Bmp-4.

An organizer-specific secreted factor, Chd, with two proteins, noggin and follistatin, secreted by the organizer, is involved in neural tissue induction and Chd is antagonized by bone morphogenesis protein-4 (bmp-4). Inhibition of bmp-4 present in the animal cap is responsible for neural differentiation. In Drosophila, the amount of neurogenic ectoderm from which the central nervous system is formed depends on the action of two molecules with antagonistic functions, decapentaplegic and short-gastrulation.