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1.  Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development 
Cell stem cell  2013;12(5):573-586.
Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. Most differentiated populations, however, 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 post-transplantation 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 seven 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.
PMCID: PMC3699205  PMID: 23642366
2.  Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice 
Stem Cell Reports  2014;2(5):662-674.
In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases.
Graphical Abstract
•3D retina was formed from ESCs/iPSCs using retinoic acid receptor antagonist•ESC/iPSC retina developed a structured layer of photoreceptors in rd1 host mice•Transplanted photoreceptors formed complete inner and outer segments•Transplanted photoreceptors morphologically made synapses with host bipolar cells
In this article, Mandai and colleagues show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. They also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases.
PMCID: PMC4050483  PMID: 24936453
3.  Cultivation of Corneal Endothelial Cells on a Pericellular Matrix Prepared from Human Decidua-Derived Mesenchymal Cells 
PLoS ONE  2014;9(2):e88169.
The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na+/K+-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.
PMCID: PMC3914933  PMID: 24505413
4.  FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation 
PLoS Genetics  2014;10(1):e1004118.
Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27Kip1, Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.
Author Summary
The ability of our brain to perceive sound depends on its conversion into electrical impulses within the cochlea of the inner ear. The cochlea has dedicated specialized cells, called inner ear hair cells, which register sound energy. Environmental effects, genetic disorders or just the passage of time can damage these cells, and the damage impairs our ability to hear. If we could understand how these cells develop, we might be able to exploit this knowledge to generate new hair cells. In this study we address an old problem: how do signals from the fibroblast growth factor (FGF) family control hair cell number? We used mice in which one of the receptors for FGF (Fgfr1) is mutated and found that the expression of a stem cell protein, Sox2 is not maintained. Sox2 generally acts to keep precursors in the cochlea in a pre-hair cell state. However, in mutant mice Sox2 expression is transient, diminishing the ability of precursors to commit to a hair cell fate. These findings suggest that it may be possible to amplify the number of hair cell progenitors in culture by tuning FGF activity, providing a route to replace damaged inner ear hair cells.
PMCID: PMC3900395  PMID: 24465223
5.  Feeder-Free Generation and Long-Term Culture of Human Induced Pluripotent Stem Cells Using Pericellular Matrix of Decidua Derived Mesenchymal Cells 
PLoS ONE  2013;8(1):e55226.
Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells’ pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.
PMCID: PMC3561375  PMID: 23383118
6.  Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture 
PLoS ONE  2012;7(12):e53024.
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.
PMCID: PMC3534089  PMID: 23300850
7.  Xenopus chordin: A Novel Dorsalizing Factor Activated by Organizer-Specific Homeobox Genes 
Cell  1994;79(5):779-790.
A Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemann’s organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.
PMCID: PMC3082463  PMID: 8001117
8.  Dorsoventral Patterning in Xenopus: Inhibition of Ventral Signals by Direct Binding of Chordin to BMP-4 
Cell  1996;86(4):589-598.
Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD 3 × 10−10 M), binding specifically to BMPs but not to activin or TGF-β1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemann’s organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.
PMCID: PMC3070603  PMID: 8752213
9.  Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model 
Journal of Clinical Investigation  2005;115(1):102-109.
Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell–derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell–derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.
PMCID: PMC539189  PMID: 15630449
10.  Relaxation-expansion model for self-driven retinal morphogenesis 
Bioessays  2012;34(1):17-25.
The generation of complex organ structures such as the eye requires the intricate orchestration of multiple cellular interactions. In this paper, early retinal development is discussed with respect to the structure formation of the optic cup. Although recent studies have elucidated molecular mechanisms of retinal differentiation, little is known about how the unique shape of the optic cup is determined. A recent report has demonstrated that optic-cup morphogenesis spontaneously occurs in three-dimensional stem-cell culture without external forces, indicating a latent intrinsic order to generate the structure. Based on this self-organizing phenomenon, we introduce the “relaxation-expansion” model to mechanically interpret the tissue dynamics that enable the spontaneous invagination of the neural retina. This model involves three consecutive local rules (relaxation, apical constriction, and expansion), and its computer simulation recapitulates the optic-cup morphogenesis in silico.
PMCID: PMC3266490  PMID: 22052700
ES cells; internal force; optic cup; retina; self-organization

Results 1-10 (10)