JC3article(2).pdf
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LETTER
doi:10.1038/nature17000
Co-ordinated ocular development from human
iPS cells and recovery of corneal function
Ryuhei Hayashi1,2, Yuki Ishikawa2, Yuzuru Sasamoto2, Ryosuke Katori2, Naoki Nomura2, Tatsuya Ichikawa2, Saori Araki2,
Takeshi Soma2, Satoshi Kawasaki2, Kiyotoshi Sekiguchi3, Andrew J. Quantock4, Motokazu Tsujikawa2 & Kohji Nishida2
The eye is a complex organ with highly specialized constituent
tissues derived from different primordial cell lineages. The retina,
for example, develops from neuroectoderm via the optic vesicle,
the corneal epithelium is descended from surface ectoderm, while
the iris and collagen-rich stroma of the cornea have a neural crest
origin. Recent work with pluripotent stem cells in culture has
revealed a previously under-appreciated level of intrinsic cellular
self-organization, with a focus on the retina and retinal cells1–5.
Moreover, we and others have demonstrated the in vitro induction
of a corneal epithelial cell phenotype from pluripotent stem cells6–9.
These studies, however, have a single, tissue-specific focus and
fail to reflect the complexity of whole eye development. Here we
demonstrate the generation from human induced pluripotent stem
cells of a self-formed ectodermal autonomous multi-zone (SEAM)
of ocular cells. In some respects the concentric SEAM mimics
whole-eye development because cell location within different zones
is indicative of lineage, spanning the ocular surface ectoderm, lens,
neuro-retina, and retinal pigment epithelium. It thus represents
a promising resource for new and ongoing studies of ocular
morphogenesis. The approach also has translational potential
and to illustrate this we show that cells isolated from the ocular
surface ectodermal zone of the SEAM can be sorted and expanded
ex vivo to form a corneal epithelium that recovers function in an
experimentally induced animal model of corneal blindness.
To generate a SEAM of ocular cells, human induced pluripotent
stem (iPS) cells were cultivated in differentiation medium in which
they spontaneously and progressively formed a primordium compris-
ing four identifiable concentric zones (Fig. 1a, Extended Data Fig. 1a).
Cell morphology in each zone was distinctive, creating a visible delin-
eation between zones (Extended Data Fig. 1b). The innermost central
area (zone 1) formed first and this was followed by the emergence
of three more radially distant concentric cell populations; zones 2–4.
(Fig. 1b, Supplementary Video). In our experiments 7.7 ± 1.8%
of human iPS cells formed colonies and 67.9 ± 4.9% of these
resulted in the generation of a SEAM (n = 5 technical replicates).
Immunolabelling for the neural cell marker class III β-tubulin
(TUBB3) was positive in zones 1 and 2, but not, more peripherally,
in zones 3 or 4 (Fig. 1c). Cells in zones 1–3 expressed the ocular cell
marker PAX6, while tho.
This study examined the role of the Sox2 gene in regulating astrocyte processes density in the mouse retina. Conditional knockout (cKO) mice lacking Sox2 specifically in astrocytes were compared to conditional wild-type (cWT) mice. Retinal images were analyzed to measure astrocyte processes frequency. While cKO mice generally showed lower average processes counts, the differences were not statistically significant due to large standard errors overlapping between groups. Further analysis found Sox2 may be more involved in regulating astrocyte processes density in the retinal periphery compared to central and middle areas, though results were still highly variable. Therefore, this study was unable to definitively determine the role of Sox2 in astrocyte networks due to significant
This study aims to characterize regulatory elements that control spatial gene expression in the skin. The researchers used P300 ChIP-seq in mouse skin to identify putative layer-specific enhancers. They developed assays to test enhancer activity, including a cell-based differentiation assay and a graft-based assay using human keratinocytes. Around 80% of putative enhancers activated reporter expression in differentiating keratinocytes in the cell-based assay. The graft-based assay showed layer-specific reporter expression driven by putative enhancers, recapitulating the spatial patterning of skin. This approach allows rapid validation of epidermal enhancers responsible for spatial gene regulation.
This study developed an in vitro model for differentiating human pluripotent stem cells into retinal neurons. The differentiation process followed identifiable stages of retinal development:
1) Undifferentiated stem cells expressed pluripotent markers.
2) After 10 days of differentiation, eye field populations expressed neural and eye field transcription factors.
3) By day 30, retinal progenitor neurospheres analogous to the optic vesicle expressed retinal progenitor markers.
4) At day 70, the stem cells yielded retinal ganglion cells and photoreceptor cells, demonstrating the ability to derive major retinal cell types.
1. There are at least two populations of Tbr2+ cells in the embryonic mouse midbrain - a lateral population and a medial "bridge" population.
2. Immunohistochemistry results from E13.5 to P0 suggest that the Tbr2+ midbrain cells are no longer mitotic, following a conserved transcription factor program of Pax6 -> Tbr2 -> Tbr1 seen in other brain regions.
3. Using Ai14/EoCreERT2 lineage tracing mice, the fate of the early Tbr2+ midbrain population can be tracked to maturity, though longer experiments are needed to identify the adult midbrain nucleus derived from these cells.
This document summarizes a study analyzing mouse embryos with a point mutation in the non-muscle myosin heavy chain II-B gene (Myh10) using high resolution 3D imaging. Mice homozygous for the R709C Myh10 mutation exhibited all features of Pentalogy of Cantrell (POC), a rare birth defect affecting the abdominal wall and heart. Heterozygous mice showed variable phenotypes. Micro-CT scans of E14.5 embryos found cardiac defects in homozygous mice consistent with POC, including double outlet right ventricle and ventricular septal defects. The homozygous mutation was embryonic lethal by E14.5 due to cardiac failure. Future studies aim
This document provides an overview of cerebellar development in mice. It discusses how signaling centers establish the cerebellar territory and define its boundaries. Two primary progenitor zones give rise to cerebellar cells - the ventricular zone and rhombic lip. The rhombic lip generates glutamatergic neurons, including granule neuron progenitors that form the external granule layer and drive cerebellar growth. The ventricular zone produces GABAergic neurons and interneurons. Finally, it notes that while mouse studies provide insights into human cerebellar development and disease, direct study of human fetal cerebella remains important due to species differences.
- There is a reduced pool of progenitor/stem cells in the skin and bone marrow of elderly mice and humans compared to young after a burn injury. This contributes to delayed healing in the elderly.
- The Wnt/β-catenin signaling pathway, important for stem cell renewal and differentiation, has increased activation in the granulation tissue and center of burns in elderly mice and human tissue compared to young.
- The reduced progenitor cell pool and disrupted Wnt/β-catenin signaling in the elderly contributes to higher mortality rates after burns compared to young patients.
1) The arrival of the trophic factor NT-3 from the thalamus into the developing neocortex is critical for dendritic outgrowth in neocortical neurons.
2) Ablating NT-3 specifically from the thalamus using a conditional knockout system resulted in decreased basal dendrite complexity in neocortical neurons, as shown through Sholl analysis.
3) Further analysis of dendritic morphology in upper layer neurons is needed to determine if thalamic NT-3 also affects dendritic development in those neurons.
This study examined the role of the Sox2 gene in regulating astrocyte processes density in the mouse retina. Conditional knockout (cKO) mice lacking Sox2 specifically in astrocytes were compared to conditional wild-type (cWT) mice. Retinal images were analyzed to measure astrocyte processes frequency. While cKO mice generally showed lower average processes counts, the differences were not statistically significant due to large standard errors overlapping between groups. Further analysis found Sox2 may be more involved in regulating astrocyte processes density in the retinal periphery compared to central and middle areas, though results were still highly variable. Therefore, this study was unable to definitively determine the role of Sox2 in astrocyte networks due to significant
This study aims to characterize regulatory elements that control spatial gene expression in the skin. The researchers used P300 ChIP-seq in mouse skin to identify putative layer-specific enhancers. They developed assays to test enhancer activity, including a cell-based differentiation assay and a graft-based assay using human keratinocytes. Around 80% of putative enhancers activated reporter expression in differentiating keratinocytes in the cell-based assay. The graft-based assay showed layer-specific reporter expression driven by putative enhancers, recapitulating the spatial patterning of skin. This approach allows rapid validation of epidermal enhancers responsible for spatial gene regulation.
This study developed an in vitro model for differentiating human pluripotent stem cells into retinal neurons. The differentiation process followed identifiable stages of retinal development:
1) Undifferentiated stem cells expressed pluripotent markers.
2) After 10 days of differentiation, eye field populations expressed neural and eye field transcription factors.
3) By day 30, retinal progenitor neurospheres analogous to the optic vesicle expressed retinal progenitor markers.
4) At day 70, the stem cells yielded retinal ganglion cells and photoreceptor cells, demonstrating the ability to derive major retinal cell types.
1. There are at least two populations of Tbr2+ cells in the embryonic mouse midbrain - a lateral population and a medial "bridge" population.
2. Immunohistochemistry results from E13.5 to P0 suggest that the Tbr2+ midbrain cells are no longer mitotic, following a conserved transcription factor program of Pax6 -> Tbr2 -> Tbr1 seen in other brain regions.
3. Using Ai14/EoCreERT2 lineage tracing mice, the fate of the early Tbr2+ midbrain population can be tracked to maturity, though longer experiments are needed to identify the adult midbrain nucleus derived from these cells.
This document summarizes a study analyzing mouse embryos with a point mutation in the non-muscle myosin heavy chain II-B gene (Myh10) using high resolution 3D imaging. Mice homozygous for the R709C Myh10 mutation exhibited all features of Pentalogy of Cantrell (POC), a rare birth defect affecting the abdominal wall and heart. Heterozygous mice showed variable phenotypes. Micro-CT scans of E14.5 embryos found cardiac defects in homozygous mice consistent with POC, including double outlet right ventricle and ventricular septal defects. The homozygous mutation was embryonic lethal by E14.5 due to cardiac failure. Future studies aim
This document provides an overview of cerebellar development in mice. It discusses how signaling centers establish the cerebellar territory and define its boundaries. Two primary progenitor zones give rise to cerebellar cells - the ventricular zone and rhombic lip. The rhombic lip generates glutamatergic neurons, including granule neuron progenitors that form the external granule layer and drive cerebellar growth. The ventricular zone produces GABAergic neurons and interneurons. Finally, it notes that while mouse studies provide insights into human cerebellar development and disease, direct study of human fetal cerebella remains important due to species differences.
- There is a reduced pool of progenitor/stem cells in the skin and bone marrow of elderly mice and humans compared to young after a burn injury. This contributes to delayed healing in the elderly.
- The Wnt/β-catenin signaling pathway, important for stem cell renewal and differentiation, has increased activation in the granulation tissue and center of burns in elderly mice and human tissue compared to young.
- The reduced progenitor cell pool and disrupted Wnt/β-catenin signaling in the elderly contributes to higher mortality rates after burns compared to young patients.
1) The arrival of the trophic factor NT-3 from the thalamus into the developing neocortex is critical for dendritic outgrowth in neocortical neurons.
2) Ablating NT-3 specifically from the thalamus using a conditional knockout system resulted in decreased basal dendrite complexity in neocortical neurons, as shown through Sholl analysis.
3) Further analysis of dendritic morphology in upper layer neurons is needed to determine if thalamic NT-3 also affects dendritic development in those neurons.
Monitoring neural activities by optical imagingMd Kafiul Islam
Monitoring neural activities by optical imaging along with the use of genetic modification provides better spatio-temporal resolution to study single neural firing and hence very useful in understanding the neural process and dynamics. This is just a glimpse of few articles reported their outcome of such imaging.
2016_Association for Research in Vision and Ophthalmology_2Mauricio Rosenfeld
BM-derived CX3CR1+ progenitors potentiate vascular repair in an ischemic retinopathy mouse model. Injection of myeloid progenitor cells may provide a potential clinical application for the treatment of retinal vascular disease. CX3CR1+/CD34+ BM cells migrated more to the retina compared to CX3CR1+/CD34- cells and decreased areas of vascular obliteration and neovascular tufts in an OIR mouse model. CX3CR1+/CD34+ cells also upregulated more proangiogenic genes than CX3CR1+/CD34- cells by qPCR analysis.
1) The study investigated the hypothesis that neural stem cells from the subventricular zone (SVZ) and dentate gyrus (DG) are recruited to injured brain sites via extracellular factors to aid repair.
2) It found that transplantation of mesenchymal stem cells into the injured cortex led to increased extracellular metalloproteinases, decreased cell death, increased host cell proliferation and regeneration, and improved motor and neurological functions.
3) This suggests stem cells may act as "bio bridges" recruiting endogenous stem cells through metalloproteinase expression to reconnect neurogenic niches to injured sites and promote repair.
The document summarizes several studies on stem cell niches. It finds that bone marrow niches for hematopoietic stem cells are regulated by osteoblasts lining bone surfaces. Deleting a BMP receptor in osteoblasts leads to more osteoblasts and more stem cells. Treating with parathyroid hormone also expands the osteoblast population and stem cell numbers by increasing Notch signaling from osteoblasts to stem cells. The microenvironment provided by osteoblasts, including cell-cell signaling via Notch ligands, plays a key role in regulating the stem cell niche in bone marrow.
This document summarizes research on the effects of misexpressing the Dip3 gene in Drosophila eye-antennal discs. The key findings are:
1. Dip3 misexpression leads to two distinct outcomes - antennal duplication, where the antennal disc splits into multiple domains each forming an antenna, and eye-to-antenna transformation, where the eye disc adopts an antennal fate and forms antenna-like structures.
2. Antennal duplication occurs when Dip3 causes underproliferation of the eye disc and overproliferation of the antennal disc, leading to its splitting. Eye-to-antenna transformation involves downregulation of eye
This paper resulted from joint research between Thai and Oz researchers at the Australian Synchrotron in 2008. It represents a on-going collaboration between Siam Photon, Suranaree University of Technology, Monash University and the Australian Synchrotron
This document summarizes a study that identified novel molecules involved in axon-glial interactions during peripheral myelination. The researchers:
1) Developed a method to isolate projections ("pseudopods") that Schwann cells extend in response to signals from axons, allowing proteomic analysis of proteins specifically present at axon-glial contacts.
2) Identified major signaling networks and novel proteins, including members of the Prohibitin family, at the glial leading edge contacting axons.
3) Found that genetic deletion of Prohibitin-2 in mice impairs axon-glial interactions and myelination, validating its importance.
This novel method provides insights into molecular organization
Cuckoo Search Optimization of Blebs in Human Embryonic Stem CellsIJMERJOURNAL
ABSTRACT: The main aim of this project is to segment the bleb from human embryonic stem cells (hESC). The behavior of bleb can be used to distinguish apoptotic bleb from the healthy bleb. The health of the human embryonic stem cells can be determined using the portion of bleb formed on the surface of the stem cells. The complete bleb formation contains bleb extraction and retraction. This paper uses the active contour algorithm for the segmentation of bleb from human embryonic stem cells. The output of the segmentation, input video and area of bleb can be used as an input to the optimization process. The cuckoo search algorithm is utilized for optimization, which inspired from the brooding parasitism will enhance the segmentation result. The proposed method attains the quick and accurate analysis in the bleb extraction process
This presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.
Loss of photoreceptor potential from retinal progenitor cell cultures, despit...Dr Reaz Vawda, MSc PhD
1) Researchers improved survival and growth rates of retinal progenitor cells (RPCs) derived from mouse retinal cells by modifying dissociation and culture methods, but photoreceptor potential was still lost.
2) Freshly dissociated retinal cells from postnatal mice that expressed rhodopsin integrated at higher rates after transplantation and formed photoreceptors, indicating these were post-mitotic photoreceptor precursors.
3) Cultured RPCs, even after attempted differentiation, did not generate photoreceptors or express rhodopsin after transplantation. Identifying cues to promote survival of specified photoreceptor precursors in vitro is needed for therapeutic use of RPCs.
- EB size affects the differentiation of hiPS cells, with larger EBs encouraging expression of the neuroectodermal marker PAX6 early on but medium EBs (500-3000 cells) best encouraging later expression of PAX6 and MiTF, a marker of ocular neuroectodermal lineage cells.
- At early timepoints, larger EBs expressed more PAX6, but at later timepoints medium EBs expressed more PAX6 and MiTF, possibly due to cell-cell interactions in larger EBs affecting PAX6 expression over time.
- The study shows EB size can be used to control differentiation of hiPS cells, with medium EBs most effectively encouraging differentiation into ocular neuroectodermal lineage cells
Akhtar and Breunig-2015-Frontiers in Cellular Neuroscience - Barriers to post...Aslam Akhtar, MS
This document summarizes barriers to postnatal neurogenesis in the cerebral cortex. It describes how the cortex develops prenatally through tightly regulated neurogenesis along radial glial scaffolds. After birth, radial glia are depleted and the "gliogenic switch" stops neurogenesis in favor of gliogenesis. As a result, the adult cortex has very little ability to replace neurons lost to injury or disease. Strategies to promote postnatal neurogenesis, such as interneuron transplantation and glial reprogramming, aim to circumvent the developmental barriers normally preventing regeneration in the adult brain.
This research article examines the organization of projections from the mouse dorsal lateral geniculate nucleus (dLGN) to the primary visual cortex (V1) using retrograde tracers. The study finds that:
1) Projection columns within the dLGN that project to V1 exhibit highly variable organization in young mice that is refined in adults, displaying profiles consistent with shell and core zones.
2) Projection column organization is disrupted in adult mice that lacked correlated spontaneous activity during development.
3) Analysis across groups suggests there may be 4-6 cryptic laminae along the length of projection columns, indicating greater complexity in dLGN organization than previously thought.
This study investigated the effects of knocking down neuroligin genes on planarian regeneration abilities. Planarians were separated into four groups - a control group with an unc22 gene knockdown and three experimental groups with neuroligin 1A/1B, 2, or 1B/2 gene knockdowns. The worms were cut and imaged over 10 days to observe eyespot regeneration and blastema tissue re-pigmentation. Results showed that single neuroligin knockdowns caused 1-2 day delays in regeneration, while the double 1B/2 knockdown caused more severe 4 day delays. This supports the hypothesis that neuroligins are important for synaptic signaling required for normal planarian regeneration.
1) Lhx2 acts as a classic selector gene that cell-autonomously specifies cortical identity and suppresses alternative fates like the hippocampal organizer fate in the developing mouse brain.
2) Using genetic mosaics and timed inactivations in mice, the study demonstrates that Lhx2 performs this function during a critical period from E8.5-E10.5 when stem cells comprise the cortical neuroepithelium.
3) In the absence of Lhx2, cells laterally adopt an antihem identity and medially become cortical hem cells, which can induce and organize ectopic hippocampal fields, showing that the cortical hem is a hippocampal organizer.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
1) Activated microglia secrete factors that promote neurogenesis from white matter cells, whereas resting microglia do not. When neonatal optic nerve cells were cocultured with activated microglia, there was a significant increase in neurons identified by TUJ-1 labeling compared to cocultures with resting microglia.
2) Oligodendrocyte progenitor cells (OPCs) generated some neurons but were not the major source. Around 70% of neurons came from A2B5-negative cells, which included astrocytes. Activated microglia increased neurogenesis from both OPCs and A2B5-negative cells.
3) Mass spectrometry identified protease ser
This document describes a new method for rapidly expanding populations of human neural precursor cells in culture over long periods of time. The researchers isolated precursor cells from human fetal brain tissue and grew them in culture as floating sphere clusters. Using traditional passaging techniques, which involve mechanically dissociating the spheres, only a 12-fold expansion of cells could be achieved over several months. However, by sectioning the spheres into quarters instead of dissociating them, cell-cell contacts were maintained and cellular trauma was minimized. This allowed for a 1.5 million-fold increase in cell number within less than 200 days. Upon differentiation, the cells formed astrocytes and neurons but no oligodendrocytes. This novel culture method provides
The Effects of Octanol on Gap Junctions and Actin of Neoblasts during Smed Pl...Marianne Gadiano
An independent project for Developmental Biology Lab (BIOL340), testing the effects of octanol exposure on the regeneration of Schmidtea mediterranea planaria. We tested to see whether or not actin was involved in the gap junction communication of migrating neoblasts during regeneration. We performed RT-PCR and Western Blot assays to detect any changes in actin levels from RNA and protein. Our results show that there seems to be little correlation between actin and gap junctions. We would like to conduct future studies on myosin, F-actin, and G-actin. The poster for this project was presented as part of the Spring 2016 Investigative Biology Labs Poster Session at the University of Maryland, Baltimore County (UMBC). Including BIOL340L, the session also features research projects from students in the Molecular and General Genetics Lab, and the Phage Hunters: Genome Analysis Lab. The session serves to inform the public about the upper-level laboratories offered by the Biological Sciences department at UMBC.
100 Original WorkZero PlagiarismGraduate Level Writing Required.docxchristiandean12115
This document provides instructions for a 1,250- to 1,400-word paper that is due on March 6, 2021. Students must choose between the topics of immigration, drug legislation, or three-strikes sentencing. For the selected topic, students must describe how each branch of the US government (executive, legislative, judicial) participates in the policy. The paper must follow APA formatting guidelines and include at least three peer-reviewed literature references, excluding sources like Wikipedia.
10.11771066480704270150THE FAMILY JOURNAL COUNSELING AND THE.docxchristiandean12115
10.1177/1066480704270150THE FAMILY JOURNAL: COUNSELING AND THERAPY FOR COUPLES AND FAMILIES / January 2005Lambert / GAY AND LESBIAN FAMILIES
❖ Literature Review—Research
Gay and Lesbian Families:
What We Know and Where to Go From Here
Serena Lambert
Idaho State University
The author reviewed the research on gay and lesbian parents and
their children. The current body of research has been clear and con-
sistent in establishing that children of gay and lesbian parents are as
psychologically healthy as their peers from heterosexual homes.
However, this comparison approach to research design appears to
have limited the scope of research on gay and lesbian families, leav-
ing much of the experience of these families yet to be investigated.
Keywords: gay men; lesbians; parenting; families
The relationships and family lives of gay and lesbian peo-ple have been the focus of much controversy in the past
decade. The legal and social implications of gay and lesbian
parents appear to have clearly affected the direction that
researchers in the fields of psychology and sociology have
taken in regard to these diverse families. As clinicians, educa-
tors, and researchers, counselors need to be aware of and
involved with issues related to lesbian and gay family life for
several reasons. First, our professional code of ethics charges
us with the ethical responsibility to demonstrate a commit-
ment to gaining knowledge, personal awareness, sensitivity,
and skills significant for working with diverse populations
(American Counseling Association, 1995; International
Association of Marriage and Family Counselors, n.d.). Coun-
selors are also in a unique position to advocate for diverse
clients and families in their communities as well as in their
practices but must possess the knowledge to do so effectively
(Eriksen, 1999). It is believed that work in this area not only
has the potential to affect the lives of our gay and lesbian cli-
ents and their children but also influences developmental and
family theory and informs public policies for the future
(Patterson, 1995, 2000; Savin-Williams & Esterberg, 2000).
This article will review the recent research regarding fami-
lies headed by gay men and lesbians. Studies reviewed in-
clude investigations of gay or lesbian versus homosexual par-
ents, sources of diversity among gay and lesbian parents, and
the personal and sociological development of the children of
gay and lesbian parents. Implications for counselors as well
as directions for future research will also be discussed.
GAY AND LESBIAN PARENTS
How Many Are Out There?
Unfortunately, accurate statistics regarding the numbers
of families headed by gay men and lesbians in our culture are
difficult to determine. Due to fear of discrimination in one or
more aspects of their lives, many gay men and lesbians have
carefully kept their sexual orientation concealed—even from
their own children in some cases (Huggins, 1989). Patterson
(2000) noted that it is es.
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2016_Association for Research in Vision and Ophthalmology_2Mauricio Rosenfeld
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1) The study investigated the hypothesis that neural stem cells from the subventricular zone (SVZ) and dentate gyrus (DG) are recruited to injured brain sites via extracellular factors to aid repair.
2) It found that transplantation of mesenchymal stem cells into the injured cortex led to increased extracellular metalloproteinases, decreased cell death, increased host cell proliferation and regeneration, and improved motor and neurological functions.
3) This suggests stem cells may act as "bio bridges" recruiting endogenous stem cells through metalloproteinase expression to reconnect neurogenic niches to injured sites and promote repair.
The document summarizes several studies on stem cell niches. It finds that bone marrow niches for hematopoietic stem cells are regulated by osteoblasts lining bone surfaces. Deleting a BMP receptor in osteoblasts leads to more osteoblasts and more stem cells. Treating with parathyroid hormone also expands the osteoblast population and stem cell numbers by increasing Notch signaling from osteoblasts to stem cells. The microenvironment provided by osteoblasts, including cell-cell signaling via Notch ligands, plays a key role in regulating the stem cell niche in bone marrow.
This document summarizes research on the effects of misexpressing the Dip3 gene in Drosophila eye-antennal discs. The key findings are:
1. Dip3 misexpression leads to two distinct outcomes - antennal duplication, where the antennal disc splits into multiple domains each forming an antenna, and eye-to-antenna transformation, where the eye disc adopts an antennal fate and forms antenna-like structures.
2. Antennal duplication occurs when Dip3 causes underproliferation of the eye disc and overproliferation of the antennal disc, leading to its splitting. Eye-to-antenna transformation involves downregulation of eye
This paper resulted from joint research between Thai and Oz researchers at the Australian Synchrotron in 2008. It represents a on-going collaboration between Siam Photon, Suranaree University of Technology, Monash University and the Australian Synchrotron
This document summarizes a study that identified novel molecules involved in axon-glial interactions during peripheral myelination. The researchers:
1) Developed a method to isolate projections ("pseudopods") that Schwann cells extend in response to signals from axons, allowing proteomic analysis of proteins specifically present at axon-glial contacts.
2) Identified major signaling networks and novel proteins, including members of the Prohibitin family, at the glial leading edge contacting axons.
3) Found that genetic deletion of Prohibitin-2 in mice impairs axon-glial interactions and myelination, validating its importance.
This novel method provides insights into molecular organization
Cuckoo Search Optimization of Blebs in Human Embryonic Stem CellsIJMERJOURNAL
ABSTRACT: The main aim of this project is to segment the bleb from human embryonic stem cells (hESC). The behavior of bleb can be used to distinguish apoptotic bleb from the healthy bleb. The health of the human embryonic stem cells can be determined using the portion of bleb formed on the surface of the stem cells. The complete bleb formation contains bleb extraction and retraction. This paper uses the active contour algorithm for the segmentation of bleb from human embryonic stem cells. The output of the segmentation, input video and area of bleb can be used as an input to the optimization process. The cuckoo search algorithm is utilized for optimization, which inspired from the brooding parasitism will enhance the segmentation result. The proposed method attains the quick and accurate analysis in the bleb extraction process
This presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.
Loss of photoreceptor potential from retinal progenitor cell cultures, despit...Dr Reaz Vawda, MSc PhD
1) Researchers improved survival and growth rates of retinal progenitor cells (RPCs) derived from mouse retinal cells by modifying dissociation and culture methods, but photoreceptor potential was still lost.
2) Freshly dissociated retinal cells from postnatal mice that expressed rhodopsin integrated at higher rates after transplantation and formed photoreceptors, indicating these were post-mitotic photoreceptor precursors.
3) Cultured RPCs, even after attempted differentiation, did not generate photoreceptors or express rhodopsin after transplantation. Identifying cues to promote survival of specified photoreceptor precursors in vitro is needed for therapeutic use of RPCs.
- EB size affects the differentiation of hiPS cells, with larger EBs encouraging expression of the neuroectodermal marker PAX6 early on but medium EBs (500-3000 cells) best encouraging later expression of PAX6 and MiTF, a marker of ocular neuroectodermal lineage cells.
- At early timepoints, larger EBs expressed more PAX6, but at later timepoints medium EBs expressed more PAX6 and MiTF, possibly due to cell-cell interactions in larger EBs affecting PAX6 expression over time.
- The study shows EB size can be used to control differentiation of hiPS cells, with medium EBs most effectively encouraging differentiation into ocular neuroectodermal lineage cells
Akhtar and Breunig-2015-Frontiers in Cellular Neuroscience - Barriers to post...Aslam Akhtar, MS
This document summarizes barriers to postnatal neurogenesis in the cerebral cortex. It describes how the cortex develops prenatally through tightly regulated neurogenesis along radial glial scaffolds. After birth, radial glia are depleted and the "gliogenic switch" stops neurogenesis in favor of gliogenesis. As a result, the adult cortex has very little ability to replace neurons lost to injury or disease. Strategies to promote postnatal neurogenesis, such as interneuron transplantation and glial reprogramming, aim to circumvent the developmental barriers normally preventing regeneration in the adult brain.
This research article examines the organization of projections from the mouse dorsal lateral geniculate nucleus (dLGN) to the primary visual cortex (V1) using retrograde tracers. The study finds that:
1) Projection columns within the dLGN that project to V1 exhibit highly variable organization in young mice that is refined in adults, displaying profiles consistent with shell and core zones.
2) Projection column organization is disrupted in adult mice that lacked correlated spontaneous activity during development.
3) Analysis across groups suggests there may be 4-6 cryptic laminae along the length of projection columns, indicating greater complexity in dLGN organization than previously thought.
This study investigated the effects of knocking down neuroligin genes on planarian regeneration abilities. Planarians were separated into four groups - a control group with an unc22 gene knockdown and three experimental groups with neuroligin 1A/1B, 2, or 1B/2 gene knockdowns. The worms were cut and imaged over 10 days to observe eyespot regeneration and blastema tissue re-pigmentation. Results showed that single neuroligin knockdowns caused 1-2 day delays in regeneration, while the double 1B/2 knockdown caused more severe 4 day delays. This supports the hypothesis that neuroligins are important for synaptic signaling required for normal planarian regeneration.
1) Lhx2 acts as a classic selector gene that cell-autonomously specifies cortical identity and suppresses alternative fates like the hippocampal organizer fate in the developing mouse brain.
2) Using genetic mosaics and timed inactivations in mice, the study demonstrates that Lhx2 performs this function during a critical period from E8.5-E10.5 when stem cells comprise the cortical neuroepithelium.
3) In the absence of Lhx2, cells laterally adopt an antihem identity and medially become cortical hem cells, which can induce and organize ectopic hippocampal fields, showing that the cortical hem is a hippocampal organizer.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
1) Activated microglia secrete factors that promote neurogenesis from white matter cells, whereas resting microglia do not. When neonatal optic nerve cells were cocultured with activated microglia, there was a significant increase in neurons identified by TUJ-1 labeling compared to cocultures with resting microglia.
2) Oligodendrocyte progenitor cells (OPCs) generated some neurons but were not the major source. Around 70% of neurons came from A2B5-negative cells, which included astrocytes. Activated microglia increased neurogenesis from both OPCs and A2B5-negative cells.
3) Mass spectrometry identified protease ser
This document describes a new method for rapidly expanding populations of human neural precursor cells in culture over long periods of time. The researchers isolated precursor cells from human fetal brain tissue and grew them in culture as floating sphere clusters. Using traditional passaging techniques, which involve mechanically dissociating the spheres, only a 12-fold expansion of cells could be achieved over several months. However, by sectioning the spheres into quarters instead of dissociating them, cell-cell contacts were maintained and cellular trauma was minimized. This allowed for a 1.5 million-fold increase in cell number within less than 200 days. Upon differentiation, the cells formed astrocytes and neurons but no oligodendrocytes. This novel culture method provides
The Effects of Octanol on Gap Junctions and Actin of Neoblasts during Smed Pl...Marianne Gadiano
An independent project for Developmental Biology Lab (BIOL340), testing the effects of octanol exposure on the regeneration of Schmidtea mediterranea planaria. We tested to see whether or not actin was involved in the gap junction communication of migrating neoblasts during regeneration. We performed RT-PCR and Western Blot assays to detect any changes in actin levels from RNA and protein. Our results show that there seems to be little correlation between actin and gap junctions. We would like to conduct future studies on myosin, F-actin, and G-actin. The poster for this project was presented as part of the Spring 2016 Investigative Biology Labs Poster Session at the University of Maryland, Baltimore County (UMBC). Including BIOL340L, the session also features research projects from students in the Molecular and General Genetics Lab, and the Phage Hunters: Genome Analysis Lab. The session serves to inform the public about the upper-level laboratories offered by the Biological Sciences department at UMBC.
Similar to JC3article(2).pdf3 7 6 N A T U R E V O L 5 3 1 .docx (20)
100 Original WorkZero PlagiarismGraduate Level Writing Required.docxchristiandean12115
This document provides instructions for a 1,250- to 1,400-word paper that is due on March 6, 2021. Students must choose between the topics of immigration, drug legislation, or three-strikes sentencing. For the selected topic, students must describe how each branch of the US government (executive, legislative, judicial) participates in the policy. The paper must follow APA formatting guidelines and include at least three peer-reviewed literature references, excluding sources like Wikipedia.
10.11771066480704270150THE FAMILY JOURNAL COUNSELING AND THE.docxchristiandean12115
10.1177/1066480704270150THE FAMILY JOURNAL: COUNSELING AND THERAPY FOR COUPLES AND FAMILIES / January 2005Lambert / GAY AND LESBIAN FAMILIES
❖ Literature Review—Research
Gay and Lesbian Families:
What We Know and Where to Go From Here
Serena Lambert
Idaho State University
The author reviewed the research on gay and lesbian parents and
their children. The current body of research has been clear and con-
sistent in establishing that children of gay and lesbian parents are as
psychologically healthy as their peers from heterosexual homes.
However, this comparison approach to research design appears to
have limited the scope of research on gay and lesbian families, leav-
ing much of the experience of these families yet to be investigated.
Keywords: gay men; lesbians; parenting; families
The relationships and family lives of gay and lesbian peo-ple have been the focus of much controversy in the past
decade. The legal and social implications of gay and lesbian
parents appear to have clearly affected the direction that
researchers in the fields of psychology and sociology have
taken in regard to these diverse families. As clinicians, educa-
tors, and researchers, counselors need to be aware of and
involved with issues related to lesbian and gay family life for
several reasons. First, our professional code of ethics charges
us with the ethical responsibility to demonstrate a commit-
ment to gaining knowledge, personal awareness, sensitivity,
and skills significant for working with diverse populations
(American Counseling Association, 1995; International
Association of Marriage and Family Counselors, n.d.). Coun-
selors are also in a unique position to advocate for diverse
clients and families in their communities as well as in their
practices but must possess the knowledge to do so effectively
(Eriksen, 1999). It is believed that work in this area not only
has the potential to affect the lives of our gay and lesbian cli-
ents and their children but also influences developmental and
family theory and informs public policies for the future
(Patterson, 1995, 2000; Savin-Williams & Esterberg, 2000).
This article will review the recent research regarding fami-
lies headed by gay men and lesbians. Studies reviewed in-
clude investigations of gay or lesbian versus homosexual par-
ents, sources of diversity among gay and lesbian parents, and
the personal and sociological development of the children of
gay and lesbian parents. Implications for counselors as well
as directions for future research will also be discussed.
GAY AND LESBIAN PARENTS
How Many Are Out There?
Unfortunately, accurate statistics regarding the numbers
of families headed by gay men and lesbians in our culture are
difficult to determine. Due to fear of discrimination in one or
more aspects of their lives, many gay men and lesbians have
carefully kept their sexual orientation concealed—even from
their own children in some cases (Huggins, 1989). Patterson
(2000) noted that it is es.
10.11771066480703252339 ARTICLETHE FAMILY JOURNAL COUNSELING.docxchristiandean12115
10.1177/1066480703252339 ARTICLETHE FAMILY JOURNAL: COUNSELING AND THERAPY FOR COUPLES AND FAMILIES / July 2003Fall, Lyons / ETHICAL CONSIDERATIONS
❖ Ethics
Ethical Considerations of Family Secret
Disclosure and Post-Session Safety Management
Kevin A. Fall
Christy Lyons
Loyola University—New Orleans
The ethical issues involved in the disclosure of family secrets in ther-
apy have been addressed in the literature, but the focus has typically
been on secrets disclosed in individual sessions. The literature
largely ignores the ethical issues surrounding in-session disclosure
and the concomitant liability of the family therapist for the post-ses-
sion well-being of the system’s members. This article explores types
of family secrets, provides a case example of in-session disclosure,
and presents ethical considerations and practice recommendations.
Keywords: family secrets; ethics; confidentiality; abuse; safety
A
family without secrets is like a two-year-old without
tantrums: a rarity. Virtually every family has secrets
involving academic problems, relationship dynamics, or even
various illegalities. Secrets permeate the family system
before therapy begins, but with the introduction of the thera-
pist, the system begins to change. The therapist ideally creates
an environment that challenges the boundaries and rules of
the system; this is the nature of therapy. As a result of the
sense of safety within the session, it is conceivable that a fam-
ily member may disclose information that has been hidden for
a wide variety of reasons. Any unearthing of hidden material
will create a disequilibrium within the system. Family thera-
pists are trained to handle the consequences of such a disclo-
sure in session and ethically lay the groundwork for timely
disclosures. Dealing with this disclosure and its impact on the
system often becomes the primary focus of the therapy, as the
perturbation caused by the disclosure can serve as a catalyst to
reorganize the system.
However, not all information is disclosed at the “perfect
time.” In fact, the idiosyncratic internal sensing of safety by
any member of the family may trigger a disclosure prema-
turely. Secrets are such an omnipresent dynamic in the life of
family systems that it seems unlikely that any family therapist
could avoid untimely disclosures. Even in these unpredict-
able moments, a disclosure creates a disequilibrium that can
be productive in the therapy process as the secret and the pro-
cess of maintaining the secret are worked through in an
atmosphere of trust and safety. The ethical question here is
two-fold: What is the therapist’s responsibility in preparing
the family members for the potential risks of counseling that
may arise from such disclosures, and what is the responsibil-
ity of the family therapist to maintain the safety of the mem-
bers after a disclosure?
Although the International Association of Marriage and
Family Counselors’ (IAMFC).
10.11770022427803260263ARTICLEJOURNAL OF RESEARCH IN CRIME AN.docxchristiandean12115
This document summarizes competing theories on whether the perceived risk of punishment deters criminally prone individuals from committing crimes. It discusses three main perspectives: 1) that all individuals are equally deterred regardless of criminal propensity, 2) that criminally prone individuals are less deterred due to their impulsivity and focus on immediate gratification, and 3) that criminally prone individuals are more deterred since socialized individuals act based on moral obligations rather than costs/benefits. The article then analyzes data from a longitudinal study in New Zealand to test the relationship between criminal propensity, perceived punishment risks, and criminal behavior.
10.11770022487105285962Journal of Teacher Education, Vol. 57,.docxchristiandean12115
10.1177/0022487105285962Journal of Teacher Education, Vol. 57, No. XX, XXX/XXX 2006Journal of Teacher Education, Vol. 57, No. XX, XXX/XXX 2006
CONSTRUCTING 21st-CENTURY TEACHER EDUCATION
Linda Darling-Hammond
Stanford University
Much of what teachers need to know to be successful is invisible to lay observers, leading to the view
that teaching requires little formal study and to frequent disdain for teacher education programs. The
weakness of traditional program models that are collections of largely unrelated courses reinforce this
low regard. This article argues that we have learned a great deal about how to create stronger, more ef-
fective teacher education programs. Three critical components of such programs include tight coher-
ence and integration among courses and between course work and clinical work in schools, extensive
and intensely supervised clinical work integrated with course work using pedagogies linking theory
and practice, and closer, proactive relationships with schools that serve diverse learners effectively
and develop and model good teaching. Also, schools of education should resist pressures to water
down preparation, which ultimately undermine the preparation of entering teachers, the reputation
of schools of education, and the strength of the profession.
Keywords: field-based experiences; foundations of education; student teaching; supervision; theo-
ries of teacher education
The previous articles have articulated a spectac-
ular array of things that teachers should know
and be able to do in their work. These include
understanding many things about how people
learn and how to teach effectively, including as-
pects of pedagogical content knowledge that in-
corporate language, culture, and community
contexts for learning. Teachers also need to un-
derstand the person, the spirit, of every child
and find a way to nurture that spirit. And they
need the skills to construct and manage class-
room activities efficiently, communicate well,
use technology, and reflect on their practice to
learn from and improve it continually.
The importance of powerful teaching is
increasingly important in contemporary soci-
ety. Standards for learning are now higher than
they have ever been before, as citizens and
workers need greater knowledge and skill to
survive and succeed. Education is increasingly
important to the success of both individuals and
nations, and growing evidence demonstrates
that—among all educational resources—teach-
ers’ abilities are especially crucial contributors
t o s t u d e n t s ’ le a r n i n g . F u r t h e r m o re , t h e
demands on teachers are increasing. Teachers
need not only to be able to keep order and pro-
vide useful information to students but also to
be increasingly effective in enabling a diverse
group of students to learn ever more complex
material. In previous decades, they were
expected to prepare only a small minority for
ambitious intellectual work, whereas they are
now expected to prep.
10.1 What are three broad mechanisms that malware can use to propa.docxchristiandean12115
10.1 What are three broad mechanisms that malware can use to propagate?
10.2 What are four broad categories of payloads that malware may carry?
10.3 What are typical phases of operation of a virus or worm?
10.4 What mechanisms can a virus use to conceal itself?
10.5 What is the difference between machine-executable and macro viruses?
10.6 What means can a worm use to access remote systems to propagate?
10.7 What is a “drive-by-download” and how does it differ from a worm?
10.8 What is a “logic bomb”?
10.9 Differentiate among the following: a backdoor, a bot, a keylogger, spyware, and a rootkit? Can they all be present in the same malware?
10.10 List some of the different levels in a system that a rootkit may use.
10.11 Describe some malware countermeasure elements.
10.12 List three places malware mitigation mechanisms may be located.
10.13 Briefly describe the four generations of antivirus software.
10.14 How does behavior-blocking software work?
10.15 What is a distributed denial-of-service system?
.
10.0 ptsPresentation of information was exceptional and included.docxchristiandean12115
10.0 pts
Presentation of information was exceptional and included all of the following elements: Identifies the role of concept analysis within theory development. Identifies the selected nursing concept. Identifies the nursing theory from which the selected concept was obtained. A nursing theory was used. Identifies the sections of the paper. Scholarly support from nursing literature was provided.
9.0 pts
Presentation of information was good, but was superficial in places and included all of the following elements: Identifies the role of concept analysis within theory development. Identifies the selected nursing concept. Identifies the nursing theory from which the selected concept was obtained. A nursing theory was used. Identifies the sections of the paper. Scholarly support from nursing literature was provided.
8.0 pts
Presentation of information was minimally demonstrated in the all of the following elements: Identifies the role of concept analysis within theory development. Identifies the selected nursing concept. Identifies the nursing theory from which the selected concept was obtained. A nursing theory was used. Identifies the sections of the paper. Limited scholarly support from nursing literature was provided.
4.0 pts
Presentation of information in one or two of the following elements fails to meet expectations: Identifies the role of concept analysis within theory development. Identifies the selected nursing concept. Identifies the nursing theory from which the selected concept was obtained. A nursing theory was used. Identifies the sections of the paper. Limited or no scholarly support from nursing literature was provided.
0.0 pts
Presentation of information is unsatisfactory in three or more of the following elements: Identifies the role of concept analysis within theory development. Identifies the selected nursing concept. Identifies the nursing theory from which the selected concept was obtained. A nursing theory was used. Identifies the sections of the paper. Limited or no scholarly support from nursing literature was provided.
10.0 pts
This criterion is linked to a Learning Outcome Definition/Explanation of Selected Concept
25.0 pts
Presentation of information was exceptional and included all of the following elements: Defines/explains the concept using scholarly literature (a dictionary maybe used for this section ONLY, and additional scholarly nursing references are required). Provides support from scholarly sources.
22.0 pts
Presentation of information was good, but was superficial in places and included all of the following elements: Defines/explains the concept using scholarly literature (a dictionary maybe used for this section ONLY, and additional scholarly nursing references are required). Provides support from scholarly sources.
20.0 pts
Presentation of information was minimally demonstrated in the all of the following elements: Defines/explains the concept using scholarly literature (a dictionary maybe used for thi.
10-K
1
f12312012-10k.htm
10-K
UNITED STATES
SECURITIES AND EXCHANGE COMMISSION
Washington, DC 20549
FORM 10-K
(Mark One)
R
Annual report pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934
For the fiscal year ended December 31, 2012
or
o
Transition report pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934
For the transition period from __________ to __________
Commission file number 1-3950
Ford Motor Company
(Exact name of Registrant as specified in its charter)
Delaware
38-0549190
(State of incorporation)
(I.R.S. Employer Identification No.)
One American Road, Dearborn, Michigan
48126
(Address of principal executive offices)
(Zip Code)
313-322-3000
(Registrant’s telephone number, including area code)
Securities registered pursuant to Section 12(b) of the Act:
Title of each class
Name of each exchange on which registered*
Common Stock, par value $.01 per share
New York Stock Exchange
__________
* In addition, shares of Common Stock of Ford are listed on certain stock exchanges in Europe.
Securities registered pursuant to Section 12(g) of the Act: None.
Indicate by check mark if the registrant is a well-known seasoned issuer, as defined in Rule 405 of the Securities Act. Yes R No o
Indicate by check mark if the registrant is not required to file reports pursuant to Section 13 or Section 15(d) of the Act. Yes o No R
Indicate by check mark if the registrant (1) has filed all reports required to be filed by Section 13 or 15(d) of the Securities Exchange Act of 1934 during the preceding 12 months (or for such shorter period that the registrant was required to file such reports), and (2) has been subject to such filing requirements for the past 90 days. Yes R No o
Indicate by check mark whether the registrant has submitted electronically and posted on its corporate Web site, if any, every Interactive Data File required to be submitted and posted pursuant to Rule 405 of Regulation S-T (§232.405 of this chapter) during the preceding 12 months (or for such shorter period that the registrant was required to submit and post such files). Yes R No o
Indicate by check mark if disclosure of delinquent filers pursuant to Item 405 of Regulation S-K (§229.405 of this chapter) is not contained herein, and will not be contained, to the best of registrant’s knowledge, in definitive proxy or information statements incorporated by reference in Part III of this Form 10-K or any amendment to this Form 10-K. R
Indicate by check mark whether the registrant is a large accelerated filer, an accelerated filer, a non-accelerated filer, or a smaller reporting company. See definitions of "large accelerated filer," "accelerated filer," and "smaller reporting company" in Rule 12b-2 of the Exchange Act. Large accelerated filer R Accelerated filer o Non-accelerated filer o Smaller reporting company o
Indicate by check mark whether the registra.
10-K 1 f12312012-10k.htm 10-K UNITED STATESSECURITIES AN.docxchristiandean12115
10-K 1 f12312012-10k.htm 10-K
UNITED STATES
SECURITIES AND EXCHANGE COMMISSION
Washington, DC 20549
FORM 10-K
(Mark One)
R Annual report pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934
For the fiscal year ended December 31, 2012
or
o Transition report pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934
For the transition period from __________ to __________
Commission file number 1-3950
Ford Motor Company
(Exact name of Registrant as specified in its charter)
Delaware 38-0549190
(State of incorporation) (I.R.S. Employer Identification No.)
One American Road, Dearborn, Michigan 48126
(Address of principal executive offices) (Zip Code)
313-322-3000
(Registrant’s telephone number, including area code)
Securities registered pursuant to Section 12(b) of the Act:
Title of each class Name of each exchange on which registered*
Common Stock, par value $.01 per share New York Stock Exchange
__________
* In addition, shares of Common Stock of Ford are listed on certain stock exchanges in Europe.
Securities registered pursuant to Section 12(g) of the Act: None.
Indicate by check mark if the registrant is a well-known seasoned issuer, as defined in Rule 405 of the Securities Act.
Yes R No o
Indicate by check mark if the registrant is not required to file reports pursuant to Section 13 or Section 15(d) of the Act.
Yes o No R
Indicate by check mark if the registrant (1) has filed all reports required to be filed by Section 13 or 15(d) of the Securities
Exchange Act of 1934 during the preceding 12 months (or for such shorter period that the registrant was required to file such
reports), and (2) has been subject to such filing requirements for the past 90 days. Yes R No o
Indicate by check mark whether the registrant has submitted electronically and posted on its corporate Web site, if any,
every Interactive Data File required to be submitted and posted pursuant to Rule 405 of Regulation S-T (§232.405 of this
Page 1 of 216F 12.31.2012- 10K
3/7/2019https://www.sec.gov/Archives/edgar/data/37996/000003799613000014/f12312012-10k.htm
chapter) during the preceding 12 months (or for such shorter period that the registrant was required to submit and post such
files). Yes R No o
Indicate by check mark if disclosure of delinquent filers pursuant to Item 405 of Regulation S-K (§229.405 of this chapter)
is not contained herein, and will not be contained, to the best of registrant’s knowledge, in definitive proxy or information
statements incorporated by reference in Part III of this Form 10-K or any amendment to this Form 10-K. R
Indicate by check mark whether the registrant is a large accelerated filer, an accelerated filer, a non-accelerated filer, or a
smaller reporting company. See definitions of "large accelerated filer," "accelerated filer," and "smaller reporting company" in
Rule 12b-2 of the Exchange Act. Large accelerated filer R Accelerated filer .
10 What does a golfer, tennis player or cricketer (or any othe.docxchristiandean12115
10 What does a golfer, tennis player or cricketer (or any other professional sportsperson) focus on to achieve high performance? They nearly always give the same answer: “Repeat my process (that is the process they have practised a million times) – replicate it under real pressure and trust in my ability” That’s why Matthew Lloyd throws the grass up under the roof at Etihad Stadium. It is why Ricky Ponting taps the bat, looks down,
looks up and mouths “watch the ball”. It’s
unnecessary for Matthew Lloyd to toss the
grass. There’s no wind under the roof – it’s
simply a routine that enables him to replicate
his process under pressure.
Ricky Pointing knows you have to watch the
ball. Ponting wants the auto pilot light in his
brain to fl ick on as he mutters “watch the ball”.
High performance in sport is achieved through focusing on your
processes, not the scores.
It is absolutely no different in local government. Our business
is governance and we need to be focusing very hard on our
governance processes. We need to learn these processes, modify
them when necessary, understand them deeply, repeat them
under pressure and trust in our capabilities to deliver. If we do
that, the scores will look after themselves.
I want to share with you my ten most important elements in
the governance process. Let me fi rst say that good governance is
the set of processes, protocols, rules, relationships and behaviours
which lead to consistently good decisions. In the end good
governance is good decisions. You could make lots of good
decisions without good governance. But you will eventually
run out of luck – eventually, bad governance process will lead
to bad decisions. Consistently good decisions come from good
governance processes and practices.
Good governance is not only a prerequisite for consistently
good decisions, it is almost the sole determinant of your
reputation. The way you govern, the ‘vibe’ in the community
and in the local paper about the way you govern is almost the
sole determinant of your reputation. Believe me, if reputation
matters to you, then drive improvements through good
governance.
So here are the ten core elements:
1. THE COUNCIL PLAN
An articulate council plan is a fundamental fi rst step to achieving
your goals. It is your set of promises to your community for a
four-year term.
Unfortunately, there are too many wrong plans:
• Claytons Plans – say too little and are too bland. Delete the
name of the council from these plans and you can’t tell whose
it is! There’s no ‘vibe’ at all.
• Agreeable Plans – where everyone gets their bit in the plan.
There’s no sense of priorities, everyone agrees with everything
in the plan and we save all the real fi ghts and confl icts to be
fought out one by one over the four-year term.
• Opposition-creating Plans – we don’t do this so often but we
sometimes ‘use the numbers’ to enable the dominant group of
councillors to achieve their goals and fail to a.
10 Research-Based Tips for Enhancing Literacy Instruct.docxchristiandean12115
10 Research-Based Tips
for Enhancing Literacy
Instruction for Students
With Intellectual
Disability
Christopher J. Lemons, Jill H. Allor, Stephanie Al Otaiba,
and Lauren M. LeJeune
Literacy
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TEACHING EXCEPTIONAL CHILDREN | SEPTEMBER/OCTOBER 2016 19
In the past 2 decades, researchers
(often working closely with parents,
teachers, and other school staff
members) have conducted studies that
have substantially increased
understanding how to effectively teach
children and adolescents with
intellectual disability (ID) to read. This
research focus has been fueled by
increased societal expectations for
individuals with ID, advocacy efforts,
and legislative priorities (e.g.,
strengthened accountability standards).
Findings from this body of work
indicate that children and adolescents
with ID can obtain higher levels of
reading achievement than previously
anticipated (Allor, Mathes, Roberts,
Cheatham, & Al Otaiba, 2014). Recent
research also suggests that the historic
focus on functional reading (e.g., signs,
restaurant words) for this population of
learners is likely too limited of a focus
for many (Browder et al., 2009).
Research outcomes suggest that
integrating components of traditional
reading instruction (e.g., phonics,
phonemic awareness) into programs
for students with ID will lead to
increases in independent reading skills
for many (Allor, Al Otaiba, Ortiz, &
Folsom, 2014). These increased reading
abilities are likely to lead to greater
postsecondary outcomes, including
employment, independence, and
quality of life. Unfortunately, many
teachers remain unsure of how to best
design and deliver reading intervention
for students with ID.
We offer a set of 10 research-based
tips for special education teachers,
general education teachers, and other
members of IEP teams to consider when
planning literacy instruction for students
with ID in order to maximize student
outcomes. For each tip, we describe our
rationale for the recommendation and
provide implementation guidance. Our
Literacy Instruction and Support
Planning Tool can be used by team
members to organize information to
guide planning. Our aim is to provide
educators and IEP team members with a
framework for reflecting on current
reading practices in order to make
research-based adjustments that are
likely to improve student outcomes.
The Conceptual Model of Literacy
Browder and colleagues (2009) proposed
a conceptual model for early literacy
instruction for students with severe
developmental disabilities. We believe
their framework provides guidance for
designing and delivering literacy
instruction for all students wit.
10 Strategic Points for the Prospectus, Proposal, and Direct Pract.docxchristiandean12115
10 Strategic Points for the Prospectus, Proposal, and Direct Practice Improvement Project
Week Two Assignment Instructions DNP 820
Please read the instructions thoroughly
Tutor MUST have a good command of the English language
The Rubric must be followed, and all the requirements met
This is a thorough professor, and she has strict requirements
I have attached the PICOT and the first 10 points (DNP 815) assignment. This is a continuation of that assignment. Please read the attachments
The following needs to be addressed:
Please note the followings: The introduction and the literature review are complete and thorough. The problem statement is written clearly PICOT is clear and very good Sample:
· How will you determine the sample size?
· What are the inclusion/exclusion criteria of the subjects? Methodology: Why is the selected methodology is appropriate? Please justify!
· Data collection approach needs to be clear. How will you collect your data? What is needed here is to describe the process of collecting data form signing the informed consent until completing the measuring.
· Data analysis-What test will you use to answer your research question?
Clinical/PICOT Questions:
“In adult patients with CVC at a Clear Lake Regional Medical Center, does interventional staff education about hub hygiene provided to RN’s who access the CVC impact CLABSI rates compared to standard care over a one-month period?”
P: Patients with Central Venous Catheters
I: Staff re-education related to Hygiene of the hub
C: Other hospitals
O: Reduce probability of CLABSIs
T: Two months
“In Patients > 65 years of age with central line catheters at a Clear Lake Regional Medical Center, how does staff training of key personnel and reinforcement of central line catheter hub hygiene after its insertion, along with the apt cleansing of the insertion site, before every approach compared with other area hospitals, reduce the incidence of CLABSIs (Central Line Associated Blood-stream Infections) over a one-month period?”
P: Patients > 65 years of age with a Central line
I: Staff training and reinforcement of Central Catheter, Hub Hygiene
C: Other area hospitals
O: Reduce probability of CLABSIs
“In adult patients, with define CVC (CVC), does interventional staff education about hub hygiene provided to RN’s who access the CVC impact CLABSI rates compared to pre and post-intervention assessments
1. I used central Missouri as an example, replace with a description of your site.
2. While you might be interested in CLASBI rates as a primary variable, there are other patient outcomes that would also be important to consider
3. Ensure you can find validity and reliability measures on CLASBI rates if you cannot, we need to determine another question to help
4. How are your two comparison groups different, as they are currently stated the groups seem very much the same, could you state, standard care instead of pre and post intervention assessments?
5. One month is the longe.
10 Most Common Errors in Suicide Assessment/Intervention
Robert Neimeyer & Angela Pfeiffer
1. Avoidance of Strong Feelings – Diverting discussions away from powerful, intense
emotion and toward a more abstract or intellectualized exchange. These responses keep
interactions on a purely cognitive level and prevent exploration of the more profound
feelings of distress, which may hold the key to successful treatment. Do not retreat to
professionalism, advice-giving, or passivity when faced with intense depression, grief, or
fear.
• Do not analyze and ask why they feel that way.
• USE empathy! “With all the hurt you’ve been experiencing it must be impossible
to hold those tears in.”
• Tears and sobbing are often met with silence of tangential issues instead of
putting into words what the client is mutely expressing: “With all the pain you’re
feeling, it must be impossible to hold those tears in.”
• “I don’t think anyone really cares whether I live or die.” Helpers often shift to
discussing why/asking questions as opposed to reflecting emotional content.
2. Superficial Reassurance – trivial responses to clients’ expressions of acute distress and
hopelessness can do more harm than good. Rather than reassuring clients, these responses
risk alienating them and deepening their feelings of being isolated in their distress.
• Attempts to emphasize more positive or optimistic aspects of the situation: “But
you’re so young and have so much to live for!”
• Premature offering of a prepackaged meaning for the client’s difficulties: “Well
life works in mysterious ways. Maybe this is life’s way of challenging you.”
• Directly contradicting the client’s protest of anguish: “Things can’t be all that
bad.”
3. Professionalism – Insulating or protecting by distancing and detaching from the brutal,
exhausting realities of clients’ lives by seeking refuge in the comfortable boundaries of role
definition. The exaggerated air of objectivity/disinterest implies a hierarchical relationship,
which may disempower the client. Although intended to put a person at ease, this can come
across as disinterest or hierarchical. Empathy is a more facilitative response.
• “My thoughts are so awful I could never tell anyone” is often met with, “You can
tell me. I’m a professional” as opposed to the riskier, empathic reply.
4. Inadequate Assessment of Suicidal Intent – Implicit negation of suicide threat by
responding to indirect and direct expressions of risk with avoidance or reassurance rather
than a prompt assessment of the level of intent, planning, and lethality. Most common
among physicians and master’s level counselors – due to time pressures, personal theories
or discomfort with intense feelings.
• What they’ve been thinking, For how long, Specific plans/means, Previous
attempts
1
• “There’s nowhere left to turn” and “I’d be better off dead” should be met with
“You sound so miserable. Are y.
10 Customer Acquisition and Relationship ManagementDmitry .docxchristiandean12115
10 Customer Acquisition and Relationship Management
Dmitry Kalinovsky/iStock/Thinkstock
Patronage by loyal customers yields 65 percent of a typical business’ volume.
—American Management Association
Learning Objectives
After reading this chapter, you should be able to do the following:
• Identify how organizational growth is best achieved by an HCO, and state the effect of the product life cycle
on an organization’s revenues.
• Discuss several approaches that an HCO can use to attract new customers, or patients.
• Delineate the premises upon which customer relationship management is based.
• Explain the advantages of database marketing, and identify ways for an organization to use a marketing
database.
• Provide examples of how an HCO can effectively manage real and virtual customer interactions.
Section 10.1Organizational Growth
Introduction
This chapter focuses on how to attract and keep patients through understanding and meeting
their needs. The long-term success of an HCO depends on its ability to attract new patients
and turn them into loyal customers who not only return for needed services, but recommend
the HCO’s services to others. This is especially important because of the nature of the life cycle
for products and services, from their introduction to their decline. Attracting new customers
and keeping existing ones involves interacting internally and externally with patients, analyz-
ing data on current patients, and managing real and virtual interactions with patients. Manag-
ing relationships with patients helps to ensure that patients stay informed and feel connected
to the HCO through its internal and external customer relationship efforts.
10.1 Organizational Growth
Most organizations have growth as a basic goal. Growth means an increase in revenue and
a greater impact on the communities served. Growth also creates opportunities for staff to
advance and take on new responsibilities. While many activities can help an HCO grow, the
most important is the development of an effective marketing plan to provide a consistent
platform for the organization’s visibility and to brand the HCO as an attractive option for
medical services. The development of an effective marketing plan was stressed in Chapter 8
as a basic marketing need for an HCO: that is, to inform new and existing customers of the
organization’s services and to persuade them to continue using or to try using these services.
Product/Service Life Cycles
Like people, products and services have a life cycle. The term product life cycle refers to the
stages that a product or service goes through from the time it is introduced until it is taken
off the market or “dies.” The stages of the product life cycle, illustrated in Figure 10.1, usually
include the following descriptions:
• Introduction—The stage of researching, developing, and launching the product or
service.
• Growth—The stage when revenues are increasing at a fast rate.
• M.
10 ELEMENTS OF LITERATURE (FROM A TO Z) 1 PLOT (seri.docxchristiandean12115
10 ELEMENTS OF LITERATURE (FROM A TO Z)
1 PLOT (series of events which make-up a story)
A 5-POINT PLOT SEQUENCE:
Exposition: initial part of a story where readers are exposed to setting and characters.
Situation: event in the story which kicks the action forward and begs for an outcome.
Complication: difficulties faced by characters as they experience internal and external conflicts.
Climax: watershed moment when it becomes apparent that major conflicts will be resolved.
Resolution: (Denouement): tying up of the loose ends of the story.
B SUB-PLOTS: PLOTS BENEATH AND AROUND THE MAJOR PLOT.
Foreshadowing: hints and clues of plot.
Flashback: portion of a plot when a character relives a past experience.
Frame story: plot which begins in the present, quickly goes to the past for story, then returns.
Episodic plot: a large plot sequence that is made up of a series of minor plot sequences.
Plausibility: likelihood that certain events within a plot can occur.
Soap Opera: multiple stories told along the sequence and spaced to sustain continual interest.
2 POINT OF VIEW (eyes through which a story is told)
C First Person major (participant major): narrator is the major character in the story.
First Person minor (participant minor): narrator is a minor character in the story.
Third Person omniscient (non-participant omniscient): narrator is outside the story and capable of
seeing into the heart, mind and motivations of all characters.
Third Person limited (non-participant limited): narrator is outside the story and capable of seeing, at
most, into the heart, mind, and motivations of one character. Narrator is
objective if not omniscient.
3 SETTING (time and place of a story, both physical and psychological)
D Physical (external) Setting: the time and place of a story, general and specific.
Psychological (internal) Setting: mood, tone, and temper of story.
E Major Tempers: Romanticism: man is free to choose against moral, spiritual backdrops. If you make
good decisions, you will be rewarded. There is a God that is in control
Existentialism: man is free to choose absent backdrops other than his own. If he feels it is right, then it is
right.
Naturalism: man is largely trapped, a cog in the impersonal machinery. He has no real way of
changing his circumstances.
Realism: eclectic view, but leaning toward the naturalistic position. Sometimes good things happen to
bad people, and sometimes bad things happen to good people. That is just the way it is.
F Other Tempers: Classicism: Man is free, but appears to be trapped due to conflicting codes.
Transcendentalism: Offshoot of romanticism, nature is a window to divine.
Nihilism: Fallout of either extreme existentialism or naturalism. Life is horrible and painful. It
lacks meaning.
4 CONFLICT (nature of the problems faced)
G Four Universal Conflicts: Person versus self
Pe.
10 ers. Although one can learn definitions favor- able to .docxchristiandean12115
10
ers. Although one can learn definitions favor-
able to crime from law-abiding individuals,
one is most likely to learn such definitions
fiom delinquent friends or criminal family
A Theory of sociation members. with These delinquent studies typically others find is the that best as-
Differential predictor of crime, and that these delinquent others partly influence crime by leading the
individual to adopt beliefs conducive to
Association crime (see Agnew, 2000; Akers, 1998; Akers and Sellers, 2004; Waw, 2001 for summaries
of such studies).
Sutherland 's theory has also inspired
Edwin H. Sutherland dnd much additional theorizing in criminology.
Theorists have attempted to better describe
Donald R. Cressey the nature ofthose definitions favorable to vi-
olation of the law (see the next selection in
Chapter 11 by Sykes and Matza). They have
Before Sutherland developed his theory, attempted to better describe the processes by
crime was usually explained in t e r n ofmul- which we learn criminal behavior from oth-
tiple factors-like social class, broken homes, ers (see the description o f social learning the-
age, race, urban or rural location, and mental ory by Akers in Chapter 12). And they have
disorder. Sutherland developed his theory of drawn on Sutherland in an effort to explain
differential association in an effort to explain group differences in crime rates (see the Wolf-
why these various factors were related to gang and Ferracuti and Anderson selections
crime. In doing so, he hoped to organize and in this part). Sutherland's theory o f differen-
integrate the research on crime u p to that tial association, then, is one of the enduring
point, as well as to guide future research. classics in criminology (for excellent discus-
Sutherlandk theory is stated in the f o m o f sions ofthe current state o f differential asso-
nine propositions. He argues that criminal ciation theory, see Matsueda, 1988, and Waw,
behavior is learned by interacting with oth- 2001).
ers, especially intimate others. Criminals
learn both the techniques of committing
crime and the definitions favorable to crime References
from these others. The s k t h proposition> Agnew Robe*. '2000. "Sources of Mminality:
which f o r n the heart of the theory, states Strain and Subcultural Theories." In Joseph F.
that 'h person becomes delinquent because of Sheley (ed.), Criminology: A Contemporary ,
an excess of definitions favorable to law vio- Handbook, 3rd edition, pp. 349-371. Belmont,
lation over definitions unfavorable to viola- CA: Wadsworth.
tion oflaw."According to Sutherland, factors Akers, Ronald L. 1998. Social Learning and So-
such as social class, race, and broken homes cia1 Structure: A General Theory of Crime and
influence crime because they affect the likeli- Deviance. Boston: Northeastern University
hood that individuals willdssociate with oth- Press.
ers who present definitions favorable to Akers, Ronal.
10 academic sources about the topic (Why is America so violent).docxchristiandean12115
10 academic sources about the topic (Why is America so violent?)
*Address all 10 academic sources in the literature review
*What have they added to the literature?
*End literature review with "What has not been addressed is.... "and with "What I'm Addressing....." (I am addressing that overpopulation is the main reason America is so violent).
*Literature review should be a minimum of 2-2 1/2 pages
Attached are my 10 academic sources.
.
Main Java[All of the Base Concepts}.docxadhitya5119
This is part 1 of my Java Learning Journey. This Contains Custom methods, classes, constructors, packages, multithreading , try- catch block, finally block and more.
हिंदी वर्णमाला पीपीटी, hindi alphabet PPT presentation, hindi varnamala PPT, Hindi Varnamala pdf, हिंदी स्वर, हिंदी व्यंजन, sikhiye hindi varnmala, dr. mulla adam ali, hindi language and literature, hindi alphabet with drawing, hindi alphabet pdf, hindi varnamala for childrens, hindi language, hindi varnamala practice for kids, https://www.drmullaadamali.com
This document provides an overview of wound healing, its functions, stages, mechanisms, factors affecting it, and complications.
A wound is a break in the integrity of the skin or tissues, which may be associated with disruption of the structure and function.
Healing is the body’s response to injury in an attempt to restore normal structure and functions.
Healing can occur in two ways: Regeneration and Repair
There are 4 phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. This document also describes the mechanism of wound healing. Factors that affect healing include infection, uncontrolled diabetes, poor nutrition, age, anemia, the presence of foreign bodies, etc.
Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
How to Setup Warehouse & Location in Odoo 17 InventoryCeline George
In this slide, we'll explore how to set up warehouses and locations in Odoo 17 Inventory. This will help us manage our stock effectively, track inventory levels, and streamline warehouse operations.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
Gender and Mental Health - Counselling and Family Therapy Applications and In...PsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
BÀI TẬP DẠY THÊM TIẾNG ANH LỚP 7 CẢ NĂM FRIENDS PLUS SÁCH CHÂN TRỜI SÁNG TẠO ...
JC3article(2).pdf3 7 6 N A T U R E V O L 5 3 1 .docx
1. JC3article(2).pdf
3 7 6 | N A T U R E | V O L 5 3 1 | 1 7 M A R C H 2 0 1 6
LETTER
doi:10.1038/nature17000
Co-ordinated ocular development from human
iPS cells and recovery of corneal function
Ryuhei Hayashi1,2, Yuki Ishikawa2, Yuzuru Sasamoto2,
Ryosuke Katori2, Naoki Nomura2, Tatsuya Ichikawa2, Saori
Araki2,
Takeshi Soma2, Satoshi Kawasaki2, Kiyotoshi Sekiguchi3,
Andrew J. Quantock4, Motokazu Tsujikawa2 & Kohji Nishida2
The eye is a complex organ with highly specialized constituent
tissues derived from different primordial cell lineages. The
retina,
for example, develops from neuroectoderm via the optic vesicle,
the corneal epithelium is descended from surface ectoderm,
while
the iris and collagen-rich stroma of the cornea have a neural
crest
origin. Recent work with pluripotent stem cells in culture has
revealed a previously under-appreciated level of intrinsic
cellular
self-organization, with a focus on the retina and retinal cells1–
5.
Moreover, we and others have demonstrated the in vitro
induction
of a corneal epithelial cell phenotype from pluripotent stem
cells6–9.
2. These studies, however, have a single, tissue-specific focus and
fail to reflect the complexity of whole eye development. Here
we
demonstrate the generation from human induced pluripotent
stem
cells of a self-formed ectodermal autonomous multi-zone
(SEAM)
of ocular cells. In some respects the concentric SEAM mimics
whole-eye development because cell location within different
zones
is indicative of lineage, spanning the ocular surface ectoderm,
lens,
neuro-retina, and retinal pigment epithelium. It thus represents
a promising resource for new and ongoing studies of ocular
morphogenesis. The approach also has translational potential
and to illustrate this we show that cells isolated from the ocular
surface ectodermal zone of the SEAM can be sorted and
expanded
ex vivo to form a corneal epithelium that recovers function in
an
experimentally induced animal model of corneal blindness.
To generate a SEAM of ocular cells, human induced pluripotent
stem (iPS) cells were cultivated in differentiation medium in
which
they spontaneously and progressively formed a primordium
compris-
ing four identifiable concentric zones (Fig. 1a, Extended Data
Fig. 1a).
Cell morphology in each zone was distinctive, creating a visible
delin-
eation between zones (Extended Data Fig. 1b). The innermost
central
area (zone 1) formed first and this was followed by the
emergence
of three more radially distant concentric cell populations; zones
3. 2–4.
(Fig. 1b, Supplementary Video). In our experiments 7.7 ± 1.8%
of human iPS cells formed colonies and 67.9 ± 4.9% of these
resulted in the generation of a SEAM (n = 5 technical
replicates).
Immunolabelling for the neural cell marker class III β-tubulin
(TUBB3) was positive in zones 1 and 2, but not, more
peripherally,
in zones 3 or 4 (Fig. 1c). Cells in zones 1–3 expressed the
ocular cell
marker PAX6, while those in zones 3 and 4 were positive for
the
epithelial/surface ectodermal markers p63 and E-cadherin (Fig.
1d, e, f ).
Thus, in a number of respects SEAM formation in two-
dimensions
mirrors whole eye development from the front of the ocular
surface
posteriorly to the retina (Fig. 1g).
Cells in zone 1 expressed neural cell-specific markers TUBB3,
SOX2
and SOX6, but no surface ectodermal markers (Fig. 2a).
Accordingly,
this central area was deemed to represent presumptive
neuroectoderm.
Zone 2 cells primarily expressed the optic vesicle marker RAX
and
the neural crest marker SOX10 (Fig. 2a). In eye development
various
cell types assume a tissue-specific arrangement at the
juxtaposition of
the embryonic anterior and posterior eye segments (Extended
Data
Fig. 1c), and perhaps this is reflected here. We also note that
4. zone 2
frequently contained retinal pigment epithelium (RPE)-like
cells, and
that SOX10+/p75+ neural crest cells were found to have
emerged in
satellite spheres in the presumptive zone 2 after two weeks in
culture
(Fig. 2b, Extended Data Fig. 1d). Differentiation of SEAMs by a
pro-
tocol which encourages retinal differentiation further
demonstrated
that CHX10+ neuro-retinal cells and MITF+ RPE cells were
present
in zone 2 towards its inner and outer margins, respectively (Fig.
2c, d).
Collectively, the findings imply that zone 2 of the ocular SEAM
is
a developmental analogue of neural eye tissues comprising the
neuro-retina, neural crest and RPE.
At the margin of zones 2 and 3 α-crystallin+ lens cell clusters
emerged after four weeks in culture and spread further through
the
SEAM by week six (Fig. 2e, Extended Data Fig. 1e). Cells in
zone 3
did not display any neural features and this region was unique
with its
PAX6/p63-double-positive phenotype, representative of ocular
surface
ectoderm. Zone 3 cells also specifically expressed ocular
surface ecto-
derm markers such as PAX6, deltaN (DN)-p63, K18, and E-
cadherin,
but no neural cell markers (Fig. 2a). Thus, cells in zone 3 are
consid-
ered to be anlages of the ocular surface epithelium. Cells in
5. zone 4
expressed epithelial genes DN-p63 and E-cadherin, and did not
express
PAX6. This points to their identity as general surface
ectodermal cells,
which will probably differentiate into epidermal keratinocytes.
The
interactions of the different cell lineages complicit in whole eye
for-
mation in situ are thus mimicked in the research described here
as
illustrated schematically in Figs 1g and 2f.
As mentioned, cells with retina-like characteristics have been
generated from human iPS cells1–5, but functional ocular
surface tissue
has not. Thus, we sought to form a transplantable corneal
epithelium;
(i) as a conceptual example of the translational potential of the
SEAM
and (ii) to demonstrate that functional anterior eye tissue can
indeed
be fashioned from human iPS cells (Fig. 3a). At two and four
weeks in
culture cells in zone 3 co-expressed PAX6 and p63, first
partially then
fully. After isolation by the manual pipetting of cells in other
zones
(Extended Data Fig. 2a, b), zone 3 cells were also found to
express K14
and cornea-specific keratin K1210 by 8–12 weeks (Fig. 3b).
This was
confirmed by qRT–PCR analysis (Extended Data Fig. 2c).
Vimentin
positive (VIM+) stroma-like cells were also present in the p63+
zone 3
7. LETTER RESEARCH
surface ectodermal commitment is caused by the inhibition of
early
developmental events induced by endogenous BMP/TGFβ.
As alluded to above, cells in SEAM zone 3 most closely
resemble
those of the presumptive ocular surface, and these were
investigated
for their translational, regenerative potential. After removal of
zones
1 and 2 from the SEAM by manual pipetting, the cells that
remained
(that is, those in zone 3 and some in zone 4) were subjected to
FACS
to isolate a specific ocular surface lineage—corneal or
conjunctival—
and the results of this are shown in Extended Data Fig. 4a.
SSEA-4 is a
common marker of pluripotent stem cells and is specifically
expressed
in corneal epithelial cells in vivo13, including stem/progenitor
cells
(Extended Data Fig. 4b). Here, the use of SSEA-4 and the basal
epithe-
lial marker ITGB4 revealed that 14.1% of cells were SSEA-
4+/ITGB4+
(P3 cells), whereas 16.6% were SSEA-4−/ITGB4+ (P2 cells)
(Fig. 3c).
Thus, the population contains both corneal and non-corneal
epithelial
cells, with the indication that most colony-forming cells are
derived
from P2 and P3 fractions (Fig. 3d). Unlike P2, however, a
8. significant
proportion of the P3 colonies expressed PAX6 (Extended Data
Fig. 4c).
P3 cells also had higher expression levels of corneal epithelial-
specific markers PAX6, K12, CLU, and ALDH3A114, but lower
levels
of the epidermal marker K10 and the mucosal epithelial marker
K13
(Extended Data Fig. 4d).
P3 cell-derived colonies mostly consisted of PAX6+/K12+
corneal
epithelial colonies along with a lower number of
PAX6+/K12low limbal
epithelial colonies, which are assumed to represent corneal
epithelial
stem/progenitor cells (Fig. 3e, Extended Data Fig. 5a). The
limbus, at
the edge of the cornea, is where corneal epithelial
stem/progenitor
cells are widely believed to reside. Stratified epithelia derived
from
0d
a
1st
1st
1st 1st 2nd
2nd
2nd
13. a, A typical SEAM of differentiated human iPS cells after 40
days of
culture. Representative of 19 independent experiments. Scale
bar, 200 µm.
b, Time-lapse microscopy of the differentiating human iPS cells
during the
first 25 days (d) of culture. Representative of six independent
experiments.
Scale bar, 200 µm. See also Supplementary Video 1. c–f,
Immunostaining
of TUBB3 (green) and p63 (red) (c, zones 1–3), PAX6 (green)
and p63
(red) (d, zones 1–3), E-cadherin (Ecad, green) and p63 (red) (e,
zones
1–3), and PAX6 (green) and p63 (red) (f, zones 2–4) in the
SEAM after
six to seven weeks of culture. Images are representative of three
or four
independent experiments. Nuclei, blue. Scale bar, 100 µm. g,
The SEAM
of human iPS cells induced different kinds of cells of
ectodermal lineage,
mimicking anterior and posterior eye development in vivo.
CNS, central
nervous system; NE, neuroectoderm; OC, optic cup; NR,
neuroretina; NC,
neural crest; LE; lens; OSE, ocular surface ectoderm; SE,
surface ectoderm;
CE, corneal epithelium; EK, epidermal keratinocyte.
a
0
2
4
6
22. CHX10
TUBB3
CRYAA
p63/Ecad
1st
2nd
3rd 4th
MITF
Merged
1st
p63
2nd 3rd
1st
SOX10/p75
p63+ Lens
Figure 2 | Characterization of cell zones in the SEAM. a, Gene
expression analysis for ectoderm-related markers in each zone
of the
SEAM (zones 1 and 2, n = 12 colonies; zones 3 and 4, n = 7
colonies
obtained from two independent experiments, respectively).
Error bars
are s.d. b, Phase-contrast image and SOX10 (red) expression
around
24. genes (Extended Data Fig. 5b), which are not expressed in cells
of the
ocular surface15,16. In the colonies of P2 cells, those
expressing HOXB4
and PAX6 existed exclusively of each other (Extended Data Fig.
5c).
Immunostaining of P2 cells further demonstrated that they
consisted
of PAX6+/K13+ conjunctival epithelial cells17 accompanied by
a major-
ity of PAX6−/K13+ non-ocular epithelial cells (Fig. 3e, f,
Extended
Data Fig. 5a). Long-term differentiation revealed that PAS-
stained,
MUC5AC+, K7+ conjunctival goblet-like cells appeared in
presump-
tive P2 cell regions (Extended Data Fig. 5d). Collectively, our
data
indicates that cells in zone 3 of the SEAM have characteristics
of cor-
neal, limbal, and conjunctival epithelial cells (Supplementary
Table 1),
raising the possibility that functional ocular surface epithelia
could be
developed for surgical use.
To investigate this prospect we generated ex vivo expanded
sheets
of corneal epithelium from FACS-sorted cells acquired from
zone 3
of the SEAM (Fig. 4a). The sheets expressed the corneal limbal
stem-
cell markers K15 and K19, along with the mucins MUC1, MUC4
and MUC16, tight junction protein ZO-1, the differentiation
marker
CX43, K3 and K12, all of which are characteristic for the
25. cornea
(Fig. 4b, Extended Data Fig. 6a). The non-corneal keratins K10
and
K18 were not expressed (Fig. 4b). Approximately 99% of the
cells in
the expanded sheets were stratified K14+ epithelial cells, 95%
were
of corneal epithelial lineage (SSEA-4+), and 70% were
differentiated
corneal epithelial cells (K12+) (Fig. 4c). Cells typically had a
smooth
apical surface with microvilli-like structures (Extended Data
Fig. 6b).
Analyses based on significantly changed genes revealed that
cells
in the expanded corneal epithelial sheet differed from human
oral
mucosal keratinocytes, dermal fibroblasts and, indeed, human
iPS
4w
Seeded on LN511E8-coated dish
DM
0
iPS cells 4w 4w
StemFit CDM
8w 10w
Pipetting
32. 1
P1 P2 P3 P4
P3 cells P2 cells
CECs
(in vitro)
Corneal epithelium Conjunctival epithelium
(PAX6+ region)
Non-ocular epithelium
(PAX6– region)
6
f
PAX6 PAX6 PAX6 PAX6
D
o
u
b
le
s
ta
in
in
g
36. 20
10
0
p
6
3
p p p p(PAX6+/K12+)
2: Conjunctival epithelium
(PAX6+/K13+/K12–)
3: Other epithelium
Figure 3 | Induction and isolation of ocular surface epithelial
cells
from the SEAM. a, Schematic representation of the strategy
used for the
generation of the ocular surface ectoderm and epithelium. CDM,
corneal
differentiation medium; CEM, corneal epithelium maintenance
medium;
CMM, corneal epithelium maturation medium; DM,
differentiation
medium; w, week(s). b, Immunostaining for ocular surface and
corneal
epithelial development-related markers, PAX6 (green), p63
(red), K14
(green), and K12 (red) during ocular surface epithelial
differentiation
culture (1–12 weeks, representative of three independent
experiments).
Nuclei, blue. Scale bar, 100 µm. c, A flow cytometric analysis
37. of SSEA-4,
ITGB4 and TRA-1-60 in the differentiated human iPS cells after
10–15 weeks of culture revealed no undifferentiated human iPS
cells
(SSEA-4+/TRA-1-60+ cells) (left panels). TRA-1-60− cells
were analysed
by SSEA-4 and ITGB4 (right panel). The four populations are
defined as
P1–P4. The data shown here are representative images of 23
independent
cell-sorting experiments. d, Colony-forming assay for the sorted
P1–P4
cell fractions. Fixed colonies were stained with rhodamine
(right upper
panel, representative of seven independent cell-sorting
experiments) and
the colony-forming efficiency were calculated (graph: seven
independent
cell-sorting experiments). Error bars are s.d. Scale bar, 5 mm. e,
The
ratios of corneal epithelial (PAX6+/K12+ or PAX6+/K12low),
conjunctival
epithelial (PAX6+/K13+/K12−) and other epithelial cell types in
the P2
and P3 colonies from five independent cell sorting experiments.
Error
bars are s.d. f, Triple-colour immunostaining for PAX6 (green),
K12
(orange), K13(magenta), and p63 (green) expression in
stratified P2
and P3 cells and cultivated human corneal limbal epithelial cells
(CECs)
(representative of three independent experiments, respectively).
Nuclei,
blue. Scale bar, 50 µm.
39. Data
Fig. 7).
To investigate the translational potential of this approach,
ex vivo expanded corneal epithelial cell sheets were cultivated
as
recoverable and translatable constructs, in which approximately
1.0% of cells maintained a colony-forming capability (Extended
Data
Fig. 8a, b). When the sheets were transplanted onto the eyes of
rabbits in an experimental model of corneal epithelial stem-cell
deficiency (Extended Data Fig. 8c–j), they successfully
recovered
a healthy corneal barrier function (Fig. 4g, Extended Data Fig.
8k)
and continued to express cornea-specific proteins (Fig. 4h).
This
advance—that is, the generation of functional SEAM-derived
ocular
surface tissue with stem/progenitor cells, which can surgically
repair
the front of the eye—is noteworthy because, although somatic
stem
cells have been used to recover the ocular surface, the long-term
clinical results have not been overly encouraging18–20. In
resolving
key purification steps for corneal epithelial stem/progenitor
cells
by applying a combination of specific antibodies to our human
iPS
cell-derived ocular SEAM, we are now in the position to initiate
first-
in-human clinical trials of anterior eye transplantation to restore
visual function.
48. o
u
n
t
95.0%
Figure 4 | Characterization and surgical use of human iPS
cell-derived corneal epithelial cells (iCECs). a, Phase-contrast
microscopy and haematoxylin and eosin (H&E) staining of the
human
iCECs on cell culture inserts (representative of four
independent
experiments, respectively). Scale bars, 100 µm (phase), 50 µm
(H&E).
b, Immunostaining for corneal epithelial functional proteins and
epithelial markers (green) in the stratified SEAM-derived
human iCEC
sheets (representative of three independent experiments).
Nuclei, red.
Scale bar, 50 µm. c, Results of flow cytometric analyses for
K14, K12 and
SSEA-4 expression in the stratified human iCECs
(representative of three
independent experiments). d, Results of a principle component
analysis
based on the global gene expression (examined by microarrays)
comparing
human iPS cells (n = 3 technical replicates), ocular surface
ectoderm
(OSE; that is, human iPS cell-derived cells after six weeks of
differentiation,
n = 3 technical replicates), human oral keratinocytes (OKs, n =
3 technical
49. replicates), human iCECs (n = 4 independent experiments),
human
dermal fibroblasts (DFs, n = 3 technical replicates) and human
corneal
epithelial cells from the limbus (CECs, n = 3 independent
experiments).
A total of 25,262 significantly changed genes (fold change >2.0,
fasle
discovery rate <0.05) were analysed. Compared to human DFs,
human
iPS cells, human OKs or the OSE, the gene expression in human
iCECs
was most similar to that of human CECs. e, Proliferation
profiles of the
human iCECs during serial passages (n = 3 independent
experiments,
average population doubling, 35.1). f, Result of a holoclone
analysis for
human iCEC colonies. Representative images of holoclone-,
meroclone-
and paraclone-derived colonies and their frequencies are shown
(n = 63
single colonies from four independent cell-sorting experiments).
Scale
bar, 10 mm. g, Barrier function assay using fluorescein staining
for
transplanted and sham-operated control corneas on days 0, 7 and
14
post-surgery (left panels). A statistical analysis of the barrier
function
interpreted as the size of the fluorescein-negative area in the
human
iCEC-sheet-transplanted and control corneas (graph). *P < 0.05;
n = 7
biological replicates, Steel’s test (Bonferroni corrected). Error
51. photoreceptors from human iPSCs. Nature Commun. 5, 4047
(2014).
4. Reichman, S. et al. From confluent human iPS cells to self-
forming neural
retina and retinal pigmented epithelium. Proc. Natl Acad. Sci.
USA 111,
8518–8523 (2014).
5. Mellough, C. B. et al. IGF-1 signaling plays an important
role in the formation of
three-dimensional laminated neural retina and other ocular
structures from
human embryonic stem cells. Stem Cells 33, 2416–2430 (2015).
6. Hayashi, R. et al. Generation of corneal epithelial cells from
induced pluripotent
stem cells derived from human dermal fibroblast and corneal
limbal
epithelium. PLoS ONE 7, e45435 (2012).
7. Shalom-Feuerstein, R. et al. Pluripotent stem cell model
reveals essential roles
for miR-450b-5p and miR-184 in embryonic corneal lineage
specification.
Stem Cells 30, 898–909 (2012).
8. Ahmad, S. et al. Differentiation of human embryonic stem
cells into corneal
epithelial-like cells by in vitro replication of the corneal
epithelial stem cell
niche. Stem Cells 25, 1145–1155 (2007).
9. Brzeszczynska, J. et al. Differentiation and molecular
profiling of human
embryonic stem cell-derived corneal epithelial cells. Int. J. Mol.
52. Med. 33,
1597–1606 (2014).
10. Lavker, R. M., Tseng, S. C. & Sun, T.-T. Corneal epithelial
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11. Liem, K. F. Jr, Tremml, G., Roelink, H. & Jessell, T. M.
Dorsal differentiation of
neural plate cells induced by BMP-mediated signals from
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Supplementary Information is available in the online version of
the paper.
Acknowledgements We thank K. Baba, Y. Oie, H. Takayanagi,
S. Hara,
Y. Yasukawa, J. Toga and M. Yagi of Osaka University and M.
Nakagawa of Kyoto
University for technical assistance and scientific discussions.
53. This work was
supported in part by the project for the realization of
regenerative medicine of
The Japan Agency for Medical Research and Development
(AMED), The Japan
Science and Technology Agency (JST) and The Ministry of
Health, Labour,
and Welfare of Japan and the Grants-in-Aid for Scientific
Research from The
Ministry of Education, Culture, Sports, Science and Technology
of Japan.
Author Contributions R.H., M.T. and K.N. designed the
research; R.H, Y.I.,
R.K. and S.A. performed the in vitro experiments and acquired
the data;
Y.S., N.N., T.I. and T.S. performed animal experiments and
acquired the data;
K.S. provided reagents (LN511E8); R.H., Y.I. and R.K.
analysed the data and
wrote the respective methods and results; S.K., K.S. and A.J.Q.
supervised the
project; and R.H., M.T., A.J.Q. and K.N. wrote the paper.
Author Information Reprints and permissions information is
available at
www.nature.com/reprints. The authors declare no competing
financial
interests. Readers are welcome to comment on the online
version of the paper.
Correspondence and requests for materials should be addressed
to
K.N. ([email protected]).
14. Estey, T., Piatigorsky, J., Lassen, N. & Vasiliou, V.
ALDH3A1: a corneal crystallin
55. http://www.nature.com/doifinder/10.1038/nature17000
http://www.nature.com/doifinder/10.1038/nature17000
http://www.nature.com/reprints
http://www.nature.com/doifinder/10.1038/nature17000
mailto:[email protected]
LETTER RESEARCH
METHODS
No statistical methods were used to predetermine sample size.
The experiments
were not randomized and investigators were not blinded to
allocation during
experiments and outcome assessment.
Human iPS cell culture. The human iPS cell lines 201B7,
253G1, and 454E2 were
obtained from the RIKEN Bio Resource Center (Tsukuba,
Japan)21,22. The 1231A3
and 1383D2 human iPS cells were provided by the Center for
iPS Cell Research
and Application, Kyoto University23. All cells were cultured in
StemFit medium
(Ajinomoto, Tokyo, Japan) on LN511E8-coated (0.5 µg cm−2)
dishes23,24. LN511E8,
produced using cGMP-banked CHO-S cells (Life Technologies,
Carlsbad, CA), was
obtained from Nippi (Tokyo, Japan). In part, LN511E8 was
produced using human
293-F cells as previously described12. The 201B7 and 454E2
human iPS cell lines
were used in the in vitro experiments, while 201B7 and 1383D2
cells were used in
the animal experiments; 253G1 and 1231A3 cells were used in
the supplementary
56. experiments, the results of which are reported in Extended Data
Fig. 7. All of the
experiments using recombinant DNA were approved by the
Recombinant DNA
Committees of Osaka University and were performed according
to our institu-
tional guidelines.
Ocular cell differentiation from human iPS cells. The
differentiation culture for
human iPS cells was performed as indicated in Fig. 3a. First,
human iPS cells were
seeded on LN511E8-coated dishes at 350–700 cells cm−2, after
which they were cul-
tivated in StemFit medium for 8–12 days. The culture medium
was then changed
to DM (differentiation medium; GMEM (Life Technologies)
supplemented with
10% knockout serum replacement (KSR; Life Technologies), 1
mM sodium pyru-
vate (Life Technologies), 0.1 mM non-essential amino acids
(Life Technologies),
2 mM l-glutamine (Life Technologies), 1% penicillin-
streptomycin solution
(Life Technologies) and 55 µM 2-mercaptoethanol (Life
Technologies) or mon-
othioglycerol (Wako, Osaka, Japan))25. In some experiments, as
indicated in the
Results section, Noggin (R&D systems, Minneapolis, MN),
LDN-193189 (Wako)
or SB-431542 (Wako) were added for the first four days. BMP4
(R&D systems)
was used in some early experiments at concentrations up to
0.125 nM. This had no
discernible effect on SEAM formation, however, so its use was
discontinued. After
four weeks of culture in DM, the medium was changed to
57. corneal differentiation
medium (CDM; DM and Cnt-20 or Cnt-PR (w/o; EGF and
FGF2) (1:1, CELLnTEC
Advanced Cell Systems, Bern, Switzerland) containing 5 ng
ml−1 FGF2 (Wako),
20 ng ml−1 KGF (Wako) 10 µM Y-27632 (Wako) and 1%
penicillin-streptomycin
solution). FGF2 in CDM was not essential for corneal epithelial
induction. During
CDM culture (around six to eight weeks of differentiation),
non-epithelial cells
were removed by manual pipetting under microscopy (Extended
Data Fig. 2a, b).
After pipetting, the medium was changed to fresh CDM. After
four weeks of culture
in CDM, the medium was changed to corneal epithelium
maintenance medium
(CEM; DMEM/F12 (2:1), Life Technologies) containing 2%
B27 supplement (Life
Technologies), 1% penicillin-streptomycin solution, 20 ng ml−1
KGF and 10 µM
Y-27632 for two to seven weeks. To achieve retinal
differentiation (Fig. 2c) after
four weeks of differentiation the medium was directly changed
to CEM. Isolated
RPE cell colonies were cultivated in CEM on separate dishes
coated with LN511E8.
Phase-contrast microscopic observations were performed with
an Axio-observer.
Z1, D1 (Carl Zeiss, Jena, Germany) and an EVOS FL Auto (Life
Technologies).
Flow cytometry and cell sorting. Differentiated human iPS cells
in CEM were
dissociated using Accutase (Life Technologies), and
resuspended in ice-cold KCM
medium (DMEM without glutamine and Nutrient Mixture F-12
58. Ham (3:1, Life
Technologies) supplemented with 5% FBS (Japan Bio Serum,
Hiroshima, Japan),
0.4 µg ml−1 hydrocortisone succinate (Wako), 2 nM 3,3′,5-
Triiodo-l-thyronine
sodium salt (MP biomedicals, Santa Ana, CA), 1 nM cholera
toxin (List Biological
Laboratory, Campbell, CA), 2.25 µg ml−1 bovine transferrin
HOLO form (Life
Technologies), 2 mM l-glutamine, 0.5% insulin transferrin
selenium solution
(Life Technologies) and 1% penicillin-streptomycin solution).
The harvested
cells were filtered with a cell strainer (40 µm, BD Biosciences,
San Diego, CA) and
then stained with anti-SSEA-4 (MC813-70, Biolegend, San
Diego, CA), TRA-1-60
(TRA-1-60-R, Biolegend) and CD104 (ITGB4; 58XB4,
Biolegend) antibodies for
1 h on ice. After being washed twice with PBS, stained cells
underwent cell sorting
with a FACSAria II instrument (BD Biosciences). For
intracellular protein staining,
a BD Cytofix/Cytoperm (BD Biosciences) kit was used. In all of
the experiments,
cells were stained with non-specific isotype IgG or IgM as
controls (Biolegend).
The data were analysed using the BD FACSDiva Software (BD
Biosciences) and
the FlowJo software program (TreeStar, San Carlos, CA).
Fabrication and harvest of human iPS cell-derived corneal
epithelial cell
(human iCEC) Sheets. Sorted human iPS cell-derived epithelial
cells obtained
from zone 3 of the SEAM (human iCECs) were seeded on
LN511E8 coated
59. (0.5 µg cm−2) cell culture inserts or temperature-responsive
dishes (UpCell,
CellSeed, Tokyo, Japan) without cell passaging, and were
cultured in CEM until
confluence26. To promote maturation, the epithelial cells were
cultivated in CMM
(corneal epithelium maturation medium; KCM medium
containing 20 ng ml−1
KGF and 10 µM Y-27632) for an additional 3–14 days after
CEM culture. The
human iCECs cultivated on temperature-responsive dishes were
released from
their substrate by reducing the temperature to 20 °C.
Quantitative real-time reverse-transcriptase PCR (qRT–PCR).
Total RNA was
obtained from differentiated human iPS cells after specific
culture periods, from
human epidermal keratinocytes (EKs (foreskin), Life
Technologies and TaKaRa
Bio, Otsu, Japan), and from human corneal limbal epithelial
cells (CECs) using the
RNeasy total RNA kit or the QIAzol reagent (Qiagen, Valencia,
CA). Reverse tran-
scription was performed using the SuperScript III First-Strand
Synthesis System
for qRT–PCR (Life Technologies) according to the
manufacturer’s protocol, and
cDNA was used as a template for PCR. qRT–PCR was
performed using the ABI
Prism 7500 Fast Sequence Detection System (Life
Technologies) in accordance
with the manufacturer’s instructions. The TaqMan MGB used in
the present study
are shown in Supplementary Table 2. The thermocycling
program was performed
60. with an initial cycle at 95 °C for 20 s, followed by 45 cycles at
95 °C for 3 s and 60 °C
for 30 s.
Immunofluorescence staining. Research grade human skin tissue
sections were
obtained from US Biomax Inc. (MD, USA) and human oral
mucosal tissue was
obtained from Science Care (Phoenix, AZ). The cells were fixed
in 4% paraform-
aldehyde (PFA) or cold methanol, washed with Tris-buffered
saline (TBS, TaKaRa
Bio) three times for 10 min and incubated with TBS containing
5% donkey serum
and 0.3% Triton X-100 for 1 h to block non-specific reactions.
They were then
incubated with the antibodies shown in Supplementary Table 3
at 4 °C overnight
or at room temperature for 3 h. The cells were again washed
twice with TBS for
10 min, and were incubated with a 1:200 dilution of Alexa Fluor
488-, 568-, 647-
conjugated secondary antibodies (Life Technologies) for 1 h at
room temperature.
Counterstaining was performed with Hoechst 33342 (Molecular
Probes) before
fluorescence microscopy (Axio Observer.D1, Carl Zeiss).
Haematoxylin and eosin staining. Fabricated human iCEC sheets
were fixed with
10% formaldehyde neutral buffer solution (Nacalai Tesque,
Kyoto, Japan). After
washing with distilled water, the human iCEC sheets were
embedded in paraffin
from which 3-µm-thick sections were cut. These were stained
with haematoxylin
and eosin following deparaffinization and hydration. The
sections were observed
61. with a NanoZoomer-XR C12000 (Hamamatsu Photonics,
Hamamatsu, Japan),
BZ-9000 (KEYENCE, Osaka, Japan) and an Axio Observer.D1.
PAS staining. Differentiated human iPS cells (more than 12
weeks of differen-
tiation) were fixed with 10% formaldehyde neutral buffer
solution, after which
PAS staining was performed with a PAS staining kit (MERCK
KGaA, Darmstadt
Germany) according to the manufacturer’s protocol. The
sections were observed
with an Axio Observer.D1.
Colony-formation assay (CFA). Epithelial cells were seeded
onto MMC-treated
NIH-3T3 feeder layers at a density of 3,000–20,000 cells per
well. These were cul-
tivated in CMM for 7–14 days. The colonies were fixed with
10% formaldehyde
neutral buffer solution and then stained with rhodamine B
(Wako). Colony for-
mation was then assessed using a dissecting microscope and the
colony-forming
efficiency (CFE) was calculated. For the holoclone analysis, a
single human iCEC
colony derived from the SEAM was cultivated on 3T3-J2
(provided by H. Green,
Harvard Medical School, Boston, MA) in CMM for 7–11 days
was picked up under
a dissecting microscope and dissociated by TrypLE Select (Life
Technologies). The
dissociated human iCECs were again seeded on a MMC-treated
3T3-J2 feeder layer
and cultivated in CMM for 10–13 days. The colonies were
scored under a micro-
scope and classified as holoclones, paraclones or meloclones
based on previously
62. reported methods27.
Microarray analysis. Human CECs were harvested from
corneoscleral rims
(Northwest Lions Eye Bank, Seattle, WA) as reported
previously28. Human CECs
and human oral keratinocytes (OKs; ScienCell, Carlsbad, CA)
along with SEAM-
derived human iCECs were cultivated on LN511E8 coated cell
culture inserts in
CEM until confluent. They were then cultivated in CMM.
Human dermal fibro-
blasts (DFs; ScienCell) were cultivated in DMEM/F12 (2:1)
containing 10% FBS.
Total RNA was obtained from human iPS cells, iCECs, CECs,
OKs, DFs, and six-
week differentiated iPS cells (that is, OSE) using the QIAzol
reagent. A microarray
analysis using Sure Print G3 human 8x60K slides (Agilent
technologies, Palo Alto,
CA) was performed at Takara Bio. The data were analysed using
the GeneSpring
GX software program (Agilent technologies). Microarray data
used in this study
are deposited in Gene Expression Omnibus under accession
number GSE73971.
Scanning electron microscopy (SEM). The cultivated epithelial
cell sheets were
fixed in 2.5% glutaraldehyde (Nacalai Tesque) at 4 °C
overnight. Subsequently,
the sheets were washed in buffer, dehydrated with ethanol and
tert-butyl alcohol
(Wako), and critical point dried (JFD-320, JEOL, Tokyo,
Japan). After sputter-coating
with platinum in an auto fine coater (JFCL-1600, JEOL), the
samples were
observed by scanning electron microscopy (JSM-6510LA,
64. Harvested human
iCEC sheets were grafted onto rabbit corneas, in which a total
epithelial limbal
stem-cell deficiency had been created following a corneal and
limbal lamellar
keratectomy (Extended Data Fig. 8c–j). After surgery, 0.3%
ofloxacin ointment
(Santen Pharmaceutical, Osaka, Japan), 0.1% betamethasone
phosphate eye drops
(Shionogi Pharmaceutical, Osaka, Japan) and 0.1% sodium
hyaluronate eye drops
(Santen Pharmaceutical) were applied three to four times per
day. Triamcinolone
acetonide (8 mg; Bristol Myers Squibb, Tokyo, Japan) was also
administered by sub-
conjunctival injection. Tacrolimus (0.05 mg kg−1 per day,
Astellas Pharma, Tokyo,
Japan) and Mizoribine (4.0 mg kg−1 per day Sawai
Pharmaceutical, Osaka, Japan)
were systemically administered using an osmotic pump
(DURECT, Cupertino,
CA). The corneal barrier function following surgery was
assessed by 0.5% fluo-
rescein eye drop instillation at day 7 and day 14 after surgery
and the fluorescein
negative area was calculated using the AxioVision software
program (Carl Zeiss).
Throughout the healing period, the cornea was observed with a
digital slit-lamp
camera (SL-7F, TOPCON, Tokyo, Japan) and 3D OCT1000
MARK II (TOPCON)
or CASIA SS-1000 (TOMEY, Nagoya, Japan) machines. If an
infection was found
or if unexpected weight loss occurred, animals were excluded
from the analysis.
65. The rabbits were euthanized by the intravenous administration
of sodium pento-
barbitone 14 days after transplantation, after which the eyes
were immediately enu-
cleated for the histological analyses. No blinding or
randomization was conducted
to allocate animals to each group.
Statistical analyses. The data are expressed as means ± standard
deviation (s.d.). The
statistical analyses were performed using the Mann–Whitney
rank sum test or Steel’s
test. Bonferroni’s correction was applied to the data in animal
experiments. All of the
statistical analyses were performed using the JMP software
program (SAS institute
Inc., Cary, NC). No statistical methods were used to
predetermine sample size.
Comprehensive technical details can be found in Protocols
Exchange, http://
dx.doi.org/10.1038/protex.2016.009.
21. Nakagawa, M. et al. Generation of induced pluripotent stem
cells without
Myc from mouse and human fibroblasts. Nature Biotechnol. 26,
101–106
(2008).
22. Takahashi, K. et al. Induction of pluripotent stem cells from
adult human
fibroblasts by defined factors. Cell 131, 861–872 (2007).
23. Nakagawa, M. et al. A novel efficient feeder-free culture
system for the
derivation of human induced pluripotent stem cells. Sci. Rep. 4,
3594 (2014).
67. LETTER RESEARCH
Extended Data Figure 1 | Differentiation of multiple ocular cells
in the
SEAM. a, A differentiated human iPS cell colony after 40 days
of culture
(macro photograph, representative of six independent
experiments). Scale
bar, 5 mm. b, Magnified views of each SEAM zone margin after
six weeks
in culture (phase-contrast and bright-field views, each
representative of
three independent experiments). Arrow heads indicate borders
between
each zone. Scale bars, 50 µm. c, Schematic for the development
of the
anterior eye. CE, corneal epithelium; CEnd, corneal
endothelium; IS, iris
stroma; NR, neuroretina; RPE, retinal pigment epithelium; LE,
lens.
d, Immunostaining for p75 (green) and SOX10 (red) in SEAM
zones 1
and 2, two weeks (w) after the start of the differentiation
culture
(representative of three independent experiments). Asterisks
indicate
SOX10+/p75+ neural crest cells. Nuclei, blue. Scale bar, 100
µm.
e, Immunostaining of lens differentiation marker α-crystallin
(green) and
epithelial marker p63 (red) in zones 2 and 3 of the SEAM after
six weeks
of culture (representative of three independent experiments).
Nuclei, blue.
Scale bar, 100 µm.
73. stratified
SEAM-derived human iCECs (representative of n = 3
independent
experiments). Magnified view of the dotted area is shown in the
lower
panel. Nuclei, red. Scale bars, 50 µm. b, Scanning electron
microscopy of
the apical surface of the stratified human iCECs (representative
of two
human iCEC sheets) and human CECs (n = 1). Scale bars, 10
µm (upper
panels) and 1 µm (lower panels). c, Results of a hierarchical
cluster analysis
based on the global gene expression as examined by
microarrays. Data are
shown for human iPS cells (n = 3 technical replicates), human
iPS cell-
derived ocular surface ectoderm (OSE; that is, human iPS cell-
derived
cells after six weeks of differentiation, n = 3 technical
replicates), human
iCECs (n = 4 independent experiments), human oral
keratinocytes (OKs,
n = 3 technical replicates), human dermal fibroblasts (DFs, n =
3 technical
replicates) and human corneal epithelial cells obtained from the
limbus
(CECs, n = 3 independent experiments). A total of 25,262
significantly
changed genes (fold change >2.0, false discovery rate <0.05)
were
analysed. d, SEAM-derived human iCECs at passage (P) 1, 3, 9
and 15
during serial passages (representative of three independent
experiments).
77. surgical use of human iPS cell-derived corneal epithelial cells
(iCECs).Extended Data Figure 1 Differentiation of multiple
ocular cells in the SEAM.Extended Data Figure 2 Enrichment
and differentiation of a SEAM-derived ocular surface
epithelium.Extended Data Figure 3 The effect of BMP/TGFβ
inhibitors on the development of ocular surface epithelium in
the SEAM.Extended Data Figure 4 Isolation of corneal
epithelial cells from the SEAM.Extended Data Figure 5
Characterization of SEAM-derived ocular surface epithelial
cells.Extended Data Figure 6 Characterization of the SEAM-
derived corneal epithelium.Extended Data Figure 7 Induction of
corneal epithelial cells from different human iPS cell
clones.Extended Data Figure 8 Transplantation of the human
iCEC sheet to repair the ocular surface.
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