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作者：Jason S. Meyera, Martin L. Katzb, Joel A. Maruniaka, Mark D. Kirka 5 z, d" a% ]" Y/ X
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【摘要】% `+ \# ^, t) ?
Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type¨Cspecific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons. ! ?$ a3 n% y3 m, C
【关键词】 Embryonic stem cell Retina Transplantation mnd mouse Differentiation Repair Neuronal ceroid lipofuscinoses
# P, {: ^7 `; r, g" N6 z INTRODUCTION
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Mammalian embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst, are pluripotent, and give rise to all specialized cell types of the body . In vitro, ES cells can be expanded indefinitely and can be engineered to secrete therapeutic factors. Consequently, ES cells are important potential tools for treatment of disease or injury.7 l4 o8 ]. F3 Y8 V
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Cultured ES cells can be induced to differentiate into unique cell types, including cells of ectodermal, mesodermal, and endodermal lineages .
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( S r a) ^5 ?0 t. _The retina is an excellent model for studying stem cell transplantation into the central nervous system (CNS). The retina arises from the same embryonic origin as the brain, but the retina is more easily accessible than other parts of the CNS. Additionally, the organization of retinal neurons is well understood, allowing detailed determination of how transplanted stem cells interact with host cells., X" H& F' W1 p: r+ w) B) g2 e
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When neural stem cells (NSCs) are transplanted into the vitreous of the eye, they can incorporate into the retina under very specific conditions .' i! T% B4 P) Z3 l8 G9 `
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The neuronal ceroid lipofuscinoses (NCLs) are the most common autosomal recessively inherited, neurodegenerative disorders of childhood, affecting the retina and entire CNS .
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In the present study, mouse ES cells were induced to a neural lineage and transplanted into the vitreous of mnd mouse eyes at an early stage of retinal degeneration. We examined whether the transplanted cells survive long-term and incorporate into the host retina and determined whether the transplanted cells differentiate within the retina. In addition, the ability of transplanted stem cells to reduce lysosomal storage body content and enhance survival of host photoreceptors was evaluated.
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8 L' Y" a$ Q( l) i7 sMATERIALS AND METHODS4 ^$ T5 T5 ^4 T8 v1 x
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Cell Culture$ a, x0 }7 @* T5 H7 j# r8 J
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Mouse ES cells (enhanced green fluorescent protein .9 k3 j- j" N. C( \
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The mnd mice (strain B6.KB2-Cln8mnd/MsrJ, C57 mouse strain background) were purchased from Jackson Laboratories (Bar Harbor, ME, http://www.jax.org) and maintained as a breeding colony at the University of Missouri¨CColumbia. Five-week-old mnd mice were used for all transplants. Animals were anesthetized with i.p. injections of 80 mg/kg ketamine, 8 mg/kg xylazine, and 1.6 mg/kg acepromazine. Neuralized ES cells were concentrated to 30,000 cells/µl, and 1.5 µl were transplanted into the vitreous of each eye behind the lens with a 10-µl Hamilton syringe and a 31-gauge needle. All animal experiments were approved by the University of Missouri-Columbia Animal Care and Use Committee and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.1 y" ? d/ ?$ O1 O: [1 K, c9 w
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Sample Preparation for Morphological Analysis
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The mice were euthanized via carbon dioxide inhalation at either 6 or 16 weeks after transplantation. The eyes were immediately enucleated and placed in a fixative consisting of 4% paraformaldehyde, 50 mM sodium cacodylate, and 8% sucrose (pH 7.4). For samples to be examined in cross-section, the corneas, irises, and lenses were removed immediately. The posterior portions of the eyes were incubated in the fixative at room temperature for 60 minutes with gentle agitation and then washed in 0.17 M sodium cacodylate buffer and equilibrated in 20% sucrose and embedded in optimal cutting temperature medium (Ted Pella Inc., Redding, CA, http://www.tedpella.com). Cryostat sections were cut at a thickness of 8 µm, and to avoid counting errors, every sixth section was sampled for cell counts. Sections were taken within 250 µm of the optic nerve.7 g( u- Q( I: N
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Whole mounts of retinas were prepared to examine the distribution of donor cells after transplantation. For these preparations, the eyes were enucleated and incubated in the 4% paraformaldehyde fixative for 10 minutes. The corneas, irises, and lenses were then removed and the eyes were incubated in 0.17 M sodium cacodylate, pH 7.4, for at least 12 hours. The neural retinas were then separated from the eyecups and incubated in the fixative for an additional 2 hours. After the second fixation, the retinas were washed again in the cacodylate buffer. A series of radial cuts were then made in the retinas to enable the tissue to lie fairly flat . The samples were then mounted on microscope slides in the cacodylate buffer. After data collection from whole mounts, the retinas were subsequently prepared for cryostat sectioning. The whole mounts were bisected in a randomly oriented plane with respect to the dorsal/ventral axis into approximately equal halves, and sections were obtained from one of the cut edges that passed through the center of the retina.
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; r+ V& e$ V6 Y! Y2 h" Z: SImmunocytochemistry% W; [; N& D/ K' H6 Y! [- y
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Fluorescence microscopy was used to determine the extent of EGFP-expressing donor cell incorporation into host retinas and to te) T7 Y+ [/ Q. j: `, `6 s
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发表于 2009-3-5 00:11
发表于 2015-6-13 18:08
