UC Irvine scientists have created retina from human embryonic stem cells (hESCs).
By Live Dr - Mon Jun 07, 2:46 pm
Gabriel Nistor1, a, Magdalene J. Seiler1, a, Fengrong Yana, David Fergusona and Hans S. Keirstead, a,
To develop three-dimensional (3D) constructs of retinal pigment epithelium (RPE) and early retina progenitor cells from human embryonic stem cells (hESCs).
3D tissue constructs were developed by culturing hESC-derived neural retinal progenitors in a matrix on top of hESC-derived RPE cells in a cell culture insert. An osmolarity gradient maintained the nutrition of the 3D cell constructs. Cross-sections through hESC-derived tissue constructs were characterized by immunohistochemistry for various transcription factors and cell markers.
hESC-derived tissue constructs expressed transcription factors characteristic of retinal development, such as pax6, Otx2, Chx10, retinal RAX; Brn3b (necessary for differentiation of retinal ganglion cells); and crx and nrl (role in photoreceptor development). Many cells expressed neuronal markers including nestin, beta-tubulin and microtubule-associated proteins.
This study shows for the first time that 3D early retinal progenitor tissue constructs can be derived from hESCs.
Keywords: Human embryonic stem cells; Retinal pigmented epithelium; Photoreceptor; Retinal progenitor differentiation; Three-dimensional culture
Abbreviations: hESC, human embryonic stem cells; RPE, retinal pigment epithelium; MAP, microtubule-associated protein; NF, neurofilament; Dkk, Dickkopf 1
- 1. Introduction
- 2. Materials and methods
- 2.1. Cell lines
- 2.2. Derivation of neural progenitor cells and RPE
- 2.3. Preparation of culture inserts
- 2.4. Media formulation
- 2.5. Co-culture—assembling the system
- 2.6. Histology
- 2.7. Immunocytochemistry
- 3.1. Extracellular matrix
- 3.2. Cell growth
- 3.3. Influence of pore size
- 3.4. Influence of the insert/container size
- 3.5. Immunohistochemistry for transcription factors and cell markers
- 4.1. Comparison to other approaches of retina-specific differentiation of hESCs
- 4.2. A unique procedure to develop layers
- 4.3. Importance of RPE co-culture
- 4.4. Expression of markers specific for eye development
Fig. 1. Experimental setup. (A) Setup of insert culture. (B) Separation of layers from membrane insert. (C) Time schedule for differentiation. *Collagen Type 1 matrix: Cell Gel (California Stem Cell). **Inserts: Corning Transwell PET or PTFE, 3 μm pore (the polycarbonate or Anapore inserts do not work).
Fig. 2. Phase-contrast images of cultures in vitro. (A) Undifferentiated hESC culture on Matrigel in phase contrast. (B and C) Island of a developing RPE layer in a mixed culture at an early stage of differentiation (30 days). (D) Stain of RPE layer cross-sections for the RPE-specific transcription factor mitf (red) and for cellular retinaldehyde binding protein (CRALBP). Nuclei are counterstained with DAPI (day 76 of differentiation). (E) RPE cells on collagen/laminin before co-culture (day 47 since induction with Dkk and LeftyA). Insert: same magnification as picture in (F). (F) Neural progenitor cells before co-culture (day 47 since induction of differentiation). (G) Co-culture of neural progenitors on top of RPE layer, 30 days after plating neural progenitors. Magnification bars = 200 μm for (A, B, F); 50 μm for (C), 100 μm for (E), 10 μm for (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. Expression of transcription factors and neuronal/glial markers within layers before co-culture. Nuclei are counterstained with DAPI (blue). (A) Strong CRALBP expression in the cytoplasm of many cells. (B) Strong staining for MAP2 (red), clear staining for transcription factor retinal RAX (green) in many large neurons. (C) Many cells express the transcription factor OTX2. (D) Many cells faintly stained and one cell strongly stained for photoreceptor-progenitor transcription factor NRL (green), almost no staining for glial marker glutamine synthetase (red). (E) Chx10/Vsx10 staining is distributed throughout layers (sample with bleach DAPI counterstain). (F) Strong expression of transcription factor NeuroD in some areas. (G) NeuN (red)/pax6 (green) staining shows partial overlap. (H) Beta-tubulin/CRX: Beta-tubulin is distributed throughout the sheet whereas CRX appears in some areas. Magnification bars = 20 μm (A–D, F–H); 10 μm (E). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 4. Expression of transcription factors and neuronal/glial markers within layers during co-culture. Nuclei are counterstained with DAPI (blue). (A) Scattered cells stain for transcription factor Brn3b (green), 1 week after co-culture. (B) Cytoplasmic staining for transcription factor NeuroD (green), 2 weeks after co-culture. (C) Many cells are strongly stained for photoreceptor-progenitor transcription factor NRL (green), almost no staining for glial marker glutamine synthetase (red), 1 week after co-culture. (D) Scattered staining for transcription factor retinal RAX (green) and neuronal marker MAP2 (red), 1 week after co-culture. (E) NeuN (red)/pax6 (green) staining shows partial overlap, 1 week after co-culture. (F and G) Double staining for OTX2 (green) and recoverin (red). Note that there is almost no recoverin staining except for few cells in (G). (F) 1 week after co-culture. (G) 2 weeks after co-culture. (H) MAP2 (green)/Vimentin. Strong Vimentin stain throughout; MAP2 concentrated in one area, 1 week after co-culture. Magnification bars = 20 μm (A, B, D–F); 10 μm (C, G, H). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Corresponding author at: Reeve-Irvine Research Center, Sue and Bill Gross Stem Cell Research Center, 2111 Gillespie Neuroscience Research Facility, School of Medicine, University of California at Irvine, Irvine, CA 92697-4265, United States. Tel.: +1 949 824 6213 begin_of_the_skype_highlighting +1 949 824 6213 end_of_the_skype_highlighting; fax: +1 949 824 5352.
UCI researchers create retina from human embryonic stem cells
Complex tissue structure – a first – offers hope to millions with degenerative eye disorders
UC Irvine scientists have created an eight-layer, early stage retina from human embryonic stem cells, the first three-dimensional tissue structure to be made from stem cells.
It also marks the first step toward the development of transplant-ready retinas to treat eye disorders such as retinitis pigmentosa and macular degeneration that affect millions.
“We made a complex structure consisting of many cell types,” said study leader Hans Keirstead of the Reeve-Irvine Research Center and the Sue & Bill Gross Stem Cell Research Center at UCI. “This is a major advance in our quest to treat retinal disease.”
In previous studies on spinal cord injury, the Keirstead group originated a method by which human embryonic stem cells could be directed to become specific cell types, a process called differentiation. Results of those studies are leading to the world’s first clinical trial using a stem cell-based therapy for acute spinal cord injury.
In this study, the Keirstead team utilized the differentiation technique to create the multiple cell types necessary for the retina. The greatest challenge, Keirstead said, was in the engineering. To mimic early stage retinal development, the researchers needed to build microscopic gradients for solutions in which to bathe the stem cells to initiate specific differentiation paths.
“Creating this complex tissue is a first for the stem cell field,” Keirstead said. “Dr. Gabriel Nistor in our group addressed a really interesting scientific problem with an engineering solution, showing that gradients of solutions can create complex stem cell-based tissues.”
The retina is the inside back layer of the eye that records the images a person sees and sends them via the optic nerve from the eye to the brain. Retinal diseases are particularly damaging to sight. More than 10 million Americans suffer from macular degeneration, the leading cause of blindness in people over 55. About 100,000 have retinitis pigmentosa, a progressive, genetic disorder that usually manifests in childhood.
“What’s so exciting with our discovery,” Keirstead said, “is that creating transplantable retinas from stem cells could help millions of people, and we are well on the way.”
The UCI researchers are testing the early-stage retinas in animal models to learn how much they improve vision. Positive results would lead to human clinical trials.
The study appears online in the Journal of Neuroscience Methods. Nistor, Magdalene J. Seiler, Fengrong Yan and David Ferguson contributed to the effort, supported by The Lincy Foundation and private donations to the Keirstead group.
About the University of California, Irvine: Founded in 1965, UCI is a top-ranked university dedicated to research, scholarship and community service. Led by Chancellor Michael Drake since 2005, UCI is among the most dynamic campuses in the University of California system, with nearly 28,000 undergraduate and graduate students, 1,100 faculty and 9,000 staff. Orange County’s largest employer, UCI contributes an annual economic impact of $3.9 billion. For more UCI news, visit www.today.uci.edu.