Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium.
For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.
Bytyqi
Afrim H
AH
Technische Universität Darmstadt, Entwicklungsbiologie & Neurogenetik, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany.
Bachmann
Gesine
G
Rieke
Matthias
M
Paraoanu
Laura E
LE
Layer
Paul G
PG
D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D000963:Antimetabolites / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002449:Cell Aggregation / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002802:Cholinesterases / Q000378:metabolism; D017077:Culture Media, Conditioned; D003627:Data Interpretation, Statistical; D005849:Gerbillinae; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D012160:Retina / Q000166:cytology* / Q000254:growth & development*; D012165:Retinal Ganglion Cells / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000502:physiology
D000963:Antimetabolites; D017077:Culture Media, Conditioned; C034477:RNA primers; D012313:RNA; D002802:Cholinesterases; D001973:Bromodeoxyuridine