Induction of retinal pigment epithelium properties in ciliary margin progenitor cells.
OBJECTIVE
Degenerative processes in the retinal pigment epithelium (RPE) are known to play a pivotal role in the development of age-related maculopathy. Substitute RPE analogue cells could be used to preserve visual function. In this paper we investigate methods for the isolation, cultivation and RPE differentiation of undifferentiated cells from the ciliary marginal zone (CMZ) of rat eyes.


METHODS
The CMZ was isolated from enucleated rat eyes, cell spheres formed in serum-free suspension culture, Bromodeoxyuridine (BrdU) incorporation indicated mitotic activity. Following baseline differentiation status assessment, directional differentiation was induced by cultivating cells in RPE-conditioned medium and vasoactive intestinal peptide (VIP). The differentiation status was analysed by immunocytochemistry. Fluorescein isothiocyanate (FITC)-labelled latex beads were used for functional evaluation.


RESULTS
CMZ-derived cells were expanded for 6-12 months. Formation of spherical cellular conglomerates, subsphere formation and expression of nestin indicated progenitor cells. Baseline levels of markers MAP-2 for neuronal and GFAP for glial properties and baseline levels of bestrophin, cytokeratins 8 and 18 and RPE 65 for RPE properties were induced by serum culture, respectively. Culture in conditioned medium with addition of VIP significantly increased RPE marker expression and reduced neuronal features, uptake of latex beads indicated phagocytosis.


CONCLUSIONS
We succeeded in isolating and cultivating cells from rodent CMZ with progenitor cell characteristics. Subsequently, these cells tested positive for neuronal, glial and RPE markers. Appropriate conditions significantly increased RPE marker expression. Unidirectional induction of differentiation makes the CMZ eligible as a source of regenerative ocular tissue for RPE-reconditioning therapy.
Vossmerbaeumer
Urs
U
Department of Ophthalmology, University Eye Hospital, Universitaets-Augenklinik, German Red Cross Blood Service of Baden-Wuerttemberg-Hessen, University of Heidelberg, Mannheim, Germany. urs.vossmerbaeumer@augen.ma.uni-heidelberg.de
Kuehl
Sandra
S
Kern
Susanne
S
Kluter
Harald
H
Jonas
Jost B
JB
Bieback
Karen
K
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation; D002924:Ciliary Body / Q000166:cytology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010587:Phagocytosis; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018874:Spheroids, Cellular; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D015415:Biomarkers; D017077:Culture Media, Conditioned; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D014660:Vasoactive Intestinal Peptide