Attachment to Cytodex Beads Enhances Differentiation of Human Retinal Progenitors in 3-D Bioreactor Culture
Retinal degenerations are the leading cause of genetically inherited blindness. One of the strategies currently being tested for the treatment is cell/tissue transplantation. As such stem cells and tissue engineered constructs are of great importance. This report describes the growth of multipotential human retinal progenitors (cell line) in a 3-D bioreactor culture vessel with (adhesive substrate) laminin coated collagen 1/cytodex beads and without adhesive substrate (beadless culture). The study demonstrates that progenitors are capable of growth and differentiation in the bioreactor with or without beads. The presence of adhesive substrate accelerates and enhances photoreceptor differentiation in the bioreactor, reflected by significantly higher level expressions of several photoreceptor specific proteins; N acetyl transferase (AaNat), rhodopsin and cone transducin GNB3. Both monomeric and dimeric forms of rhodopsin are expressed in cells attached to beads, whereas, only the monomeric form is expressed in beadless culture. Similarly, a different isomeric form of tyrosine hydroxylase (a doublet) is expressed in cell bead attached cultures. Co-culturing retinal progenitors with retinal pigment epithelium (RPE) in cell bead cultures further stabilizes the photoreceptor phenotype and rhodopsin expression. Most of the retinal neuronal phenotypes are confirmed by an expression of specific proteins. The adhesive substrate in the form of collagen 1, laminin coated cytodex beads, could be just an effector for stabilization or a positive signal, modulating extracellular matrix (ECM) molecules and/or neurotrophins. In the future, the bioreactor culture system could be utilized to grow retina-like structures from ciliary epithelium by incorporating biodegradable substrates.
Keywords: Human retinal progenitors, bioreactor, cytodex beads, differentiation, photoreceptors, electronic stimulation, transplantation of chips, pharmacological rescue, gene therapy
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