Human Retinal Progenitor Cells Grown as Neurospheres Demonstrate Time‐Dependent Changes in Neuronal and Glial Cell Fate Potential

Abstract

The spatiotemporal birth order of the seven major classes of retinal cells is highly conserved among vertebrates. During retinal development, long projection neurons (ganglion cells) are produced first from resident progenitors, followed by the appearance of retinal interneurons, photoreceptors, and Muller glia. This sequence is maintained through the complex orchestration of cell-intrinsic and cell-extrinsic events and factors, including local influences between neighboring cells. Here we asked whether cultures of human prenatal retinal cells might also yield different ratios of cell types based on gestational age and time spent in vitro, thus recapitulating in vivo development. An established chopping technique was used to passage human prenatal retinal cells as neurospheres, avoiding the use of proteases and preserving cell-cell contacts and native microenvironments present in vivo. Retinal neurospheres cultured in this manner demonstrated specific patterns of growth over a limited time period, possibly reflecting trends in normal retinal development. Upon differentiation, immunocytochemical analysis revealed that retinal neurospheres produce predominantly glial cells with increasing gestational age and time in culture. Conversely, the percentage of betaIII tubulin-positive neurons declined over time. This provides information for optimizing culture systems aimed at the study of human retinal development and the generation of specific retinal cell types for therapeutic use or drug testing.

Structure and cell composition of spheres cultured from human fetal retina

“…However, the cell capacity to division decreased with fetal age. We found numerous dividing cells in all studied spheres from 15-and 18-week retinas, which contradicts the data on drastically decreasing proliferation during culturing of cells from 10-13-week human retina [9]. Virtually no proliferating cells were seen in spheres derived from 22-24-week retina, except solitary spheres with large numbers of dividing cells, indicating the differences in the cell composition of spheres.…”

“…Experimental data on the production of cloned neurospheres from rodent and human fetal retina are contradictory. The development of human neurospheres ceased during the second passage [9,23]. However, it seems possible to obtain them in experiments on mice [11,15].…”

Human Retinal Development in an in situ Whole Eye Culture System

“…In vitro experiments on the developing human retina have usually been in the form of cell suspensions focusing on the development of individual cells. These reports highlight the importance of cell-to-cell contact for proper development of retinal cell subtypes [18,19,20]. A few short-term culture experiments on isolated full-thickness retinas derived from second-trimester fetuses have been reported [21,22].…”

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

“…The provided chopping protocol allows for the production of large-scale banks from one fetal sample greater than passage 10, an unlikely feat using standard passaging methods. While this method for passaging hNPCs is unconventional, it is growing in popularity and was recently, published with other cell types such as neural stem cells derived from human embryonic and induced pluripotent stem cells, enabling large scale expansion for various applications including in vitro disease modeling [41][42][43][44][45][46] 1. The two most important factors to address before chopping is sphere diameter and media conditioning or color.…”