Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

Cuenca N1, Fernández-Sánchez L, McGill TJ, Lu B, Wang S, Lund R, Huhn S, Capela A.
Source: Invest Ophthalmol Vis Sci
Publication Date: (2013)
Issue: 54(10): 6745-56
Research Area:
Stem Cells
Basic Research
Cells used in publication:
Neural stem cell (NSC), human
Species: human
Tissue Origin: brain
Culture Media:

For generation of HuCNS-SC the authors use X-VIVO15 as follows: Briefly, donated second trimester (16–20 gestation weeks) human brain tissue was dissected, treated enzymatically, and labeled with CD133 and CD24 antibodies. The CD133þCD24/lo target population was sorted aseptically. Sorted cells were cultured as a neurosphere suspension in a chemically defined, serum-free culture medium composed of X-VIVO (Lonza, Walkersville, MD) 15 medium supplemented with N2, heparin, N-acetyl cysteine (NAC), basic fibroblast growth factor (FGF2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) at a density of 10exp5 cells/ml. When neurosphere size reached 200 to 250 lm, cultures were passaged by collagenase treatment and replated in the same medium


PURPOSE: Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. METHODS: The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). RESULTS: The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. CONCLUSIONS: This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.