Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

Abstract

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of- in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy. Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking ), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

Thrombospondin-1 Promotes Circuit-Specific Synapse Formation via β1-Integrin

“…To mechanistically examine the role of TSPs in retinal synapse formation, we used purified rat retinal ganglion cells (RGCs) in culture, which have been extensively utilized to discover the molecular underpinnings of astrocyte-induced synapse formation (Barres et al, 1988;Christopherson et al, 2005;Ullian et al, 2001). RGCs, which are purified from P7 rat retinas, form very few synapses when cultured alone in a serum-free growth media of known composition, but addition of astrocyte or retinal glial feeder layers or astroglia-conditioned media robustly increases the number of excitatory synapses (Christopherson et al, 2005;Koh et al, 2018). The synapses formed on the RGCs are identified by immunohistochemical methods by quantifying the colocalization of pre-(Bassoon, green) and post-(Homer, red) synaptic markers.…”

Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration

“…3-5 maximum intensity projections were averaged to obtain the number of colocalized puncta per field (dots on graphs). The border between OFF and ON sublaminae was defined based on histologic landmarks (Koh et al, 2018). Published protocols were followed to count synapses based on the colocalization of presynaptic and postsynaptic markers (Ippolito and Eroglu, 2010;Koh et al, 2018).…”

“…The border between OFF and ON sublaminae was defined based on histologic landmarks (Koh et al, 2018). Published protocols were followed to count synapses based on the colocalization of presynaptic and postsynaptic markers (Ippolito and Eroglu, 2010;Koh et al, 2018). In brief, the projected images were divided into individual channels (presynaptic and postsynaptic markers); background was subtracted for each channel (rolling ball radius = 50); thresholds were adjusted manually to detect individual puncta while minimizing background; and puncta smaller than 4 pixels were filtered out.…”

Umbilical Cord Blood and Serum for the Treatment of Ocular Diseases: A Comprehensive Review

“…Subretinal transplantation of cells derived from umbilical tissue was shown to rescue visual function by preserving retinal synaptic connectivity and attenuating glial reactivity. Multiple injections provided enhanced effects, thus confirming the potential therapeutic application of these cells in the setting of human retinal degeneration [19]. Recently, ischemic retinopathies such as diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion have been treated using vasoregenerative cell therapy [20][21][22][23][24].…”

“…could be related to the secretion of trophic factors such as BDNF and glial cell-derived neurotrophic factor[19,24,25].Table 1summarizes the main published studies on UCB use in ophthalmic practice.…”