Dynamic transcriptomic remodeling in grafted human neural progenitor cells uncovers mechanisms for vision preservation in a rat model of retinitis pigmentosa

Domain: chemistry
Published: 2026-03-06
Editorial outcome: major_revision

Abstract
Human neural progenitor cells (hNPCs) show promise in slowing retinal degeneration and are currently being tested in clinical trials for retinitis pigmentosa (RP). However, the long-term fate of grafted hNPCs and their interaction with host retinal cells remain unclear. Here, we used single-cell transcriptomics to investigate temporal gene expression changes in grafted hNPCs and host retinal cells following subretinal injection into a rodent model of RP, revealing dynamic transcriptomic changes in the degenerative retinal environment. Grafted hNPCs primarily differentiate into an astroglial phenotype and mature over time, contributing to photoreceptor protection through trophic factor secretion, metabolic modulation, suppression of apoptosis, oxidative stress, and inflammation, alongside extracellular matrix remodeling. CellChat analysis revealed a decline in intercellular signaling and communication strength over time, correlating with weakened hNPC-host interactions. These findings suggest that enhancing trophic factor support, particularly MANF, and improving the host retinal environment are critical for sustaining long-term vision preservation.

Introduction
Retinitis pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive vision loss due to mutations in photoreceptors and retinal pigment epithelium (RPE). More than 80 genes with over 3000 mutations have been linked to RP1. Gene therapy targeting specific mutations, such as RPE65, has been approved for treating RP patients2,3. Given the high genetic diversity of RP, pursuing a mutation-independent therapeutic strategy to slow disease progression and preserve vision is a logical and promising approach.

Stem cell-based therapies offer promising avenues for treating retinal degenerative diseases through two main strategies: cell replacement and preservation of host cells. Replacement therapies, such as RPE cell transplants for age-related macular degeneration (AMD), have shown mixed outcomes in animal models and clinical trials4. Similarly, photoreceptor transplants at the advanced stages of retinal degeneration have shown promising results in animal studies; however, establishing synaptic connections with host retinal cells remains challenging5,6. Therapies that target preservation of host retinal cells have employed various stem cells in both preclinical and clinical settings, including retinal progenitor cells (RPCs)7, mesenchymal stem cells (MSCs)8–10, and human neural progenitor cells (hNPCs)11–13. hNPCs have been tested in multiple preclinical models, including rd1 and USH2A mutant mice14,15, RGC-depleted glaucoma mice16, and spinal cord injury models17, where they consistently demonstrated neuroprotective and anti-inflammatory effects. Our preclinical studies demonstrate that hNPCs delivered to the subretinal space of animal models survive long-term, migrate extensively, and protect vision by providing trophic support, regulating immune response, and enhancing antioxidant defense mechanisms by upregulating Nrf212,18,19. These findings have led to the delivery of clinical-grade hNPCs (termed CNS10-NPC) to RP patients in an ongoing phase I/2a clinical trial (NCT04284293).

While stem cell-based therapies hold promise for retinal disease, long-term protection is compromised due to efficacy deterioration over time, as widely reported from both preclinical and clinical studies. Key questions remain about the behavior of grafted stem cells and the host retina in a progressively degenerative environment, including how grafted cells adapt to their environment, how preserved photoreceptors evolve over time, and how grafted stem cells interact with host retinal cells. Here, we investigated the changes in hNPCs and host retinal cells over time following subretinal injection into the Royal College of Surgeons (RCS) rat, a well-characterized model for retinal degeneration. Using single-cell RNA sequencing (scRNA-seq), along with histological and functional assessments, we analyzed transcriptomic remodeling and intercellular interactions between grafted hNPCs and host retinal cells. Fourteen hNPC subpopulations with distinct transcriptional states were identified, indicating dynamic changes in grafted cells over time. Top-regulated genes, including mesencephalic astrocyte-derived neurotrophic factor (MANF), myeloid-derived growth factor (MYDGF), midkine (MDK), and pleiotrophin (PTN), were identified in hNPCs. The host retina showed temporal changes in phototransduction, visual perception, glial responses, and metabolic processes. Grafted hNPCs protect vision through multiple modalities, including trophic factor release, extracellular matrix (ECM) remodeling, metabolic regulation, and suppression of apoptosis, oxidative stress, and inflammation. Notably, the signaling interaction and strength between hNPCs and host retinal cells declined over time. These findings provide insight into the dynamic changes in grafted hNPCs and host retinal cells and highlight the importance of combining stem cell therapy with improving host retinal environment to sustain long-term vision preservation.

Results
Grafted hNPCs mature into astrocytic cells in the degenerative retina
To study the molecular insights of therapeutic effect and gradual changes of the grafted hNPCs over time in the degenerative retinal environment, hNPCs were delivered to the subretinal space of RCS rats at postnatal day (P) 21, followed by visual function evaluation at P60 and P90 (Supplementary Fig. 1a) using our previously published protocols19,20. After overnight dark adaptation, animals were prepared under dim red light. Scotopic ERGs were recorded using 5 ms white-light flashes at 25 cd·s/m² with 10-second interstimulus intervals to assess retinal function. Under these conditions, a-waves were not detectable at P60 and P90, due to rod bleaching and light adaptation. Our previous study demonstrated that rod function in RCS rats declines early21,22 however, rod-driven a-waves were detectable at P100 following hNPC transplantation when ERGs were recorded using rod-optimized conditions with longer dark-recovery intervals11. Despite this, ERG b-wave amplitudes remained significantly higher for hNPC-treated eyes compared to untreated controls at P60 and P90, indicating preservation of cone and inner retinal activity, consistent with our previous findings19,22. Notably, while visual acuity for hNPC-treated eyes remained significantly higher than untreated controls and stable over time, ERG b-wave amplitudes decreased from P60 to P90, reflecting progressive decline of retinal function (Supplementary Fig. 1b-c). Histological evaluation of retinal sections also revealed a reduction in the outer nuclear layer (ONL) of hNPC-treated retinas at P90 (6–8 layers) compared to P60 (8–10 layers) (Supplementary Fig. 1d-g). Immunofluorescence staining for recoverin, which labels both rods and cones, revealed a preserved ONL in the graft-associated areas, while untreated retinas showed only a single layer of photoreceptors (Supplementary Fig. 2a-b). Cone-arrestin staining further demonstrated well-organized cones with inner and outer segments (IS and OS), and pedicles in regions associated with the hNPC grafts. In contrast, age-matched untreated retinas displayed a degenerative cone morphology with diminished cone-arrestin signals (Supplementary Fig. 2c-d). hNPC treatment also maintained the structural integrity of photoreceptor IS and OS by preserving the level of interphotoreceptor matrix proteoglycans 2 (IMPG2), as evidenced by intense immunofluorescence staining in the hNPC-treated retina at P90, compared to the age-matched untreated control, where no positive staining was detected due to loss of both rod and cone segments (Supplementary Fig. 2e-f). IMPG2 is a key component of the interphotoreceptor matrix, which provides essential structural support to photoreceptors and facilitates nutrients and metabolic waste exchange between photoreceptors and RPE23. Moreover, immunofluorescence staining for RPE65 and collagen IV showed a well-organized layer of RPE cells in the hNPC-protected regions, whereas the untreated control showed a disorganized RPE layer with diminished RPE65 and collagen IV staining (Supplementary Fig. 2g-j). These findings suggest that hNPC treatment contributes to photoreceptor preservation, at least in part, by supporting RPE cell integrity. However, despite these protective effects, progressive photoreceptor loss was still evident over time in hNPC-treated retinas, indicating that while hNPCs slow disease progression, they do not completely halt the degenerative process. It is noted that sham surgery was not included in the current study, since the focus of this study was to investigate the interaction between grafted hNPCs and host retinal cells. Sham surgery would not address this goal, as it would not provide relevant mechanistic insight into donor cell behavior, survival, integration, or paracrine effects. However, sham-related transient effects have been demonstrated across multiple animal models24–26, including in our recent dose-ranging study, which shows that vision preservation is dose-dependent, with transient effects observed in vehicle-treated controls and limited efficacy at low cell doses12. In addition, removal of subretinal debris at the peak of accumulation in RCS rats has been shown to provide long-term visual protection27, underscoring the importance of distinguishing non-specific surgical effects from cell-type specific therapeutic mechanisms.

To understand the molecular underpinnings of these changes over time at single-cell resolution, the grafted hNPCs and host retinal cells were collected at P60 and P90 and processed for scRNA-seq. High-quality cells (in vitro: 5502; in vivo P60: 4943; in vivo P90: 5536, Supplementary Fig. 3a-d) were obtained by in silico sorting from the merged scRNA-seq datasets of all time points (Supplementary Fig. 3e-g). Next, we identified the distinct cell-type clusters using known human and retinal cell-specific marker genes (Supplementary Fig. 3h). Six clusters were present for hNPCs, photoreceptors (PR), Müller glia, retinal ganglion cells (RGCs), bipolar cells, and microglia (Fig. 1a). Subsequently, we subset 8,088 high-quality hNPCs (in vitro hNPCs: 5502; in vivo P60 hNPCs: 1575; in vivo P90 hNPCs: 1011) and performed unsupervised clustering on the batch-corrected, integrated transcriptomic dataset, resulting in 14 distinct subpopulations (Fig. 1b), which were profiled and annotated based on the expression of well-established marker genes (Fig. 1c, Supplementary Fig. 3i-j).

The 14 transcriptomically distinct hNPC subpopulations were classified as SOX9+/NES+/HOPX/SLC1A3+ astrocyte progenitor cells (APC; Clusters 0, n=1316; 7, n=531, and 10, n=224), CD44+/AQP4+/GJA1+/FLT+ astrocyte precursors (APs; Clusters 4, n=711; 6 n=533; and 9, n=337), GFAP+/S100B+/FABP7+ immature astrocytes (IASC; Cluster 11, n=185), GFAP+/AQP4+/GJA1+ mature astrocytes (ASC; Cluster 1 n=1042), PDGFRA+/OLIG1+/OLIG2+/SLC1A3+/S100B+ oligodendrocyte astrocyte progenitor cells (OAPCs; Cluster 2, n=1042), PDGFRA+/OLIG1+/OLIG2+ oligodendrocyte precursor cells (OPC; Clusters 3, n=755; and 5, n=708), small subpopulation of SOX10+/MBP+/MYRF+ oligodendrocytes (OL; Cluster 13, n=114), DCX+/SOX4+/SOX11+ immature neurons (IN1; Cluster 8, n=505) and DDX17+/FTX+ immature neurons (IN2; Cluster: 12; n=166) (Fig. 1c, Supplementary Fig. 3k-l).

To explore the impact of the host degenerative retinal environment on the grafted hNPCs over time, the cluster-specific gene expression profile of hNPCs both before grafting and at P60 and P90 after transplantation was compared (Fig. 1d–h, Supplementary Fig. 4a-f). We found that clusters 0, 2, 5, 7, and 10 of grafted hNPCs expressed progenitor marker genes (NES, SOX2) over time (Fig. 1d). We also observed that grafted hNPC clusters associated with astrocyte lineage (clusters: 0, 1, 7, 10, and 11) expressed mature astrocyte marker genes (AQP4, GJA1) compared to in vitro hNPCs with astrocyte lineage, suggesting a prevalence of mature astrocyte states of grafted hNPCs in vivo (Fig. 1g). Interestingly, we also noticed that cluster 2 of grafted hNPCs is enriched for both astrocyte- (S100B, GFAP) and oligodendrocyte-specific (PDGFRA, OLIG1, OLIG2) genes (Supplementary Fig. 4c). Further, we found that clusters 3 and 5 of grafted hNPCs are enriched for oligodendrocyte precursor markers (PDGERA, OLIG1, OLIG2) compared to clusters 3 and 5 of hNPCs in vitro, suggesting the acquisition of an early oligodendrocyte fate of grafted hNPCs in the host degenerative retinal environment (Fig. 1e, Supplementary Fig. 4b). Like previous studies28,29, both clusters 3 and 5 (oligodendrocyte precursor cell clusters: OPC 1 and 2) also showed the enrichment of genes involved in neuron differentiation and development (HES6, SOX11, DCX; Supplementary Fig. 4d-e), indicating that they may have the capability to differentiate into neurons in vivo. Oligodendrocyte fate acquisition of grafted hNPCs is further supported by the presence of a small oligodendrocyte cluster enriched with mature oligodendrocyte genes (MBP, MOB), identified as cluster 13 only in grafted hNPCs, but absent in the in vitro hNPCs (Fig. 1f). Gene ontology (GO) term enrichment analysis of upregulated genes in cluster 13 also revealed the enrichment of biological processes related to oligodendrocyte differentiation, myelination, and galactolipid biosynthesis (Supplementary Fig. 4f). Clusters 8 and 12 were enriched with early-developing neuronal genes (Cluster 8: SOX4, SOX11, DCX and Cluster 12: SOX11, DDX17) in grafted hNPCs compared to in vitro hNPCs (Fig. 1c, h, Supplementary Fig. 4b). Clusters 4, 6, and 9, which are enriched with marker genes of different astrocyte maturation stages (CD44, HEPACAM, ALDOC, SLC16A3) were greatly reduced in grafted hNPCs (Fig. 1c, Supplementary Fig. 4a-b).

We further validated these transcriptomic findings at the protein level by performing immunofluorescence staining on hNPC-treated retinal sections (Fig. 1i). Grafted hNPCs formed a distinct cell layer in the subretinal space and were associated with photoreceptor protection, consistent with our previous studies12,19. These cells showed strong immunoreactivity for neural progenitor markers, Nestin and SOX2, and robust expression of astrocytic markers GFAP and AQP4. A small subset of OLIG2+ OPC-like cells was observed, while only a limited number of cells expressed neuronal markers MAP2 and DCX. Although scRNA-seq identified a small MBP+ mature oligodendrocyte cluster, MBP protein expression was not detected in tissue sections, likely reflecting the low abundance of these cells in vivo. Overall, these findings indicate that the majority of grafted hNPCs predominantly adopt an astrocytic fate and gradually mature over time within the host retinal environment.

hNPCs secrete trophic factors for survival and retinal protection
We have previously shown that hNPCs provide retinal protection by secreting various neurotrophic factors, including insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2)11. Changes in the therapeutic profile over time may be partially due to reduced secretion of neurotrophic factors. Here, we found that in addition to FGF, hNPCs expressed mesencephalic astrocyte-derived neurotrophic factor (MANF), myeloid-derived growth factor (MYDGF), midkine (MDK), and pleiotrophin (PTN) both in vitro and in vivo as well as the expression of their receptors (Fig. 2a–c). Notably, MANF, MYDGF, MDK, and PTN, as well as their receptors, were expressed at relatively high levels across the subpopulations of hNPCs (Supplementary Fig. 5). Interestingly, there was a significantly higher expression of MDK, PTN, and their receptors (PTPRZ1, CSPG5) in P60 compared to P90 grafted hNPCs (Fig. 2a). MDK and PTN have been shown to promote differentiation, migration, and survival of neural stem and progenitor cells30–32. These trophic factors provide neuroprotection, reduce oxidative stress, and have anti-apoptotic effects30,31,33. MDK and PTN may act on hNPCs via their receptors in an autocrine manner to promote their stability, survival, and migration in the degenerative retinal environment. Additionally, secreted MDK and PTN might act paracrinely on Müller glia through their receptors and protect photoreceptors34. We also found a significant increase in MANF, MYDGF, and their receptor KDELR1 in grafted hNPCs at P90 compared to P60 (Fig. 2b). MANF has significant neuro-regenerative potential and protects photoreceptors from degeneration35–37. MYDGF promotes NPC survival by maintaining their stemness38,39. Additionally, autocrine and paracrine effects of MANF and MYDGF can provide neuroprotection by inhibiting apoptosis, oxidative stress, and inflammation, and enhancing metabolic activity36–38,40–45. Hence, increased expression of MANF, MYDGF, and their receptors in grafted hNPCs at P90 compared to P60 may suggest their autocrine protection of hNPCs, and protection of photoreceptors via KDELR through paracrine action.

We validated our findings using immunofluorescence of MANF, MYDGF, and their receptor KDELR in grafted hNPCs at P60 and P90. Double immunolabeling using human-specific nuclear marker HuNu and MANF demonstrated cytoplasmic and perinuclear puncta staining of MANF at both P60 and P90 (Fig. 2d, e, upper panel). Similarly, double immunofluorescence staining of HuNu and MYDGF demonstrated intense cytoplasmic, perinuclear, and nuclear staining of MYDGF in grafted hNPCs (Fig. 2d, e, middle panel). Next, double immunofluorescence using HuNu and KDELR demonstrated intense cytoplasmic staining of KDELR in hNPCs (Fig. 2d, e, lower panel). Altogether, these findings suggest that MDK and PTN may promote grafted hNPC survival and migration at P60, while at P90, MANF and MYDGF may sustain hNPC survival and function via inhibiting cellular oxidative stress, inflammation, and immune response as degeneration progresses.

Grafted hNPC adaptation in a degenerative retinal environment
Grafted hNPCs undergo extensive biological changes over time in response to the host degenerative retinal environment. To investigate the adaptation and functional shifts, we compared transcriptomic profiles of grafted hNPCs at P60 and P90 with in vitro hNPCs (Fig. 3a, b). At P60, grafted hNPCs displayed increased expression of genes involved in differentiation, neuron projection morphogenesis, synaptic signaling, and survival pathways, reflecting their integration within the host retina (Fig. 1i). By P90, fewer genes showed differential expression, indicating stabilization and maturation of hNPCs in the host environment (Fig. 3a–d). Common pathways were enriched across P60 and P90, including cellular processes supporting differentiation, migration, axonogenesis, endocytosis, and autophagy, as well as suppression of immune activation (e.g., TGF-β regulation). In contrast, biological processes related to neurotrophin and mTOR signaling, negative regulation of apoptosis, protein processing in ER, ErbB signaling, and metabolic pathways, including glycolysis and pyruvate metabolism, were consistently downregulated, suggesting a shift in energy utilization and metabolic activity over time (Fig. 3e).

Temporal analyses revealed distinct transcriptomic changes in P60 and P90 hNPCs. At P60, grafted hNPCs upregulated genes linked to neuron development, adhesion, and axonogenesis while downregulated genes related to metabolic pathways, reflecting early adaptation to the host retina (Supplementary Fig. 6a-h). By P90, there was increased expression of genes associated with glial differentiation and immune responses, along with further suppression of metabolic pathways, suggesting a shift toward immune and stress-responsive phenotypes as degeneration progressed (Supplementary Fig. 6i-o). These changes reflect ongoing adaptation and maturation of hNPCs within a progressively degenerative environment.

To understand the impact of the degenerative retinal environment on the transcriptomic remodeling of grafted hNPCs over time, we conducted a comparative transcriptomic analysis between P60 and P90 hNPCs (Fig. 3f). At P90, hNPCs exhibited enhanced expression of genes related to phagocytosis, energy metabolism (e.g., mitochondrial ATP synthesis coupled electron transport, oxidative phosphorylation), and cellular stress and death (e.g., response to oxidative stress, apoptosis) (Fig. 3g), reflecting increased metabolic demands and stress responses. Notably, the upregulation of lysosomal and phagosome-related genes (e.g., CTSD, LAMP1, LAMP2) suggests active clearance of photoreceptor outer segments (POS) from the subretinal space, which is critical for photoreceptor survival in the RCS rats (Fig. 3h)19. However, the downregulation of pathways such as FoxO signaling, TGF-β signaling, and neurotrophin signaling, coupled with increased oxidative stress and immune activation, indicates a probable gradual shift of hNPCs toward a stress-responsive phenotype as degeneration progresses (Fig. 3g–i, Supplementary Fig. 7a-l).

Retinal transcriptomic remodeling following hNPC transplantation
To assess the impact of hNPC treatment on the host degenerative retinal cells, we performed scRNA-seq from untreated and hNPC-treated retinas at P60 and P90, alongside wild-type (WT) controls. A total of 118,478 high-quality cells, encompassing all major retinal cell types, were analyzed (Supplementary Fig. 8a-g). These included rods (14,675), cones (4341), rod bipolar cells (RBCs: 40,882), cone bipolar cells (CBCs: 9650 cells), Müller glia (MULG: 14,614), retinal ganglion cells (RGCs: 9654), amacrine cells (ACs: 2577), as well as microglia (MGs: 15,084), macrophages (MO: 519), and endothelial cells (ECs: 6482) (Supplementary Fig. 8h-i). Retinal degeneration in RP is marked by progressive loss of rods and cones, with corresponding changes in Müller glia and microglia. Müller glia, known to support photoreceptor health through neurotrophic and metabolic functions, exhibited altered transcriptional profiles, reflecting their response to retinal stress46. Similarly, microglia showed signs of activation and infiltration, consistent with their role in degenerative processes47. Hence, we focused on rods, cones, Müller glia, and microglia for further downstream analysis.

Grafted hNPCs modulate photoreceptor dysfunction and drive rescue
To understand the biological changes in photoreceptors during retinal degeneration, we analyzed transcriptional signatures in rods and cones at P60 and P90. In untreated retinas, 1,811 and 1,685 differentially expressed genes (DEGs) were identified in rods at P60 and P90, respectively, while cones exhibited 1,634 and 2,664 DEGs (Fig. 4a–c). Gene ontology (GO) analysis revealed downregulation of photoreceptor signaling, phototransduction pathway, G protein transmits signals (Gnb1, Cnga1, Pde6b) and TOR signaling (Ddit4, Lars1) in rods at P60, alongside progressive declines in genes essential for visual perception, photoreceptor maintenance, and glucose metabolism by P90, compared to WT (Fig. 4d, f). Concurrently, untreated rods showed upregulation of genes associated with synapse organization, axonogenesis (Lrrtm4, Nrxn3, Gnb3), calcium channel activity (Calm1, Cacna2d1, Robo2), ATP synthesis, neurotransmitter secretion/transport and downregulation of genes related to glycolysis (Hk2, Ldha), indicating metabolic dysfunction and neuronal remodeling (Fig. 4e, g). In untreated retinas, cones displayed similar transcriptional trends, with reduced expression of genes related to phototransduction, visual perception, and ATP metabolism alongside increased expression of synaptic signaling and axon outgrowth genes (Fig. 4e, g). These findings suggest rods and cones experience metabolic stress and compromised function during degeneration, with compensatory neuronal reorganization to maintain connectivity.

To elucidate the photoreceptor protective mechanism after hNPC treatment, we conducted transcriptomic analysis between hNPC-treated and untreated rods and cones at P60 and P90. In rods, 688 differentially expressed genes were identified at P60 and 2130 at P90 following hNPC treatment (Fig. 4a). Of these, 457 DEGs at P60 and 1,198 at P90 were shared between untreated/WT and hNPC-treated/untreated. Importantly, among these shared DEGs, only 382 at P60 and 985 at P90 in hNPC-treated rods showed expression patterns similar to WT rods, defining them as rod-specific rescue genes (Fig. 4b, c). Further analysis of time point-specific DEGs revealed upregulation of photoreceptor maintenance, photoreceptor cilium, retinal homeostasis, and glycolysis (Hk2, Ldha) and downregulation of oxidative phosphorylation and ATP synthesis coupled to electron transport in P60- and P90 hNPC-treated rods (Fig. 4d–g). Additionally, P90 hNPC-treated rods showed upregulation of genes involved in photoreceptor inner/outer segments, visual and sensory perception (Cnga1, Rho, Pde6b), highlighting enhanced photoreceptor integrity and function (Fig. 4d, f, h, j). These data strongly demonstrated that hNPCs protect rods and slow down the progression of retinal degeneration (Fig. 4, Supplementary Figs. 1d-g, 2a-b), consistent with our previous studies12,19.

Next, we evaluated the transcriptomic changes in cones at P60 and P90 following hNPC treatment. We identified 1,912 DEGs at P60 and 1,254 DEGs at P90 in hNPC-treated cones compared to untreated (Fig. 4a). Among these, 761 at P60 and 592 at P90 were shared between untreated/WT and hNPC-treated/untreated cones. Intriguingly, only 172 DEGs in P60 and 289 in P90 hNPC-treated cones displayed expression patterns similar to WT cones, annotated as cone-specific rescue genes (Fig. 4b-c). GO analysis of time point-specific DEGs revealed that at P60, hNPC-treated cones exhibited increased expression of genes associated with anterograde trans-synaptic signaling and GTPase-mediated signal transduction (Fig. 4d, j), while at P90 hNPC-treated cones demonstrated upregulation of genes involved in synapse organization and lipid metabolism (Fig. 4f, k). P60 and P90 untreated cones showed upregulated genes involved in autophagic cell death, consistent with previous findings suggesting that dysregulated autophagy contributes to photoreceptor cell death in rodent models for retinal degeneration19,48. Moreover, P60 untreated cones also showed upregulation of genes associated with light detection and oxidoreductase activity compared to hNPC-treated cones (Fig. 4e, Supplementary Fig. 9c). Notably, untreated P90 cones retained some functionality, with higher expression of phototransduction and light response genes compared to hNPC-treated cones, suggesting residual light responsiveness despite degeneration (Fig. 4f, Supplementary Fig. 9d). These results align with recent studies indicating that cones retain partial functionality even as degeneration progresses49.

hNPC treatment also significantly increased the expression of Nxnl1, encoding the rod-derived cone viability factor (RDCVF)50–52, which reduces oxidative stress and promotes aerobic glycolysis to support cone survival53,54, underscoring the therapeutic potential of hNPCs (Fig. 4l–o, Supplementary Fig. 10). Interestingly, untreated cones at P60 also exhibited elevated Nxnl1 expression compared to hNPC-treated cones, potentially reflecting an intrinsic compensatory response to rod loss. However, downregulation of mitochondrial genes in hNPC-treated photoreceptors highlights a need to address energy deficits to enhance long-term photoreceptor survival55,56. These findings demonstrate that hNPC treatment preserves retinal integrity by promoting photoreceptor function and maintenance despite limitations in sustaining mitochondrial energy production at the late stage of degeneration.

Grafted hNPCs remodel glial cell transcriptomes
Our previous studies demonstrated suppression of Müller cell gliosis and microglial activation following hNPC transplantation in the RCS rats19. However, how hNPCs modulate the transcriptomic signatures of Müller glia and microglia remains unknown. To explore these changes, we analyzed Müller glial and microglial transcriptomes at P60 and P90, revealing distinct temporal and functional remodeling patterns (Fig. 5a–k).

By comparing untreated Müller glia with WT, we first investigated the disease-associated changes in these cells over time. We identified 1,710 differentially expressed genes at P60 and 1,685 at P90 (Fig. 5a). Untreated Müller glia displayed increased expression of genes associated with reactive gliosis (Gfap, Lcn2) at both early (P60) and late (P90) stages of the disease, compared to WT, concordant with other retinal degenerative diseases (Fig. 5d–g)57. Specifically, at P60, untreated Müller glia exhibited downregulation of early responsive genes (Egr1, Fos, Jun) and upregulation of genes involved in inflammation, defense response, and apoptosis (Ifi27, Cp, Cst3, Clu), indicating the activation of stress and immune responses at the early stage of retinal degeneration. Additionally, at P60, untreated Müller glia also showed increased expression of genes (Nlgn1, Ephb1, Nrxn3) related to neurotransmitter transport and synapse organization, suggesting their compensatory role in remodeling and stabilizing synapses to support retinal connections at the early stage of disease. However, downregulation of genes involved in visual perception, photoreceptor cell maintenance, cellular response to calcium ions, and retinal homeostasis reflects impaired support for photoreceptor function at the early stage. As the disease progresses, untreated Müller glia exhibited further upregulation of genes associated with glial cell proliferation (Vim, Gfap, Clu), innate immune response (Apoe, C1s, Lcn2), and autophagy (Gabarap, Ctsd, Sqstm1), coupled with downregulation of genes related to oxidative phosphorylation and mitochondrial ATP synthesis coupled electron transport, indicating metabolic dysfunction at the late stage of disease. Consistent with previous studies, these findings suggest a shift in the Müller glial reactive status, transitioning from an early-stage compensatory to a late-stage decompensatory state with retinal degeneration progression.

Differential gene expression analysis identified 1,801 DEGs at P60 and 1,707 DEGs at P90 in untreated microglia compared to WT (Fig. 5a). At both time points, untreated microglia exhibited upregulation of genes involved in antigen processing and presentation (B2m, Cd74) and integrin-mediated signaling (Tyrobp, Itgb1). At P90, additional upregulation of genes related to the adaptive immune response (B2m, C3), interleukin-10 production (Plcg2, Cd84), and autophagosome membrane docking (Vamp8, Calm1) was observed (Fig. 5a). However, genes associated with phototransduction, visual perception, photoreceptor cell maintenance, and retinal homeostasis were downregulated (Fig. 5d, f). Together, these findings may indicate a dynamic transition of microglia to a reactive state to mitigate cellular stress by enhancing autophagy and adaptive immune response, along with disease progression58.

Comparative analysis between hNPC-treated and untreated Müller glia identified 478 DEGs at P60 and 461 DEGs at P90 (Fig. 5a). Among these, 265 DEGs at P60 and 299 at P90 were shared across untreated/WT and hNPC-treated/untreated groups, with 159 (P60) and 261 (P90) displaying expression patterns similar to WT. These were classified as Müller glia-specific rescue genes (Fig. 5b, c). GO analysis revealed that hNPC treatment at P60 upregulated genes associated with synapse organization (Nlgn1, Nrxn3), cell adhesion (Sema5a, Neo1), axon guidance (Ephb1, Epha5), and glutaminergic synapse (Nlgn1, Gria2), highlighting a neuroprotective role in maintaining synaptic stability and retinal integrity (Fig. 5d, h). By P90, upregulated genes were linked to cytokine production (Egr1, Csf1r), cell migration (Ccl4, Jun), as well as immune, inflammatory, and defense responses (C1qa, Nfkbia), indicating adaptation to the progressively degenerative environment (Fig. 5f, i). Interestingly, at P60, hNPC-treated Müller glia exhibited downregulation of genes related to phototransduction and light response, while at P90, genes related to cellular metal ion homeostasis and calcium ion transport (Mt2A, Smdt1, Calm1) were downregulated (Fig. 5e, g). However, key reactive gliosis genes (Gfap, Vim) were consistently downregulated in hNPC-treated Müller glia at both time points, indicating suppression of reactive gliosis following hNPC treatment (Fig. 5j), consistent with our previous studies19,20.

At P60, hNPC-treated microglia showed increased expression of genes related to oxidative phosphorylation, ATP synthesis, and NAD(P)H dehydrogenase (quinone) activity (Mt-nd3, Mt-atp8) (Fig. 5d), reflecting enhanced homeostatic functions such as surveillance and phagocytosis, which rely on oxidative metabolism59. By contrast, in inflammatory microglia, metabolic remodeling suppresses their oxidative phosphorylation60. However, by P90, hNPC-treated microglia showed upregulation of disease-associated microglial (DAM) genes61 related to inflammatory and immune responses (Icam1, Clec7a, Apoe, Il1b, Spp1) (Fig. 5f, k) along with genes involved in the homeostatic process (Igf1, Nfe2l), lipid metabolism (Apoe, Abca1, Trem2), and negative regulation of TNF production (Cd274, Tnfaip3), suggesting an adaptive response to stabilize retinal homeostasis, modulate inflammation, and mitigate damage. Conversely, hNPC-treated microglia at P60 showed downregulation of genes involved in peptide metabolism (Rpl30, Rps12), while at P90, genes related to ribosome biogenesis (Rplp1, Rps2), protein folding (Hsp90aa1, Hsph1), and complement cascades (C1qa, C3) were downregulated. These changes reflect metabolic and functional remodeling in microglia in response to a progressively degenerating retinal environment.

In sum, our findings suggest that hNPC treatment provides neuroprotection over time via dynamic transcriptomic remodeling of glial cells within the progressively degenerating retinal environment.

Temporal transcriptomic shifts reveal hNPC-mediated retinal protection
To explore how hNPC-treated retinal cells change their transcriptomes and assess their functional stability in a progressively degenerating retinal environment over time, we compared the transcriptomic profiles of P60 with P90 hNPC-treated rods, cones, Müller glia, and microglia. We found a total of 890 DEGs in rods, 1,075 in cones, 691 in Müller glia, and 757 in microglia (Fig. 6a, b). Gene set overlap analysis demonstrated that up- and down-regulated DEGs with the largest effect sizes were shared across these cells (Fig. 6c, d). At P60, rods and cones showed increased expression of genes related to photoreceptor inner/outer segments (Gngt1, Cnga1, Rho), phototransduction (Rcvrn, Pde6g), and visual perception (Pdc, Guca1a, Rgs9, Cngb3) and decreased expression of apoptosis (Selenos, Btg2) and phagocytosis-related genes (Cst3, Ctsd) (Fig. 6e–g, Supplementary Fig. 11a-b). Additionally, at P60, hNPC-treated cones also displayed upregulation of genes essential for photoreceptor maintenance (Crb1, Cep290), cone development (Gnat2, Thrb), mitochondrial bioenergetics pathways [NADH dehydrogenase (quinone) activity and ATP synthesis coupled electron transport (Mt-nd6, Mt-nd5)] (Fig. 6e, h), indicating higher metabolic activity, important for cone function and maintenance. Conversely, at P90, cones showed increased expression of genes related to ROS metabolic processes (Nfe2l2/Nrf2, Cyba), while rods upregulated genes linked to calcium ion homeostasis (Kcnma1, Calm1), growth factor responses (Nfkb1, Egr1, Fos), and synapse formation (Kif1a, Nrxn3), indicative of adaptive responses to oxidative and metabolic stress in the progressively degenerating retinal environment.

GO analysis of DEGs between P60 and P90 hNPC-treated Müller glia showed upregulation of genes associated with oxidative phosphorylation (Mt-nd3, Mt-nd5), synapse assembly, axonal guidance (Sema5a, Sema3a) and cAMP-mediated signaling (Pde4d, Pde10a) (Fig. 6e, i, Supplementary Fig. 11c) at P60. Conversely, by P90, they showed increased expression of genes related to phagocytosis (Cyba, Aif1), glial cell activation (Jund, C1qa), TNF signaling (Selenos, Zfp36), synapse pruning (C1qa, C1qb), apoptosis (Aif1, Ctsd), metabolic process (Ckb, Rpsa) and glucocorticoid response (Icam1, Zfp36) (Fig. 6f). These findings suggest a transition from neuroprotective to reactive state over time, aiming to mitigate stress and support retinal homeostasis during degeneration.

At P60, microglia exhibited upregulation of genes related to cell projection assembly (Nrp1, Slit2, Robo2) (Fig. 6e), while by P90, microglia increased expression of genes related to lysosome formation (Tpp1, Ctsd), antigen processing and presentation (Ctss, Rt1-Db1), inflammatory response (Cebpb, Nlrp3), T cell activation (Cd74, Icam1), leukocyte mediated immunity (Nlrp3, C3, Il1b), and superoxide metabolic processes (Nfe2l2, Ncf1) (Fig. 6f, j, Supplementary Fig. 11d). Additionally, the upregulation of genes related to lipid transport (Abca1, Apoe) and defense response (Cebpb, Grn, Il1b) highlights their role in stress management. These findings indicate that microglia play a supportive role in maintaining retinal neuronal connectivity and function at the early stage and are involved in immune surveillance, inflammation, and stress management by removing cellular debris to maintain retinal homeostasis as retinal degeneration progresses.

Progressive loss of hNPC–retinal cell crosstalk impairs protection
To investigate how grafted hNPCs interact and communicate with host retinal cells over time, we analyzed cell–cell communication dynamics across the combined datasets. We first integrated single-cell datasets from grafted hNPCs with hNPC-treated host retinal cells by mapping high-confidence one-to-one orthologous genes using ENSEMBL multiple species comparison tool62. We then generated a joint UMAP using the Seurat v4 workflow and applied Harmony for batch correction63 (Fig. 7a). Next, we applied CellChat to identify and compare the signaling pathways mediating the interaction between hNPCs and retinal cells, based on the expression of ligand-receptor pairs. This analysis revealed a decreased number of signaling interactions (Fig. 7b) and intercellular communication strength (Fig. 7c) between hNPCs and host retinal cells at P90 compared to P60 (Fig. 7d-e). Further, we applied unsupervised machine learning-based approach to analyze the functional changes in the landscape of intercellular communication of specific signaling pathways (Fig. 7f) and compute the relative information flow between P60 and P90 retinas (Fig. 7g). This analysis revealed downregulation of trophic factor, synaptic, and cell adhesion signaling pathways at P90 compared to P60 (Fig. 7h-i, Supplementary Figs. 13–16). Notably, MANF, FGF, BMP, and JAM signaling were significantly downregulated at P90, suggesting a reduction in neuroprotective support.

MANF and FGF-2 are known to protect photoreceptors35,64, and our analysis revealed decreased Manf and Fgf2 in rods at P90, while increased Fgf1 (Fig. 7j, Supplementary Fig. 13d, 17a). Conversely, cones exhibited increased Manf and Fgf2 with decreased Fgf1 at P90 (Fig. 7j, Supplementary Fig. 12d, 17b). Notably, MANF receptors (Kdelr1, Kdelr2) and Fgfr1 were upregulated in rods, whereas cones showed increased Kdelr1 and reduced Kdelr2, with no change in Fgfr1 expression (Fig. 7j, Supplementary Fig. 13d, 17b). MANF interacts with NPTN to mitigate inflammation and apoptosis by inhibiting NF-κB signaling43,65, and has demonstrated immunomodulatory properties that promote tissue repair and regeneration in the retina36. Additionally, MANF has been shown to exert neuroprotective effects in the brain following ischemic injury by increasing the number of phagocytic innate immune cells66. Interestingly, double immunostaining of IBA1 and MANF confirmed significantly higher MANF expression and increased MANF+ microglia in the hNPC-treated retina at P60 compared to P90 (Fig. 7k-m). Furthermore, overall increased MANF expression was detected in P60 hNPC-treated retina compared to P90 (Fig. 7k-l), reinforcing that MANF plays a critical role in photoreceptor protection and immune modulation, with its decline at P90 potentially contributing to reduced retinal protection. Further, while BMP can induce retinal regeneration and provide neuroprotection by upregulated FGF signaling67,68, the downregulation of BMP signaling at P90 (Supplemenaty Fig. 13g-h) underscores the progressive weakening of neuroprotective mechanisms over time.

Cell adhesion molecule (CAM) signaling, like NECTIN, NRXN, and NCAM1 was significantly upregulated at P60 (Supplementary Fig. 14a-f), contributing to synaptic stability and visual signal transmission. Immunofluorescence staining and quantitative analysis of NCAM1 further revealed a marked reduction in NCAM1 expression in grafted hNPCs and across multiple retinal cell layers in P90 hNPC-treated retina compared to P60 (Supplementary Fig. 14g-h). Notably, NECTIN signaling was detected across all retinal cell types except rods, hNPCs, and microglia. By P90, CAM signaling declined, potentially weakening synaptic connectivity between retinal neurons and disrupting visual signals transmission, ultimately compromising visual function69–71. Additionally, COLLAGEN and LAMININ signaling, essential for ECM remodeling, structural integrity, and cell-ECM interactions72–74 were significantly downregulated at P90 (Fig. 8a–d), indicating weakened ECM integrity and reduced structural support for retinal cells during disease progression. Protein level validation confirmed these findings, showing a significant decrease in Collagen IV (Col IV) and IMPG2 expression in P90 hNPC-treated retina compared to P60 (Fig. 8e–g). Furthermore, Tenascin C (TNC), an ECM glycoprotein that creates a specialized and supportive environment and promotes the migration of hNPCs75 was markedly reduced and exhibited a disorganized pattern in P90 hNPCs, whereas P60 hNPCs displayed a well-organized TNC+ ECM layer (Fig. 8h). Despite these declines, CADM signaling-based communications emerged at P90 between hNPCs and retinal neurons, including rods, cones, and CBCs, which were absent at P60 (Supplementary Fig. 15a-b). CADM signaling (CADM1 and CADM3) plays a key role in visual circuit formation by regulating axon guidance, neurite growth, and synapse formation69,76. Similarly, CDH signaling was reduced at P90 but showed newly established communications between rods and other retinal cells, including cones, RBCs, CBCs, and ACs, which may partially be compensating for declining synaptic pathways (Supplementary Fig. 15c-d). Interestingly, PTN signaling remained stable between hNPCs and retinal cells over time (Supplementary Fig. 13e-f).

Together, these data indicate a progressive decline in critical trophic and growth factors, cell adhesion, and ECM-related signaling pathways, leading to reduced photoreceptor protection and synaptic stability in the hNPC-treated retinas over time.

Methods
Ethical statement
All animal experimental procedures were approved by Cedars-Sinai Medical Center’s Institutional Animal Care and Use Committee (IACUC 7611) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Animals
The Royal College of Surgeons (RCS) rats were maintained in a 12 h light/dark cycle at 20–25°C and controlled humidity (30–70%) in a pathogen-free facility with ad libitum access to food and water. Both male and female rats were used for all experiments. The rats between postnatal day (P) 21-23 were randomly assigned to two groups: one group received a subretinal injection of human neural progenitors (hNPCs) (n=65), while the others served as untreated control (n=44). To minimize the immune response to human-derived cells in this xenograft model, cyclosporine A was administered via drinking water (210 mg/L), starting 1 day prior to transplantation and continuing until sacrifice. The experiment was repeated 3 times for P60 (untreated: n=24; hNPC-treated: n=30) and 2 times for P90 (untreated: n=20; hNPC-treated: n=35) time points.

Cell preparation and transplantation
The generation and expansion of hNPCs were conducted by Svendsen et al. in 199887. Briefly, human neural progenitor cells were isolated from the developing human cortex and grown in culture as sphere-shaped clusters. The spheres were sectioned into quarters, and cell-cell contacts were maintained. This simple and novel culture method allows rapid expansion of large numbers of hNPCs. Research-grade hNPCs were used in this study under the Stem Cell Research Oversight Committee (Pro00025772) at Cedars-Sinai Medical Center. Cells were prepared according to our published protocol20. Briefly, hNPC vials were rapidly thawed in a 37°C water bath after removal from liquid nitrogen, followed by resuspension in prewarmed Stemline medium (Sigma, St. Louis, Missouri) supplemented with rhEGF (100 ng/mL; Millipore, Billerica, MA) and rhLIF (100 ng/mL; Millipore). The cell suspension was centrifuged at 200 × g for 5 min, and the pellet was resuspended in Balanced salt solution (BSS, Alcon). Cells were then labeled using PKH26 Red Fluorescent Cell Linker Kits (Sigma, St. Louis, Missouri) according to the manufacturer’s protocol and finally resuspended at a concentration of 30,000 cells/μL in BSS. Cells were kept on ice until transplantation. Cell injections were performed following a previously established protocol20. Briefly, 2 μL of the hNPC suspension (6 × 10^4 cells/eye) was injected into the subretinal space of both left and right eyes through a small scleral incision using a fine glass pipette (internal diameter 75–150 μm) attached via tubing to a 25-μL syringe (Hamilton). To reduce intraocular pressure and minimize cell efflux, the cornea was punctured prior to injection. Immediately after injection, the fundus was examined for retinal damage or signs of vascular distress. Animals showing such issues were excluded from further study.

Efficacy evaluation
Optokinetic response (OKR). OKR testing provides a non-invasive method for assessing spatial visual acuity, measured in cycles per degree (c/d). Visual acuity was evaluated at P60 and P90, following our previously published protocol11,20. Animals were tested using a virtual reality–based OKR system consisting of an elevated circular platform surrounded by four computer monitors and a downward-facing overhead camera (OptoMotry HD, Cerebral Mechanics, Lethbridge, Canada). The monitors displayed rotating vertical grating patterns generated and controlled by OptoMotry HD software, with spatial frequencies ranging from 0.003 to 0.6 c/d. The movement of this grating induces an optokinetic response, where the rodents’ eyes and head track the movement involuntarily. Using the OKR OptoMotry system, measurements are limited to 0.6 c/d because reliability decreases at higher spatial frequencies. Due to lateralized visual processing and the rodent preference for temporal-to-nasal motion, each eye was assessed independently by presenting clockwise rotation to preferentially stimulate the left eye and counterclockwise rotation to stimulate the right eye. OKR testing was terminated when animals no longer exhibited tracking responses to the visual stimulus. The OKR results were observed and recorded by two independent, blinded investigators. Data were analyzed after incorporating our previous study datasets.

Electroretinography (ERG). Electroretinography (ERG) measures the overall electrical activity of the retina in response to light stimulation. ERG recordings were performed at P60 and P90 according to our previously established protocol19,20. In brief, following overnight (14–16 h) dark adaptation, animals were prepared under dim red light. Scotopic ERGs were recorded by stimulating the eyes with 5 ms white-light flashes at an intensity of 25 cd·s/m² with 10 s interstimulus intervals using a computer-controlled Espion system (Diagnosys LLC). A total of 20–30 sweeps were recorded for each animal, and the averaged responses were used to determine the response amplitude and assess rod-mediated retinal function. Data were analyzed after incorporating our previous study datasets.

Isolation of grafted hNPCs and rat retinal cells. Rats were sacrificed at P60 and P90 time points after visual function tests. Eyes were enucleated, and retinas were dissected in AMES solution (equilibrated with 95% O2/5% CO2). Under a fluorescent dissecting microscope, red fluorescent region containing the injection site (dorsal-temporal part) was dissected from the retina. For untreated retinas, the dorsal-temporal part corresponding to the same location in the cell-treated eye was dissected. Retinas from 8-15 rats were pooled, digested with papain, and dissociated into single-cell suspensions through manual trituration in ovomucoid solution88. Single-cell suspensions from hNPC-treated and untreated retinas were labeled with DAPI. PKH26-labeled hNPCs, along with hNPC-treated and untreated retinal cells, were sorted using a FACS sorter (FACSAria III, BD Biosciences, Franklin Lakes, NJ) and collected into 1.7 mL RNase-free tubes (Eppendorf, Hamburg, Germany) containing 2% BSA in PBS. From hNPC-treated cell suspension, PKH26-labeled hNPCs and treated retinal cells were collected in separate tubes.

Single-cell RNA sequencing and library preparation. The sorted grafted hNPCs, rat retinal cell suspensions, and in vitro hNPCs were immediately processed for single-cell RNA sequencing following the 10x Chromium v3 single-cell reagent kit protocol for library preparation. cDNA library quantification and quality were assessed with D1000 or D5000 HS kit for the Agilent TapeStation 4200. Libraries were sequenced using Illumina Novaseq 6000 S4 platform using 100 bp paired-end sequencing for a sequencing depth of 50,000 read pairs/cell.

Histology and immunofluorescence staining. Eyes were enucleated from P60 and P90 time points, fixed in 4% paraformaldehyde, and embedded in OCT for cryostat sections. Retinal cryostat sections were placed on a series of 5 slides, with 4 sections per slide. The first slide of each series was used for cresyl violet staining to visualize retinal lamination and donor cell location. The remaining slides were then used for immunofluorescent staining following previously described protocols20,89. Briefly, retinal sections were rehydrated in 1× PBS and blocked for 1 h in 2.5% BSA solution containing 5% serum (respective to the antibodies). Sections were incubated with primary antibodies at 4°C overnight with following antibodies: rabbit anti-MANF (1:200; Sigma, HPA011175), rabbit anti-MYDGF (1:200; Proteintech, 11353-1-AP), rabbit anti-KDELR (1:200; Thermo Fisher Scientific, PA5-101011), mouse anti-human nuclear marker (1:300; Millipore, MAB1281), goat anti-Iba1 (1:1000; FUJIFILM Wako Pure Chemical Corporation, 019-197441), goat anti-doublecortin (1:200; Santa Cruz Biotechnology, sc-8066), mouse anti-MAP2 (2a + 2b) (1:500; Sigma, M1406), mouse anti-RPE65 (1:500; Sigma, MAB5428), mouse anti-AQP4 (1:500; R&D Systems, MAB11659-SP), rabbit anti-collagen IV (1:200; Abcam, ab19808), mouse anti-tenascin C (1:500; Abcam, ab86182), rabbit anti-cone arrestin (1:1000; Millipore, AB15282), rabbit anti-GFAP (1:1000; Dako–Agilent, Z0334), rabbit anti-nestin (1:2000; Millipore, ABD69), rabbit anti-NCAM1 (1:200; Proteintech, 14255-1-AP), rabbit anti-IMPG2 (1:200; Abcam, ab272558), rabbit anti-oligodendrocyte-specific protein (1:100; Abcam, ab7474), rabbit anti-recoverin (1:2000; Millipore, AB5585), rabbit anti-SOX2 (D6D9) XP (1:400; Cell Signaling Technology, 3579), and rabbit anti-TXNL6 (1:200; Proteintech, 11203-1-AP). Following incubation with the primary antibodies, sections were washed three times for 5 min each in 1× PBS and incubated with anti-mouse or -rabbit or -goat secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 for 1 h at room temperature. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted using Fluorescent Mount media (Sigma-Aldrich). Images were taken with a Leica or Nikon microscope.

Image analysis. Differences in immunoreactivity of MANF, Col IV, IMPG2, and NCAM1 between untreated and hNPC-treated retina at P60 and P90 were assessed as fluorescent intensities (FI) with our previous protocol88. In brief, for FI measurements, MANF, Col IV, IMPG2, and NCAM1 positive region of interest (ROI) was selected using ImageJ freehand tool. Fluorescent intensities of ROIs were measured after normalizing to the background. Fluorescent intensity values were averaged (4 sections per retina and 8-9 retinas per group; each section 40 μm apart) to determine signal density. The FI values were presented as arbitrary units. The processing and analysis were kept consistent between images. Statistical analysis was performed on average FI values (mean ± SEM). MANF+ microglia were counted manually (4 sections per retina and 10 retinas per group; each section 40 μm apart). Image analysis was performed blindly and separately by 2 observers.

Preliminary processing scRNA-seq raw data of hNPCs and retinal cells. Cell Ranger software (version 4.0.0) (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/4.0/what-is-cell-ranger) was used to perform preliminary processing of single-cell sequencing data, including aligning sequencing reads to the reference transcriptome (hNPCs: GRCh38; rat: Rnor6), estimating and filtering, and generating feature-barcode matrices. Wild-type rat sequencing data from Wang et al. were downloaded from the GEO database (GSE209872: GSM6403183 and GSM6403184).

Removing low-quality cells and identification of cell types. Seurat (version 4.4.0) software was used to perform downstream quality control, integration, identification of cell types, and subsequent differential expression gene analysis on the gene-cell expression matrices obtained above90. For hNPC datasets, cells with less than 250 and greater than 20,000 UMIs, less than 200 genes, and mitochondrial gene percent greater than 20% were excluded as low-quality cells. For rat retinal cell datasets, cells with counts, genes, mitochondrial gene percentage, and ribosomal gene percentage outside of three standard deviations from the mean were excluded as low-quality cells.

For hNPCs, the ‘LogNormalize’ function was used for normalization and scaling of the expression matrix for each sample, and then the ‘SelectIntegrationFeatures’ and ‘FindIntegrationAnchors’ functions were used to select the features and anchors for downstream integration. These anchor points were then used to integrate all datasets of the hNPCs through the ‘IntegrateData’ function. Next, the ‘ScaleData’ and ‘RunPCA’ functions were used for scaling and calculating principal component analysis (PCA) dimensions in the hNPC integrated datasets. Unsupervised clustering was then performed using the ‘FindNeighbors’ and ‘FindClusters’ functions, grouping the datasets into distinct clusters. Dimensionality reduction was conducted using the ‘RunUMAP’ function. Clusters were annotated based on known retinal cell type markers and hNPC-specific gene markers. Subsequently, hNPC clusters were subset and reprocessed following the same steps described above. The ‘FindAllMarkers’ and ‘FindConservedMarkers’ functions were used to identify and annotate specific markers for the hNPC clusters.

Similarly, for retinal cells, the expression matrix for each sample was normalized and scaled using the ‘SCTransform’ function91. This was followed by feature and anchor selection with the ‘PrepSCTIntegration’ and ‘FindIntegrationAnchors’ functions. The identified anchors were then used to integrate all datasets from the same tissue using the ‘IntegrateData’ function. Subsequently, the ‘ScaleData’ and ‘RunPCA’ functions were employed to scale the data and perform principal component analysis (PCA) on the integrated datasets of hNPCs and rat retinal cells. Unsupervised clustering of the datasets into distinct clusters was performed using the ‘FindNeighbors’ and ‘FindClusters’ functions. Dimensionality reduction was carried out using the ‘RunUMAP’ function. To investigate the gene expression profile of each cluster, the ‘FindAllMarkers’ function (with avg_log2FC > 0.4) was used to identify highly expressed genes (marker genes) that are specific to each cluster and correspond to distinct cell types based on marker gene expression.

Differential gene expression and functional analysis. To identify differentially expressed genes (DEGs) between in vitro and in vivo hNPCs at P60 and P90, the ‘FindMarkers’ function with the MAST92 test in Seurat was applied. DEGs between wild-type, untreated, and hNPC-treated retinal cells, across different retinal cell types, were identified using the ‘FindMarkers’ function with the Wilcoxon test. For hNPCs, we used the EnrichR93 R package (version 3.2) and the ‘gseGO’ and ‘gseKEGG’ functions from the clusterProfiler94 R package (version 4.10.1) to identify enriched biological pathways and processes in our sets of DEGs, as well as in DEGs shared across different differential expression analyses. For rat retinal cells, we employed the ‘gseGO’ and ‘gseKEGG’ functions from the clusterProfiler R package to determine enriched biological pathways and processes in our DEG sets.

Cell-cell ligand-receptor interaction analysis. We performed cell-cell ligand-receptor interaction in our scRNA-seq integrated rat retinal cell and hNPC datasets with CellChat95 (v1.6.1), using cell type labels. The human CellChatDB ligand-receptor interaction database was used for this analysis. To facilitate downstream comparisons of the signaling networks in untreated versus hNPC-treated and P60 versus P90 hNPC-treated groups, we ran the CellChat workflow separately on each dataset from different time points. The CellChat object was created using the normalized gene expression matrix and the cell type annotations, removing any cell groups with fewer than 10 cells. We then ran the recommended CellChat workflow using the following functions: identifyOverExpressedGenes, identifyOverExpressedInteractions, projectData, computeCommunProb, filterCommunication, subsetCommunication, computeCommunProbPathway, aggregateNet, and netAnalysis_computeCentrality. We separately merged P60 and P90 untreated and hNPC-treated and P60 and P90 hNPC-treated CellChat objects into one object using the mergeCellChat function. We compared the signaling networks separately at P60 and P90 between untreated and hNPC-treated groups and then between P60 and P90 hNPC-treated groups both functionally and structurally using the computeNetSimilarityPairwise function. Further, we used the rankNet function to compute the relative information flow changes between the groups across all signaling pathways. We identified differentially expressed ligands and receptors as well as their signaling pathways using the identifyOverExpressedGenes function, visualizing results with the netAnalysis_signalingRole_heatmap.

Statistics and reproducibility. No statistical method was used to predetermine sample size. Investigators were blinded to experimental group allocation during data collection and analysis, where applicable. Single-cell RNA sequencing data were analyzed with appropriate batch correction methods. Visual function tests were performed on multiple animals per group. Histological analyses were performed at least three times to ensure reproducibility. Statistical analyses were performed using GraphPad Software’s PRISM (version 10), and the results are presented as the mean ± SEM. Results were analyzed by One-way ANOVA coupled with a Tukey’s multiple comparisons test for more than 2-group. For ERG and OKR, One-way ANOVA, Kruskal-Wallis test, coupled with Dunn’s Multiple comparison test were used. P-value < 0.05 was considered statistically significant. No data were excluded from the analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors employed scRNA-seq to comprehensively analyze dynamic transcriptomic remodeling in grafted human neural progenitor cells. They made several key findings such as temporal gene expression changes in grafted cells and retinal cells, upregulation of novel trophic factors, cell fate of grafted cells, intercellular signaling changes, etc. The findings revealed dynamic transcriptomic changes in the degenerative retinal environment and helped our understanding of the rescue mechanism of grafted hNPCs. However, several issues need to be addressed in the paper.\nMajor concerns:\n1. ERGs were used to assess the retinal function after hNPC treatment. However, the description of ERG results was very sketchy. It was not clear whether the ERG results were recorded under the scotopic or photopic conditions. Therefore, it was not clear how the treatments affect the rod and cone function, respectively. The authors used b-wave amplitudes to determine the rescue effect. Although b-wave amplitudes may indirectly reflect the function of photoreceptors, a-wave amplitudes should be used to gauge the function of photoreceptors, particularly rod photoreceptors.\n2. Figure 4o: There was a much smaller size of con cell population in the treated retina than in the untreated retina. By contrast, the rod population in the treated retina is larger than in the untreated. Does that suggest that the hNPC treatment only protects rods but accelerates cone degeneration? If this is the case, it needs interpretation. Figure 4m has a similar trend, but less significant than in 4o, which could be due to the shorter duration of treatment. This should be reflected in the functional test results, such as ERGs.\n3. Figure 5k. The heatmap shows selected 729 microglial homeostatic and activation. The comparison was made between three groups. It was not clear how the comparison was made. What was the reference expression level for each group of the sample when the upregulation or downregulation was determined?\n4. In lines 362-364, gene expression profiles in Muller glia cells were compared between different groups. Downregulation of genes involved in visual 363 perception, photoreceptor cell maintenance was found. Muller glia cells are not known to express a significant amount of genes involved in visual 363 perception, photoreceptor cell maintenance. These results presented here indicate that the Muller glia cell clusters were contaminated with other photoreceptor cells. The authors should estimate whether the contamination obscured the overall conclusion.\n5. Retinal degeneration in RCS rats stems from RPE malfunction. How hNPC transplantation affects the function of RPE and the contribution of gene expression profile changes in RPEs should be assessed.", "reviewer_2": "The manuscript by Shahin et al. presents an extensive single cell transcriptomic analysis of hNPCs at two time points (P60 and P90) following placement into the subretinal space of the RCS rat model of retinal degeneration at a single time point (P21) and compares the results to untransplanted hNPCs. Emphasis was placed on DEGs related to a number of cellular processes attributed to the donor cells or the surrounding host retina. The hPSC cells of interest are currently being tested in a clinical trial, and thus they have shown sufficient promise and safety in preclinical models to warrant examining their therapeutic potential in humans.\nWhile the effort is admirable and the results are of interest given the ongoing clinical trial, the study has numerous major limitations. More specifically, the manuscript is 1. one-dimensional with regard techniques and models (single cell transcriptomics alone with minimal immunocytochemistry and use of the RCS rat only), 2. insufficiently controlled (no sham or non-hPSC cell transplant control; no cyclosporine A-only control), 3. presented in a selectively positive manner with essentially all highlighted findings being considered a strength of the hNPC treatment, and 4. exclusively descriptive and correlative with broadly qualified conclusions without demonstration of causation – i.e., there are no direct experimental manipulations to test any of the proposed mechanisms or interactions that are speculated. Lastly, most of the findings are predictable (e.g., donor cells proliferate and then mature; host photoreceptors are preserved, which is a known effect, etc.) and do not broadly advance the field of cellular transplantation. Other comments are listed below.\n1. The RCS rat has a known sham effect with photoreceptor survival occurring with any subretinal intervention. A sham control and a non hNPC cell transplant control is needed to show specific effects of hNPCs.\n2. No evaluation of the RPE is provided, and the RCS rat harbors a primary mutation in the RPE cells with a secondary effect on photoreceptors. How does the transplant affect the health of the host RPE?\n3. All of the single cell transcriptomic analyses are broadly presented and speculated to have causal relationships when no such conclusions can be drawn from the studies presented.\n4. The ICC shown suggests that the majority of transplanted cells do not integrate in the host retina (contrary to the comments in the introduction that state that they migrate extensively within the host retina). Instead, they stay mostly as a mass between the RPE and retina. No effort to discern the fate of cells within the host retina or adjacent to it is provided.\n5. Cells were delivered only at one timepoint in the RCS rat – assuming that patients will be treated at different stages of their disease, results obtained from an injection at only one early time point significantly reduces the impact of the results.\n6. The rats were immunosuppressed with cyclosporine A but there was no control provided for the effect of this drug alone (in the absence of cell transplantation).", "reviewer_3": "This study unveils the dynamic transcriptomic changes of grafted stem cells within the host’s degenerative environment over time, which fill in the knowledge gap in regenerative medicine. The findings will have broad implications for the field of stem cell therapy for degenerative diseases such as Parkson’s disease and ALS. Furthermore the study strongly suggests that both engineering stem cells with additional trophic factor(s) such as MANF for modulating microglia and Muller glia, and improving host’s degenerative environment are essential for ensuring long-term efficacy in regenerative medicine. In summary, stem cells and trophic factors as add-onto improve host environment are needed for long-term vision protection.\nOverall, the design is sound, experimental data are interesting and conclusions are well supported.\nI have some minor concerns.\n1.This study only covered two time points (P60 and P90), early and later time points are needed to capture the temporal dynamics of grafted hNPC and host retinal changes.\n2.Injection at P21, early time of retinal degeneration, later time points of grafting should be performed.\n3.It would be interesting to present additional study using hNPCengineered to release trophic factor MANF to see whether the therapeutic effect will be enhanced; how hNPC-MANF modulates host retinal environment.\n4.The graft-associated effect on host photoreceptors, Muller glia and microglia is estimated since we cannot dissect precisely grafted-related area. Spatial transcriptomics would be ideal to address directly host retinal cells affected by graft."}, "author_response": "We are very grateful for reviewers’ thoughtful and constructive feedback. Below, we provide point-to-point responses to each concern, outlining the steps we have taken to strengthen the study and clarify its scope:\nReviewer #1 (Remarks to the Author):\nThe authors employed scRNA-seq to comprehensively analyze dynamic transcriptomic remodeling in grafted human neural progenitor cells. They made several key findings such as temporal gene expression changes in grafted cells and retinal cells, upregulation of novel trophic factors, cell fate of grafted cells, intercellular signaling changes, etc. The findings revealed dynamic transcriptomic changes in the degenerative retinal environment and helped our understanding of the rescue mechanism of grafted hNPCs. However, several issues need to be addressed in the paper.\nResponse: Thank you for your thoughtful summary and constructive feedback. To our knowledge, this is the first study to comprehensively characterize the dynamic transcriptomic remodeling of both grafted human neural progenitor cells (hNPCs) and the host retinal cells over time in a degenerative environment. Our findings demonstrate that the efficacy of a single stem cell-based therapy may decline as retinal degeneration progresses. This highlights the need for a combinatorial therapeutic strategy, integrating stem cell transplantation with interventions targeting the degenerative host retinal environment to achieve sustained vision preservation.\nMajor concerns:\n1. ERGs were used to assess the retinal function after hNPC treatment. However, the description of ERG results was very sketchy. It was not clear whether the ERG results were recorded under the scotopic or photopic conditions. Therefore, it was not clear how the treatments affect the rod and cone function, respectively. The authors used b-wave amplitudes to determine the rescue effect. Although b-wave amplitudes may indirectly reflect the function of photoreceptors, a-wave amplitudes should be used to gauge the function of photoreceptors, particularly rod photoreceptors.\nResponse: Thank you for this insightful and critical comment. We fully agree that clarifying the ERG methodology and distinguishing between rod and cone function is important for interpreting ERG data. In the RCS rat model, rod photoreceptor function declines rapidly and is compromised by postnatal day (P) 21, leaving cones as the primary contributors to residual/remaining retinal function. Our prior studies have consistently demonstrated that while stem cell therapy does not restore lost rod function, it can preserve cone-mediated responses (e.g., Girman et al. 2005, PMID: 15607350; Lu et al. 2023, PMID: 37743503; Shahin et al. 2023, PMID: 37786347).\nWe have revised the manuscript to clarify the ERG recording conditions in the Method section under the heading Electroretinography (ERG) and interpretation in the Results section:\nMethod section (lines 931-936): \"In brief, following overnight (14-16h) dark adaptation, animals were prepared under dim red light. Scotopic ERGs were recorded by stimulating the eyes with 5 ms white-light flashes at an intensity of 25 cd/m² with 10 s interstimulus intervals using a computer-controlled Espion system (Diagnosys LLC). A total of 20-30 sweeps were recorded for each animal, and the averaged responses were used to determine the response amplitude and assess rod-mediated retinal function.”\nResults section (lines 120-131): \"After overnight dark adaptation, animals were prepared under dim red light. Scotopic ERGs were recorded using 5 ms white-light flashes at 25 cd/m² with 10-second interstimulus intervals, to assess rod-mediated retinal function. As expected, a-waves were not detectable at P60 and P90, due to loss of rod photoreceptor outer segment activity, which is known to be compromised as early as P21 in the RCS model, as reported in our previous study17. However, ERG b-wave amplitudes were significantly higher for hNPC-treated eyes compared to untreated controls at P60 and P90 (Supplementary Fig.1c). These b-wave responses are primarily cone-driven, demonstrating preservation of inner retinal activity, consistent with our previous findings15,17. Notably, while visual acuity (tested under photopic condition) for hNPC-treated eyes was significantly higher than untreated controls and remained stable, ERG b-wave amplitudes decreased over time, reflecting progressive deterioration of retinal function (Supplementary Fig.1b-c).\"\nWe acknowledge the reviewer's point that a-wave amplitudes most directly reflect photoreceptor (particularly rod) function. However, due to the early rod degeneration in RCS rats, a-wave measurements provide negligible functional information at later stages, such as P60 and P90 time points in our study. Our focus on b-wave amplitudes is therefore justified and aligns with established methodology in this RP model. The scotopic b-wave in the RCS rat reflects the activity of inner retinal cells (bipolar and Müller cells) that remain responsive to surviving photoreceptor input.\n2. Figure 4o: There was a much smaller size of con cell population in the treated retina than in the untreated retina. By contrast, the rod population in the treated retina is larger than in the untreated. Does that suggest that the hNPC treatment only protects rods but accelerates cone degeneration? If this is the case, it needs interpretation. Figure 4m has a similar trend, but less significant than in 4o, which could be due to the shorter duration of treatment. This should be reflected in the functional test results, such as ERGs.\nResponse: Thank you for this thoughtful comment. We emphasize that hNPC treatment protects both rods and cones, as demonstrated in our previous studies (Wang et al. 2011, PMID: 18579765; Lu et al. 2023, PMID: 37743503; Shahin et al. 2023, PMID: 37786347). To strengthen this conclusion, we incorporated new supporting data (new Supplementary Fig. 2) confirming photoreceptor protection. Specifically, recoverin immunostaining, which labels both rods and cones, revealed a preserved outer nuclear layer in the graft-protected areas, whereas only a single layer of photoreceptors remained in the untreated retina. Cone-arrestin staining further confirmed well-organized cones with inner and outer segments in regions associated with hNPC-grafted area. In contrast, age-matched untreated retinas displayed degenerative cone morphology with diminished cone-arrestin signals. These histological findings are consistent with functional outcomes. ERG recordings demonstrated significantly higher b-wave amplitudes in hNPC-treated eyes compared to untreated controls, reflecting preserved cone-driven retinal responses. Together, these data confirm that hNPCs protect both rod and cone photoreceptors.\nWe have incorporated all these findings in the result section of the revised manuscript (lines 133-153).\nThe apparent reduction in cone numbers in Fig. 4o is likely due to sampling variability rather than real cone loss. This is further supported by cone-arrestin staining and ERG results, which demonstrate clear preservation of cone structure and function in treated retinas.\n3. Figure 5k. The heatmap shows selected 729 microglial homeostatic and activation. The comparison was made between three groups. It was not clear how the comparison was made. What was the reference expression level for each group of the sample when the upregulation or downregulation was determined?\nResponse: Thank you for pointing out the need for clarification. In Fig. 5k, each row (i.e., each gene) was z–score normalized across the three experimental groups at a given time point (WT, Untreated, and hNPC-Treated). Specifically, at P60 (and likewise at P90), we first calculated the pseudo-bulk average expression of each gene within each group. We then computed the mean (μ) and standard deviation (σ) of these three values, and derived the z-score for each group i as:\n𝐸 −𝜇\n𝑧 = 𝑖\n𝑖\n𝜎\nwhere E is the average expression of the gene in group i. Thus, the “reference” is the mean expression of that gene across all three groups at the corresponding time point. Positive z-scores (reddish) indicate expression above the overall mean, whereas negative z-scores (bluish) indicate expression below it. This normalization enables direct pattern comparison across groups while controlling for baseline differences.\n4. In lines 362-364, gene expression profiles in Muller glia cells were compared between different groups. Downregulation of genes involved in visual 363 perception, photoreceptor cell maintenance was found. Muller glia cells are not known to express a significant amount of genes involved in visual 363 perception, photoreceptor cell maintenance. These results presented here indicate that the Muller glia cell clusters were contaminated with other photoreceptor cells. The authors should estimate whether the contamination obscured the overall conclusion.\nResponse: We thank the reviewer for raising this important point. We agree that under physiological conditions, Müller glia are not major contributors to pathways involved in visual perception or photoreceptor maintenance. However, in the context of retinal degeneration, Müller glia are known to upregulate genes typically associated with photoreceptors as part of a reactive or compensatory program (Tomita et al., 2021, PMID: 34799683).\nTo address this, we have clarified the manuscript (lines 431-435): “Of note, Müller glia upregulate genes involved in phototransduction and light stimulus in response to retinal degeneration. However, in hNPC-treated Müller glia, downregulation of genes related to these biological processes suggests that hNPCs may modulate Müller glia reactivity, shifting their transcriptional profile away from compensatory photoreceptor-like responses and toward a supportive, neuroprotective state.”\nImportantly, we carefully re-examined the clustering and marker gene expression profiles and found no evidence of photoreceptor contamination within the Müller glia clusters. The cell identities remain consistent with established Müller glial markers, and the overall conclusions are robust to this analysis. We are therefore confident that the observed changes represent genuine Müller glial responses to hNPC treatment rather than artifacts of misclassification or contamination.\n5. Retinal degeneration in RCS rats stems from RPE malfunction. How hNPC transplantation affects the function of RPE and the contribution of gene expression profile changes in RPEs should be assessed.\nResponse: Thank you for raising this important point. In our study, we observed that RPE cell integrity, as indicated by RPE65 and collagen IV immunostaining, was preserved in regions associated with grafted hNPC distribution (new Supplementary Fig. 2 and new Fig. 8). Additionally, we did not observe the characteristic subretinal debris accumulation typically seen in RCS rats, which results from RPE dysfunction and failure to phagocytose photoreceptor outer segments (OS), as previously reported (Gamm et al. 2007, PMID: 17396165, Shahin et al. 2023, PMID: 37786347). Both our current and prior studies demonstrate that grafted hNPCs are capable of phagocytosing OS, thereby compensating for the defective RPE and helping to maintain a healthy subretinal environment.\nWhile we did not directly assess RPE transcriptomes, the preserved RPE morphology, absence of subretinal debris, and evidence of hNPC-mediated phagocytosis together suggest that hNPCs indirectly support RPE function, which likely contributes to the observed photoreceptor protection.\nIn this study, we did not isolate RPE cells for transcriptomic analysis, as our primary goal was to recover grafted hNPCs and retinal cells (especially photoreceptors and glial cells) from a large number of eyes while preserving RNA quality. We agree with the reviewer that future studies focusing on RPE-specific gene expression changes in response to hNPC transplantation will be important to further elucidate their contribution to therapeutic efficacy.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Shahin et al. presents an extensive single cell transcriptomic analysis of hNPCs at two time points (P60 and P90) following placement into the subretinal space of the RCS rat model of retinal degeneration at a single time point (P21) and compares the results to untransplanted hNPCs. Emphasis was placed on DEGs related to a number of cellular processes attributed to the donor cells or the surrounding host retina. The hPSC cells of interest are currently being tested in a clinical trial, and thus they have shown sufficient promise and safety in preclinical models to warrant examining their therapeutic potential in humans.\nWhile the effort is admirable and the results are of interest given the ongoing clinical trial, the study has numerous major limitations. More specifically, the manuscript is 1. one-dimensional with regard techniques and models (single cell transcriptomics alone with minimal immunocytochemistry and use of the RCS rat only), 2. insufficiently controlled (no sham or non-hPSC cell transplant control; no cyclosporine A-only control), 3. presented in a selectively positive manner with essentially all highlighted findings being considered a strength of the hNPC treatment, and 4. exclusively descriptive and correlative with broadly qualified conclusions without demonstration of causation – i.e., there are no direct experimental manipulations to test any of the proposed mechanisms or interactions that are speculated. Lastly, most of the findings are predictable (e.g., donor cells proliferate and then mature; host photoreceptors are preserved, which is a known effect, etc.) and do not broadly advance the field of cellular transplantation. Other comments are listed below.\nResponse: We thank the reviewer for these critical and constructive comments. We recognize the importance of addressing these limitations directly and have revised the manuscript accordingly. Below, we provide point to point responses to each concern, outlining the steps we have taken to strengthen the study and clarify its scope:\n1. One-dimensional with regard techniques and models (single cell transcriptomics alone with minimal immunocytochemistry and use of the RCS rat only).\nResponse: We acknowledge the reviewer’s concern regarding the scope of study. Our decision to focus on the well-characterized RCS rat model was deliberate, as it allowed us to build directly upon our previous IND-enabling study (Lu et al. 2023, PMID: 37743503). While we agree that future investigations comparing grafted hNPC behavior across multiple retinal degeneration models would provide valuable insights, the depth of our single-cell transcriptomic analysis in this highly relevant model offers a unique and unprecedented view of the dynamic cellular interactions between grafted hNPCs and the host retina. Furthermore, our previously published work has already demonstrated the limited proliferation of grafted hNPCs (Shahin et al. 2023, PMID: 37786347; Lu et al. 2023, PMID: 37743503), which is a key safety feature emphasized by the FDA.\nWe also fully agree that validation of key transcriptomic findings at the protein level is essential. To address this, we have significantly revised our manuscript by performing extensive new immunohistochemistry analyses and incorporating five new figures. These additional analyses strengthen and validate our key scRNA-seq findings at the protein level. Below, we outline the steps we have taken to strengthen the study and clarify its scope.\nFig.1i: Characterization of grafted hNPCs at the protein level\nOur transcriptomic data suggested that grafted hNPCs predominantly adopt a glial-like fate. This finding is now confirmed by immunofluorescence staining, which demonstrates robust expression of the astrocytic markers GFAP and AQP4. Grafted hNPCs also retained key neural progenitor markers like Nestin and SOX2. We observed only a few OLIG2- and DCX-positive cells, which precisely corroborates the low abundance of OPC-like and neuronal populations identified in our scRNA-seq dataset.\nFindings are incorporated in the results section of the revised manuscript (lines 203-213).\nSupplementary Fig. 2: Protection of photoreceptors and RPE integrity by hNPCs\nWe have incorporated histological evidence demonstrating that grafted hNPCs preserved both rod and cone photoreceptors as well as RPE integrity. Recoverin staining confirmed preservation of both photoreceptor types in areas corresponding to hNPC grafts, whereas untreated controls showed only a single remaining photoreceptor layer. Cone-arrestin staining revealed well-organized cones with intact inner and outer segments in graft-associated regions, in contrast to the degenerative cone morphology and diminished staining seen in untreated controls. Additionally, RPE65 and Collagen IV staining confirmed RPE preservation in hNPC-treated areas, while both markers were weak or absent in untreated retinas.\nFindings are incorporated in the results section of the revised manuscript (lines 133-153).\nSupplementary Fig. 10: Validation of RdCVF (Nxnl1/Txnl6) expression in the RCS retina following hNPC treatment\nhNPC-treated retinas showed increased expression of Txnl6 (Nxnl1), compared to untreated controls. However, P90 hNPC-treated retinas exhibited reduced Txnl6 (Nxnl1) expression compared to P60 hNPC-treated retinas.\nFig. 8: Expression of Extracellular Matrix Molecules\nOur scRNA-seq data indicated that grafted hNPCs express extracellular matrix (ECM) molecules involved in supportive functions. We have now validated this finding at the protein level, demonstrating that grafted hNPCs express ECM molecules such as Tenascin-C. In addition, we also show that grafted hNPCs preserve host retinal ECM integrity by maintaining the levels of collagen IV and IMPG2, which provide support to RPE cells and maintain the structural stability of the photoreceptor outer segment.\nFindings are incorporated in the results section of the revised manuscript (lines 550-555).\nSupplementary Fig. 14g-h: Validation of NCAM signaling\nOur scRNA-seq data indicated that grafted hNPCs upregulate cell adhesion molecule (CAM) signaling, including NCAM1, which contributes to synaptic stability and visual signal transmission. We now validated this finding at the protein level, demonstrating increased NCAM1 expression in both grafted hNPCs and the host retina at P60 compared to P90.\nFindings are incorporated in the results section of the revised manuscript (lines 541-543).\nTogether, we believe, these additions significantly broaden the scope of our study by providing complementary protein-level validation, while preserving the unique depth of insight offered by scRNA-seq.\n2. insufficiently controlled (no sham or non-hPSC cell transplant control; no cyclosporine A-only control).\nResponse: We included untreated (non-hNPC transplant) control groups at both P60 and P90 time points for comparison. All experimental animals, including controls, received cyclosporine A in their drinking water following our previously established protocols (Gamm et al. 2007, PMID: 17396165; Lu et al. 2015, PMID: 25694843), as cyclosporine A has been reported to influence visual function in the RCS rat model (Cooper et al. 2016, PMID: 27008099). Our design thus ensured that any observed differences were attributable to the presence or absence of hNPC transplantation rather than the effects of immunosuppression alone.\nOur study was designed to examine the biological impact of grafted hNPCs and their interactions with the host retina, rather than to evaluate the surgical procedure itself. As such, we did not include a sham-operated group (surgery without cell delivery), since our focus was not on isolating the effects of the surgical intervention. We acknowledge that inclusion of sham or non-hPSC cell transplant controls will be an important consideration in proof-of-concept study. Our previously published studies, which included sham-operated and non-hPSC cell transplant controls, demonstrated that the surgical procedure alone does not provide the therapeutic effects observed with hNPCs (Shahin et al, 2023, PMID: 37786347; Lu et al., 2023, PMID: 37743503). Based on these foundational findings, we focused the present study on a direct comparison between hNPC-treated and untreated retinas to specifically address the molecular mechanisms underlying the therapeutic benefits of the hNPCs themselves.\n3. presented in a selectively positive manner with essentially all highlighted findings being considered a strength of the hNPC treatment\nResponse: We thank the reviewer for this insightful comment. We fully agree that it is important to present both the strengths and limitations of hNPC treatment transparently for a balanced and rigorous interpretation of our findings. In the revised manuscript, we have clarified that hNPC transplantation does not halt retinal degeneration but rather slows disease progression. For example, we explicitly note that while ERG b-wave amplitudes remain significantly higher in hNPC-treated eyes compared to controls, they still decline over time, and some photoreceptor loss continues by P90. This reflects a slowing, rather than a complete prevention of degenerative processes.\nWe also expanded the Limitations section to emphasize the descriptive and hypothesis-generating nature of our single-cell and CellChat analyses. We acknowledged that further functional studies are required to establish causality for the pathways identified. These revisions ensure that while we highlight the meaningful neuroprotective role of hNPCs, our presentation remains scientifically balanced, reinforcing the idea that this approach is a valuable component of a broader therapeutic strategy to modify the degenerative retinal environment.\n4. exclusively descriptive and correlative with broadly qualified conclusions without demonstration of causation – i.e., there are no direct experimental manipulations to test any of the proposed mechanisms or interactions that are speculated.\nResponse: We thank the reviewer for this important comment. This study was designed as a descriptive and hypothesis-generating investigation, and we acknowledge that no direct experimental manipulations were performed to establish causation for the proposed mechanisms and interactions. Retinal degenerative disease is a highly complicated process involving dynamic remodeling of multiple cell types, and our goal was to capture these temporal transcriptomic changes, understand how grafted hNPCs evolve in a progressive degenerative environment, and identify potential therapeutic targets to enhance hNPC therapeutic efficacy. Our findings highlight several promising avenues for intervention, including engineering hNPCs to overexpress trophic factors such as MANF and targeting key retinal support cells such as Müller glia and microglia, as adjunctive strategies to enhance photoreceptor survival. These targets will be systematically validated in follow-up studies using genetic and pharmacological approaches.\n5. Lastly, most of the findings are predictable (e.g., donor cells proliferate and then mature; host photoreceptors are preserved, which is a known effect, etc.) and do not broadly advance the field of cellular transplantation.\nResponse: We respectfully disagree with the suggestion that the findings are predictable or lack broader significance. First, our data, consistent with previous reports from our group (Shahin et al, 2023, PMID: 37786347; Lu et al., 2023, PMID: 37743503) demonstrate that grafted hNPCs exhibit minimal or no proliferation after transplantation. This limited proliferative capacity is not only non-trivial but also critically important from a translational and regulatory standpoint, as non-proliferation is a key safety requirement outlined by the FDA for cell-based therapies.\nWhile photoreceptor preservation has been observed in earlier studies involving stem cell transplantation, the mechanisms underlying this protective effect remain poorly understood. Our study is, to our knowledge, the first to systematically examine both graft and host retinal transcriptomic remodeling over time, revealing that stem cell therapy initiates a dynamic and bidirectional molecular cross-talk between transplanted cells and the degenerating retina.\nImportantly, our findings show that:\n• A single delivery of stem cells may not have long-last effects on vision preservation.\n• Long-term neuroprotection requires addressing the degenerative host microenvironment, especially by modulating key retinal support cells such as Müller glia and microglia.\n• These insights not only refine the understanding of stem cell behavior in the retina but also have broader implications for regenerative medicine strategies targeting other neurodegenerative diseases, such as Parkinson’s disease and ALS, where glial and degenerative host environment significantly influence therapeutic outcomes.\nThus, our study moves beyond simply documenting known outcomes and instead provides a conceptual framework and actionable insights for improving stem cell–based therapies in retinal and CNS degenerative diseases.\nOther comments’ responses are listed below.\n1. The RCS rat has a known sham effect with photoreceptor survival occurring with any subretinal intervention. A sham control and a non hNPC cell transplant control is needed to show specific effects of hNPCs.\nResponse: We agree with the reviewer that the RCS rat exhibits a well-documented sham effect, where short-term photoreceptor preservation can occur following subretinal intervention, independent of the transplanted cell type. In our previous studies (Gamm et al. 2007, PMID: 17396165; Lu et al. 2015, PMID: 25694843; Shahin et al, 2023, PMID: 37786347; Lu et al., 2023, PMID: 37743503), we directly addressed this issue by including sham-operated and untreated control groups, which clearly demonstrated the specific therapeutic effects of hNPCs beyond the sham response. These data formed the foundation for the ongoing clinical trial.\nThe current study builds on that body of work by shifting focus toward understanding the temporal dynamics of grafted hNPCs and their molecular interactions with host retinal cells. Our goal was not to re-demonstrate efficacy over sham but rather to explore how both graft and host undergo transcriptomic and cellular remodeling over time. These insights provide a mechanistic understanding of the limitations of single-dose therapy and point to new opportunities to enhance long-term efficacy through modulation of the host retinal environment.\n2. No evaluation of the RPE is provided, and the RCS rat harbors a primary mutation in the RPE cells with a secondary effect on photoreceptors. How does the transplant affect the health of the host RPE?\nResponse: We appreciate the reviewer’s insightful comment regarding the importance of the retinal pigment epithelium (RPE) in the RCS rat model, which indeed carries a primary mutation in Mertk that impairs RPE phagocytosis, leading to secondary photoreceptor degeneration.\nIn the current study, we evaluated RPE integrity using RPE65 and Collagen IV immunostaining, which showed preserved RPE structure in regions associated with grafted hNPCs (new Supplementary Fig. 2 and new Fig. 8). These findings are consistent with our previous studies (Gamm et al. 2007, PMID: 17396165; Shahin et al, 2023, PMID: 37786347) showing reduced subretinal debris accumulation in hNPC-protected areas. In the RCS model, undigested outer segments (OS) accumulate in the subretinal space due to defective RPE phagocytosis. Our data support that grafted hNPCs can compensate for this dysfunction by directly phagocytosing OS (Shahin et al, 2023, PMID: 37786347), thereby helping to maintain a healthier subretinal environment.\nWhile we did not isolate host RPE cells for transcriptomic analysis in this study, due to our emphasis on recovering high-quality RNA from grafted hNPCs and host retinal cells across a large number of eyes, we agree that a focused evaluation of RPE-specific gene expression changes in response to hNPC transplantation would provide important mechanistic insight. We plan to pursue this in future studies using targeted approaches such as RPE-specific single-cell transcriptomics.\n3. All of the single cell transcriptomic analyses are broadly presented and speculated to have causal relationships when no such conclusions can be drawn from the studies presented.\nResponse: We thank the reviewer for this important point. Below we clarify how our manuscript frames all single‐cell and CellChat analyses as correlative and hypothesis‐generating, and we highlight our language choices and explicit “Limitations” statements that underscore the need for functional validation.\n1. Observational Nature of scRNA-seq and CellChat Analyses\n• Throughout the Results (e.g., lines: 102–113), we describe dynamic shifts in gene expression, such as upregulation of MANF, MDK, PTN, MYDGF, and their receptors in grafted hNPCs, and declines in intercellular communication strength over time. These sections present fold-changes and pathway enrichments but do not claim causation.\n• Similarly, our CellChat analyses (Fig. 7, lines: 504–555) identify changes in ligand–receptor “communication probability” and “information flow,” which we characterize as predictive network inferences rather than demonstrations of functional effect.\n2. Careful Language in Discussion\nIn the Discussion, we consistently use verbs such as suggest, indicate, and highlight the potential when referring to identified pathways (lines: 576–587), thereby framing these as hypotheses to be tested, not proven causal mechanisms. For eg.,\n“These findings suggest that enhancing trophic factor support, particularly MANF, … are critical for sustaining long-term vision preservation.”\nHere, “suggest” explicitly signals that follow-up studies are required.\n3. Explicit Limitations and Call for Functional Follow-Up\nWe dedicate a section, “Limitations of the study” (lines: 654–665) to acknowledge that our work is descriptive and that functional perturbation experiments (e.g., genetic manipulation of therapeutic targets, time-course intervention) are needed to establish causality.\nText excerpt:\n“While this study provides valuable insights into the transcriptomic remodeling of grafted hNPCs and host retinal cells over time, additional later time points and loss- or gain-of-function experiments will be required to test the precise mechanistic roles of identified trophic factors and signaling pathways.”\nIn summary, we fully acknowledge that our single-cell and CellChat data are correlative and presented as such. We have used precise, hypothesis-generating terminology throughout and explicitly state the need for functional validation in our Limitations section. We trust this clarification fully addresses the reviewer’s concern.\n4. The ICC shown suggests that the majority of transplanted cells do not integrate in the host retina (contrary to the comments in the introduction that state that they migrate extensively within the host retina). Instead, they stay mostly as a mass between the RPE and retina. No effort to discern the fate of cells within the host retina or adjacent to it is provided.\nResponse: Thank you for your insightful comments. Our findings indicate that the majority of grafted hNPCs did not integrate into the host retina but rather remained in the subretinal space, where they adopted a glial-like fate. As demonstrated in our previously published studies (Gamm et al. 2007, PMID: 17396165; Shahin et al, 2023, PMID: 37786347; Lu et al., 2023, PMID: 37743503), these cells are distributed in two forms in the subretinal space: a lump of cells near the injection site or in the form of a continuous line (2-3 layers) within the subretinal space between the RPE and photoreceptors (Gamm et al. 2007, PMID: 17396165; Shahin et al, 2023, PMID: 37786347; Lu et al., 2023, PMID: 37743503). In our revised manuscript, we have now added images (new Fig. 1i) showing the characterization and migratory distribution of hNPCs in the subretinal space.\n5. Cells were delivered only at one timepoint in the RCS rat – assuming that patients will be treated at different stages of their disease, results obtained from an injection at only one early time point significantly reduces the impact of the results.\nResponse: We appreciate the reviewer’s important observation. Indeed, most preclinical studies, including ours, have historically initiated cell transplantation at early stages of disease progression to maximize the opportunity for preservation of retinal structure and function. However, as the reviewer correctly points out, patients typically present at varying stages of retinal degeneration, and it is essential to understand how disease stage influences treatment efficacy.\nWe recognize this limitation and have explicitly stated it in the limitation of the study. Importantly, in our previous studies, we have evaluated the efficacy of different cell types, including hNPCs, when delivered at mid or late stages of retinal degeneration (Wang et al. 2011, PMID: 18579765; Shahin et al, 2023, PMID: 37786347), showing stage-dependent differences in treatment outcomes.\nThe current study was specifically designed to examine temporal molecular and cellular changes following hNPC transplantation, and thus focused on a single, early time point for delivery to ensure consistent analysis of graft–host interactions over time. Moving forward, we fully agree that it will be critical to extend this approach to include cell transplantation at multiple disease stages, in order to better reflect clinical reality and optimize therapeutic strategies for patients at different stages along the degenerative spectrum.\n6. The rats were immunosuppressed with cyclosporine A but there was no control provided for the effect of this drug alone (in the absence of cell transplantation).\nResponse: We appreciate the reviewer’s attention to the study design. However, we respectfully note that the primary objective of this study was to investigate the biological interactions between grafted hNPCs and the host retina, particularly the dynamic transcriptomic changes occurring over time. To achieve this, it was necessary to maintain immune compatibility and minimize graft rejection by administering Cyclosporine A (CsA) to all animals that received hNPCs, consistent with our previous protocols (Gamm et al. 2007, PMID: 17396165; Lu et al. 2015, PMID: 25694843).\nWhile we recognize that cyclosporine A can have biological effects on its own, we designed the study such that all animals, both transplanted and untreated controls received identical immunosuppression, allowing us to isolate the impact of hNPC transplantation. Given that our aim was to investigate dynamic transcriptomic changes and interaction between grafted hNPCs and the host retina, a CsA-only group would not have provided additional mechanistic insight into the questions we sought to address. Nevertheless, we agree that the independent effects of immunosuppression could be an interesting focus for future studies.\nReviewer #3 (Remarks to the Author):\nThis study unveils the dynamic transcriptomic changes of grafted stem cells within the host’s degenerative environment over time, which fill in the knowledge gap in regenerative medicine. The findings will have broad implications for the field of stem cell therapy for degenerative diseases such as Parkson’s disease and ALS. Furthermore the study strongly suggests that both engineering stem cells with additional trophic factor(s) such as MANF for modulating microglia and Muller glia, and improving host’s degenerative environment are essential for ensuring long-term efficacy in regenerative medicine. In summary, stem cells and trophic factors as add-on to improve host environment are needed for long-term vision protection.\nOverall, the design is sound, experimental data are interesting and conclusions are well supported.\nResponse: Thank you very much for your positive and encouraging comments. We agree that the dynamic transcriptomic insights provided by this study fill an important knowledge gap in regenerative medicine. We also believe that our findings have broad implications beyond retinal degeneration, potentially informing stem cell therapies for neurodegenerative diseases such as Parkinson’s disease and ALS. We appreciate your recognition of the study design and data, and we look forward to further advancing this field by exploring strategies to optimize both graft function and the host environment for long-term therapeutic benefit.\nI have some minor concerns.\n1.This study only covered two time points (P60 and P90), early and later time points are needed to capture the temporal dynamics of grafted hNPC and host retinal changes.\nResponse: We completely agree with the reviewer’s point. Expanding the analysis to include additional early and later time points will be critical to fully capture the temporal dynamics of grafted hNPCs and host retinal responses. Our selection of P60 and P90 was deliberate. This allowed us to focus on two key phases of retinal degeneration in the RCS rat model: P60, a time point when photoreceptor degeneration is well-established but not complete, and P90, a later stage of severe degeneration. This provided a focused snapshot of the hNPC-mediated molecular and cellular changes during the critical intermediate and late phases of disease progression.\nWe plan to incorporate such comprehensive longitudinal studies in future work to further elucidate the progression of graft–host interactions over time.\n2.Injection at P21, early time of retinal degeneration, later time points of grafting should be performed.\nResponse: Thank you for raising this important question. We agree that the timing of cell transplantation is a critical factor, as patients present at the clinic with different stages of retinal degeneration. In our previous studies, we have shown encouraging results following cell transplantation at later stages of retinal degeneration (Wang et al. 2011, PMID: 18579765; Shahin et al, 2023, PMID: 37786347). The current study was specifically designed to provide a detailed and foundational understanding of the temporal dynamics of grafted hNPCs and their interactions with the host retina. To achieve this, we performed a comprehensive time-course analysis from a single, well-defined starting point. We fully agree with the reviewer that capturing the dynamic changes of grafted stem cells and host retina at later time points will be more directly relevant to the clinical setting, and we plan to pursue this in future studies.\n3.It would be interesting to present additional study using hNPC engineered to release trophic factor MANF to see whether the therapeutic effect will be enhanced; how hNPC-MANF modulates host retinal environment.\nResponse: We appreciate the reviewer’s insightful suggestion. Our previous studies have demonstrated that hNPCs engineered to express GDNF provide enhanced vision protection (Gamm et al. 2007, PMID: 17396165; Shahin et al, 2023, PMID: 37786347). We have also successfully engineered hNPCs to express MANF, and other trophic factors, and ongoing studies in our laboratory are currently investigating how hNPC-MANF modulates the host retinal environment and whether it can further improve therapeutic outcomes. We look forward to reporting these results in the near future.\n4.The graft-associated effect on host photoreceptors, Muller glia and microglia is estimated since we cannot dissect precisely grafted-related area. Spatial transcriptomics would be ideal to address directly host retinal cells affected by graft.\nResponse: Thank you for this insightful comment. We fully acknowledge that the retinal dissections inevitably encompass both graft-adjacent and more distal host regions, limiting the ability to localize transcriptomic changes exclusively to cells in direct proximity to the graft.\nWe agree that spatial transcriptomics represents the ideal approach to directly map host retinal cells affected by hNPC grafts and to delineate the microenvironmental interactions driving these changes. While such technology was not available at our institute during the execution of this study, we view it as an essential next step and are actively pursuing its integration in follow-up work.\nImportantly, despite this limitation, our combined scRNA-seq and CellChat analyses captured consistent and biologically meaningful remodeling of Müller glia, microglia, and photoreceptors in hNPC-treated retinas. These findings provide strong evidence that grafting alters host retinal cell states and intercellular communication, establishing a valuable foundation upon which future spatially resolved studies can build to more precisely define graft–host dynamics."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of the concerns. The question regarding ERGs persists. The previous report showed that the a-wave amplitude of scotopic for RCS rats at P21 was normal (Sauve Y et al., PMID: 14599567), contrasted to what was described by the authors in the revised manuscript. The a wave was pretty evident in P51 treated animals, although absent from untreated. The reason that the authors did not observe a wave in their P60 treated animals is probably due to the inappropriate condition used for recordings. Under the condition (25 cd·s/m², 10 s interval, and up to 30 traces for the scotopic ERGs) described in the paper, rods could have been bleached and become light-adapted, leaving only cone-driven responses, but not rod responses out there. Therefore, the ERG data shown in the paper only indicate preservation of cone function. To obtain rod ERG signals, it is recommended the interval is increased to 2 min and traces reduced to one or two if 25 cd·s/m² is used.\nMinor point:\n1. The unit for light intensity is cd·s/m², rather than cd/m².", "reviewer_2": "In this revision, the authors acknowledge some of the limitations of the study – most notably that it is purely descriptive. They also added some IHC to the manuscript, confirming the identity of cell types within the graft (primarily astrocytes) and expression of a few of the transcripts identified by sc-RNAseq analysis. No other changes or additions of significance are apparent. As such, this reviewer's prior comments largely remain.\nIt remains to be seen if any of the effects of these specific donor cells on host photoreceptor preservation in the RCS rat would extend to other animal models. In this manuscript, there is a lot of theorizing about how changes in the expression of specific genes by scRNA-seq may have led to preservation of host photoreceptors. However, as previously mentioned, no experimental manipulations were performed to evaluate these theories (for example, knocking down any of the identified upregulated growth factors). As such, no underlying mechanisms have been determined, and it remains to be seen if changes in any specific genes were directly responsible for host photoreceptor preservation. As this is a purely descriptive paper and not mechanistic, I suggest removal of all of the mechanistic theorizing from the results section, and just simply describe the gene expression changes (that is, all of the speculation should go in the discussion). As it currently is written a large portion of the results section reads like a discussion section. Some examples are below:\n235-239: “Increased expression of MANF, MYDGF, and their receptors in grafted hNPCs at P90 compared to P60, suggest their autocrine neuroprotection to hNPCs, supporting their self-protection, stability, and survival in the progressive degenerative retinal environment. Moreover, increased secretory MANF and MYDGF, alongside their autocrine protection to hNPCs, may also protect photoreceptors via KDELR through paracrine action.”\n247-252: “Altogether, these findings suggest that MDK and PTN promote grafted hNPC migration and survival through their autocrine and paracrine action, while MANF and MYDGF promote grafted hNPC survival and function via inhibiting cellular oxidative stress, inflammation, and immune response as degeneration progresses. Given their critical roles in hNPC stability and neuroprotection, these trophic factors could serve as therapeutic targets to enhance the efficacy of hNPC-based therapies for retinal degenerative diseases.”\nAnd lastly, from new text incorporated into the revision:\n430-434: “Of note, Müller glia upregulate genes involved in phototransduction and light stimulus in response to retinal degeneration. However, in hNPC-treated Müller glia, downregulation of genes related to these biological processes suggests that hNPCs may modulate Müller glia reactivity, shifting their transcriptional profile away from compensatory photoreceptor-like responses and toward a supportive, neuroprotective state.”\nThis new text exemplifies the point of data overinterpretation. This is just one of many potential theories that could explain the changes in the host Muller glia gene expression profile (one should also rule out the presence of photoreceptor transcripts in Muller glia as a result of phagocytosis in the non-transplanted degenerate retina).\nLastly, throughout the manuscript, preservation of host photoreceptors is attributed to gene expression changes in the transplanted cells or changes in gene expression patterns in the host retina (as a direct result of the transplanted cells). However, no direct evidence is provided to support these theories. At the end of the day, it could very well be that any donor cell type in the RCS subretinal space (or any sort of physical separation of the RPE from the neural retina in this model) would/could lead to a preservation of host PRs. These and other plausible, nonspecific mechanistic explanations need to be included in the interpretation of the data. The inclusion of such alternative explanations is especially important in the continued absence of a sham surgery control.", "reviewer_3": "all my concerns have been satisfactorily addressed."}, "author_response": "Reviewer’s Comments:\nReviewer #1 (Remarks to the Author):\nThe authors have addressed most of the concerns. The question regarding ERGs persists. The previous report showed that the a-wave amplitude of scotopic for RCS rats at P21 was normal (Sauve Y et al., PMID: 14599567), contrasted to what was described by the authors in the revised manuscript. The a-wave was pretty evident in P51 treated animals, although absent from untreated. The reason that the authors did not observe a wave in their P60 treated animals is probably due to the inappropriate condition used for recordings. Under the condition (25 cd·s/m², 10 s interval, and up to 30 traces for the scotopic ERGs) described in the paper, rods could have been bleached and become light-adapted, leaving only cone-driven responses, but not rod responses out there. Therefore, the ERG data shown in the paper only indicate preservation of cone function. To obtain rod ERG signals, it is recommended the interval is increased to 2 min and traces reduced to one or two if 25 cd·s/m² is used.\nResponse: Thank you for these insightful comments. Indeed, preservation of the ERG a-wave following stem cell treatment has been demonstrated previously using ARPE19 (Sauvé et al. 2004, PMID: 14599567) and human neural progenitor cells (hNPCs) derived from fetal cortex (hNPCCTX) (Gamm et al. 2007, PMID: 17396165). The same donor cell type (hNPCs) was used in our current study. In these studies, a 25 cd·s/m² flash intensity was used, with up to three averaged traces per condition and 2-minute intervals between double flashes to allow nearly full rod recovery. Gamm et al. demonstrated that both the a- and b-waves were preserved at approximately P100 in RCS rats following hNPC transplantation (below).\nAs the reviewer correctly pointed out, short inter-flash intervals can induce rod bleaching and light adaptation, which likely contributed to the absence of detectable a-waves at P60 and P90 in our current study.\nTo address this clearly and transparently, we have revised the manuscript text (Lines 126-130) as:\nUnder these conditions, a-waves were not detectable at P60 and P90, due to rod bleaching and light adaptation. Our previous study demonstrated that rod function in RCS rats declines early17, however, rod-driven a-waves were detectable at P100 following hNPC transplantation when ERGs were recorded using rod-optimized conditions with longer dark-recovery intervals11.\nMinor point:\n1. The unit for light intensity is cd·s/m², rather than cd/m².\nResponse: Thank you. It has been corrected.\nReviewer #2 (Remarks to the Author):\nIn this revision, the authors acknowledge some of the limitations of the study – most notably that it is purely descriptive. They also added some IHC to the manuscript, confirming the identity of cell types within the graft (primarily astrocytes) and expression of a few of the transcripts identified by sc-RNAseq analysis. No other changes or additions of significance are apparent. As such, this reviewer's prior comments largely remain.\nResponse: We thank reviewer for their comments. In our previous revision, we performed additional experiments and provided immunohistochemical validation of key genes at their protein levels using immunofluorescence. We conducted immunohistochemical validation of the most relevant genes identified by transcriptomic analyses and added five new figures: Fig. 1I, Fig. 8, and Supplementary Figs. 2, 10, and 14.\nThese new data provide:\n• Graft cell fate validation: Immunostaining confirms that most transplanted hNPCs adopt an astroglial-like phenotype while retaining limited progenitor characteristics (Nestin+, SOX2+, GFAP+, AQP4+), consistent with scRNA-seq findings.\n• ECM validation: Protein-level confirmation of key neuroprotective and ECM-related molecules (e.g., Collagen IV, IMPG2, Tenascin-C, NCAM1), supporting the transcriptomic findings.\n• Functional and anatomical preservation: Histological evidence of photoreceptor and RPE preservation in hNPC-protected areas, supporting the functional rescue observed by ERG and visual acuity testing.\nThese data directly confirm protein-level changes in the grafted hNPCs and host retina. We believe this comprehensive validation provides strong support for our conclusions. In our previous revision, we agreed that our study is descriptive and hypothesis-generating, and we clearly stated this in the ‘Discussion and Limitations’ sections.\nIt remains to be seen if any of the effects of these specific donor cells on host photoreceptor preservation in the RCS rat would extend to other animal models.\nResponse: Thank you for your comments. Human neural progenitor cells (hNPCs) have been tested in multiple preclinical CNS and ocular models, including rd1 and USH2A mutant mice (Li et al. 2016, PMID: 27067610; Lu et al. 2010, PMID: 19959642), RGC-depleted glaucoma mice (Divya et al. 2017, PMID: 28979193), and spinal cord injury models (Martin et al. 2024, PMID: 39626671), where they consistently demonstrated neuroprotective and anti-inflammatory effects, supporting their broader therapeutic potential, and have progressed to FDA-reviewed INDs and ongoing clinical trials, including our Phase I/II trial for retinitis pigmentosa (NCT04284293).\nWe have revised the manuscript (Lines 86-89). HNPCs have been tested in multiple preclinical models, including rd1 and USH2A mutant mice14,15, RGC-depleted glaucoma mice16, and spinal cord injury models17, where they consistently demonstrated neuroprotective and anti-inflammatory effects.\nIn this manuscript, there is a lot of theorizing about how changes in the expression of specific genes by scRNA-seq may have led to preservation of host photoreceptors. However, as previously mentioned, no experimental manipulations were performed to evaluate these theories (for example, knocking down any of the identified upregulated growth factors). As such, no underlying mechanisms have been determined, and it remains to be seen if changes in any specific genes were directly responsible for host photoreceptor preservation.\nResponse: We appreciate the reviewer’s thoughtful comments regarding interpretation of gene expression data. We fully agree that mechanistic validation is important; however, the primary objective of this study was to define the dynamic donor–host interactions and molecular correlates of photoreceptor preservation following hNPC transplantation, rather than to dissect single-gene mechanisms.\nOur scRNA-seq analysis identifies patterns of trophic, inflammatory, and extracellular matrix (ECM) modulation associated with slowed degeneration, not complete rescue to a wild-type state.\nKnockdown of individual trophic factors (e.g., MANF, MYDGF) in hNPCs would not adequately test the complex, multifactorial nature of neuroprotection in retinal degeneration, which involves integrated regulation of trophic support, oxidative stress, inflammation, and ECM remodeling. Instead, a more mechanistically informative approach—now underway in our laboratory—is a gain-of-function study using hNPCs engineered to overexpress MANF to determine whether enhanced trophic support confers greater or more durable protection.\nFinally, we emphasize that our study was designed to provide a comprehensive temporal and molecular framework for how donor cells influence the degenerating retina, thereby laying the foundation for these follow-up mechanistic studies.\nAs this is a purely descriptive paper and not mechanistic, I suggest removal of all of the mechanistic theorizing from the results section, and just simply describe the gene expression changes (that is, all of the speculation should go in the discussion). As it currently is written a large portion of the results section reads like a discussion section. Some examples are below:\nResponse: We appreciate the reviewer’s suggestion. We respectfully note that our results section integrates both descriptive findings and brief interpretative statements to provide biological context and clarity for the reader. This approach is consistent with standard practice in experimental and translational studies, where interpretation helps link molecular and cellular observations to their biological significance. Nonetheless, we have reviewed the Results section carefully and moved extended mechanistic speculation to the Discussion to ensure a clear separation between data presentation and interpretation.\n235-239: “Increased expression of MANF, MYDGF, and their receptors in grafted hNPCs at P90 compared to P60, suggest their autocrine neuroprotection to hNPCs, supporting their self-protection, stability, and survival in the progressive degenerative retinal environment. Moreover, increased secretory MANF and MYDGF, alongside their autocrine protection to hNPCs, may also protect photoreceptors via KDELR through paracrine action.”\nResponse: Thank you for the instructive suggestion. We have revised the text accordingly, avoiding most of the mechanistic theorizing except some to conclude the results.\n247-252: “Altogether, these findings suggest that MDK and PTN promote grafted hNPC migration and survival through their autocrine and paracrine action, while MANF and MYDGF promote grafted hNPC survival and function via inhibiting cellular oxidative stress, inflammation, and immune response as degeneration progresses. Given their critical roles in hNPC stability and neuroprotection, these trophic factors could serve as therapeutic targets to enhance the efficacy of hNPC-based therapies for retinal degenerative diseases.”\nResponse: Thank you for the instructive suggestion. We have revised the text accordingly.\nAnd lastly, from new text incorporated into the revision:\n430-434: “Of note, Müller glia upregulate genes involved in phototransduction and light stimulus in response to retinal degeneration. However, in hNPC-treated Müller glia, downregulation of genes related to these biological processes suggests that hNPCs may modulate Müller glia reactivity, shifting their transcriptional profile away from compensatory photoreceptor-like responses and toward a supportive, neuroprotective state.”\nThis new text exemplifies the point of data overinterpretation. This is just one of many potential theories that could explain the changes in the host Muller glia gene expression profile (one should also rule out the presence of photoreceptor transcripts in Muller glia as a result of phagocytosis in the non-transplanted degenerate retina).\nResponse: Thank you for the instructive suggestion. We have revised the text accordingly in discussion (Lines 618-638).\nLastly, throughout the manuscript, preservation of host photoreceptors is attributed to gene expression changes in the transplanted cells or changes in gene expression patterns in the host retina (as a direct result of the transplanted cells). However, no direct evidence is provided to support these theories. At the end of the day, it could very well be that any donor cell type in the RCS subretinal space (or any sort of physical separation of the RPE from the neural retina in this model) would/could lead to a preservation of host PRs. These and other plausible, nonspecific mechanistic explanations need to be included in the interpretation of the data. The inclusion of such alternative explanations is especially important in the continued absence of a sham surgery control.\nResponse: We thank the reviewer for continued comments on the “sham surgery” concern. We have added sentences in the text (Lines 156-163): It is noted that sham surgery was not included in the current study, since the focus of this study was to investigate the interaction between grafted hNPCs and host retinal cells. Sham surgery would not address this goal, as it would not provide relevant mechanistic information about donor cell behavior, survival, integration, or paracrine effects. However, sham-related transient effects have been demonstrated in multiple animal models24-26, including our recent dose-ranging study, which shows vision preservation is dose-dependent, vehicle-treated, and even low cell doses have limited efficacy12.\nWe acknowledge that subretinal implantation or injection can induce local trophic responses (e.g., FGF2 upregulation) and transient, localized photoreceptor preservation (Wen et al. 1995, PMID: 7472491; Cao et al. 1997, PMID: 9268592; Pardue et al. 2005, PMID: 15671299; Lu et al. 2023, PMID: 37743503; Shahin et al. 2023, PMID: 37786347). However, the magnitude, duration, anatomical distribution, and donor-cell–dependent molecular signatures observed in our work argue that the preservation we report cannot be explained solely by a nonspecific injection/implant injury.\nThe primary focus of this study was to characterize the dynamic changes of donor hNPCs and their interactions with the host retina over time. Sham surgery would not address this goal, as it does not provide information on donor cell survival, integration, or paracrine effects. Therefore, a sham control, while critical for testing cell-specific effects, would not meet the objectives of our study.\nOur recent dose-dependent study (Lu et al., 2023, PMID: 37743503) further supports this conclusion. In RCS rats, there was no difference between untreated and vehicle-injected controls, whereas hNPC-treated groups exhibited dose-dependent preservation of vision as measured by OKR and ERG, with significantly improved outcomes at higher cell doses (60K and 400K). These findings indicate that the observed neuroprotective effects are dependent on viable donor cell presence and dose, rather than a nonspecific effect of subretinal manipulation. Note: CNS10-NPC is GMP-grade of hNPC.\nFigure 1. CNS10-NPC preserves vision in a dose–response fashion. (A and B) OKR shows CNS10-NPC protects visual acuity in a dose-dependent manner, with 60 K being the optimal dose (n = 10/treatment). Visual acuity remained unchanged over time in 60K and 400 K groups. (C and D) ERG reveals that the optimal dose offers significantly higher b-wave amplitude compared with the low dose and control groups. There is no significant difference between the 60K and 400 K groups. Data are represented as mean ± SEM. One-way ANOVA with Tukey’s test was used for multiple comparisons. **p < 0.01; *** p < 0.0001; ns: no significance.\nFigure 2. CNS10-NPC protect photoreceptors in a dose–response fashion. (A) Retinal P90 sections stained with cresyl violet show extensive photoreceptor preservation with 60 K and 400 K doses compared with other groups. Photoreceptor protection is associated with donor cell distribution (Triangles in 60 K and 400 K), with 6–8 layers of photoreceptors (Arrows) preserved, compared with 2–3 layers (Arrows) in 6 K group, some donor cells contained pigmental granules (Triangles in 60 K and 400 K). (B) Retinal sections from vehicle and 60 K treated groups were stained with Stem 121 (brown color, arrows in 60 K) and cone arrestin (dark purple, chevron showing cones in b’ and b’’, downward triangles showing cone pedicles in b’’). Cone photoreceptors from 60 K group with segments (Chevrons) and pedicles (Triangles) were observed compared with vehicle treatment. b’ and b’’ are high power images from the corresponding outlines. (C) The length of preserved ONL (more than 2 nuclei thickness) against the whole retinal length by ImageJ showed 60 K and 400 K groups had significantly greater ONL protection than other groups. Data are represented as mean ± SEM. One-way ANOVA with Tukey’s test was used for multiple comparisons. *** p < 0.001; ns: no significance. Scale bar = 50 μm.\nINL inner nuclear layer, ONL outer nuclear layer, RGC ganglion cell layer, RPE retinal pigment epithelium.\nReviewer #1 (Remarks to the Author):\nNo more questions.\nResponse: Thank you.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed some concerns including adding additional figures confirming the glial identity of most of the transplanted cells, and have provided other histology and immunostaining data. Some effort was also made in terms of tempering the language around data interpretation, although it still reads like a mechanism-focused paper even though the authors acknowledge it's a purely a descriptive study. In addition, my final comment was not addressed. The authors have not taken into account that photoreceptor preservation in this RCS model could arise from a physical separation of the RPE from the neural retina. Their response and new text in the manuscript is focused on sham surgeries, not the observed neuroprotective effect of nonspecific cell transplantation that could have a prolonged, physical mechanism of preservation, rather than a biochemical mechanism. They mention from previous work that sham surgery visual response preservation is transitory, and that there is a dose response for nNPC injection. That doesn't fully address the point, however. Yes, the sham effect is transitory, and yes there is somewhat of a dose response (noted by the authors and in other studies), but the short-term preservation response in shams could be due to the transitory physical separation of RPE from neural retina, whereas with nonspecific cell transplantation a dose response could arise because more cells = greater physical separation and more prolonged physical separation. Ultimately, many interventions lead to a similar photoreceptor preservation response in this model. While a lot of work was done in this study to describe the molecular signature of the transplanted cells and host retina, without any actual experimental manipulations, all of those interpretations are speculation only. The fact that the photoreceptor preservation response is achieved across many different donor cell types/scaffolds/implants suggests that there is a shared mechanism that may have little to do with the specific molecular signature of hNPCs. The authors should add a discussion around this point, and not just focus on sham controls.\nResponse: We respectfully disagree with the suggestion that photoreceptor preservation in the RCS rat arises from a physical separation of the RPE from the neural retina, or that an apparent dose–response effect reflects greater or more prolonged physical separation due to increased cell numbers. Retinal injury induced by surgical procedures, such as sham surgery, is well established to trigger the release of trophic factors that can transiently protect photoreceptors (24–26). In addition, removal of subretinal debris in the RCS rat has been shown to provide long-term visual protection (Lorach et al., 2018).\nImportantly, the surgical procedure not only induces trophic factor release but also facilitates clearance of toxic debris, thereby improving nutrient diffusion from the choroid and enhancing metabolic support to photoreceptors through closer functional coupling with the RPE. In contrast, excessive physical separation of photoreceptors from the RPE by injected cells has been shown to result in reduced or absent photoreceptor protection, in part due to impaired nutrient transport from the choroid (Lu et al., 2023, PMID: 37743503).\nArrows in C indicate thick layers of grafted cells, while there are limited photoreceptors; arrows in C’ indicate much thinner layers of grafted cells, while there are 4-5 layers of photoreceptors preserved.\nConsistent with this interpretation, recent comparative studies demonstrate that only hiPSC-derived RPE cells maintain therapeutic efficacy at 8 weeks post-injection, whereas hAFSCs, hBMSCs, and hDPSCs confer only short-term effects, and undifferentiated hiPSCs provide minimal visual protection in the RCS rat (Lu et al., 2023, PMID: 37743503). Together, these findings indicate that photoreceptor preservation cannot be attributed to nonspecific physical effects of cell injection, and that therapeutic efficacy is highly dependent on cell type–specific biological mechanisms rather than mechanical separation alone.\nWe have modified the text: However, sham-related transient effects have been demonstrated across multiple animal models (Cao et al., 1997, PMID: 9268592; Pardue et al., 2005, PMID: 15671299; Wen et al., 1995, PMID: 7472491), including in our recent dose-ranging study, which shows that vision preservation is dose-dependent, observed in vehicle-treated controls, and that low cell doses confer limited efficacy (Lu et al., 2023, PMID: 37743503). In addition, removal of subretinal debris at the peak of accumulation in RCS rats has been shown to provide long-term visual protection (Lorach et al., 2018, PMID: 30054542), underscoring the importance of distinguishing nonspecific surgical or debris-clearing effects from cell type–specific therapeutic mechanisms.\nReviewer #3 (Remarks to the Author):\nAll of my concerns have been satisfactorily addressed\nResponse: Thank you."}]

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