(208b) Engineered Chemokine Gradients across the Retina Improve the Integration of Human Stem Cell-Derived Retinal Neurons in Mice

Glaucoma is the leading cause of irreversible blindness worldwide, with the increasing life expectancy expected to increase its incidents further.¹ Glaucoma and other optic neuropathies are characterized by progressive loss of retinal ganglion cells (RGCs), the neurons in the retina that form the optic nerve and transfer all visual information from the eye to the brain. The mammalian retina does not possess the capacity to regenerate; thus, RGC death leads to irreversible vision loss.² While available treatments can reduce intraocular pressure to slow disease progression,³ there are currently no therapies to restore lost visual function and restore lost RGCs.

Cell transplantation strategies have been proposed to restore RGCs lost in glaucoma. However, as with other neuron transplantation studies, one of the significant barriers to successful RGC integration into the existing neural circuitry is cell migration toward their natural position in the retina, the ganglion cell layer (GCL).^4–6 Moreover, the inner limiting membrane (ILM) has recently been identified as a significant barrier to donor cell migration into the GCL.⁷

During development, RGCs avoid the ILM altogether and migrate via somal translocation (ST) from the apical to the basal surface of the retina into the GCL.⁸ ST migration is the preferred mode of early-born neurons, but RGCs can migrate through multipolar (MP) migration if ST is inhibited.⁸ MP migration does not rely on the extension and attachment of neural processes to reach their final location and is the preferred migratory mode for late-born neurons that navigate through developed tissues.^9,10 It is unknown if RGCs are capable of MP migration in mammals, and it is not clear if the same rules apply to donor RGCs in the transplant setting. Being an “approachable part of the brain,” the eye provides a unique opportunity to study donor neuron fate, migration, and integration within the central nervous system.

Methods:

Here, we present a methodology to control stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. To identify these microenvironment modifiers that can control donor neuron behavior, we describe a framework for identifying, selecting, and applying chemokines to direct cell migration in vivo within the retina.

We first analyzed the single-cell transcriptome of the developing human retina in silico to identify six receptor-ligand candidates to direct stem cell-derived migration: ADGRG1, CXCR4, DCC, FZD3, NR2F1, and ROBO2 (Fig. 1A-B). The lead candidates were then tested in functional in vitro transwell assays for their ability to drive stem cell-derived RGC recruitment (Fig. 1C-D). RGCs were differentiated from Brn3b-tdTomato hESC for this and other experiments using retinal organoid cultures.^11,12 After identifying SDF1 as the most potent chemokine for RGC recruitment, we transplanted human stem cell-derived RGCs subretinally and delivered recombinant SDF1 protein intravitreally to establish a chemokine gradient across the retina (Fig. 1E). This approach allows us to overcome the ILM barrier while directing donor RGC migration in a developmentally revenant way.

To investigate the mechanisms of chemoattraction and the modes of donor RGC migration, we applied small-molecule inhibitors to donor RGCs before transplantation for 1) the CXCR4 receptor – AMD3100, 2) somal translocation (ST) – CK666, and 3) multipolar migration (MP) – roscovitine. Retinas were stained for donor and host neurons three days post-transplantation and evaluated by tracking the position of each donor RGC in 3D reconstructions of retinal flat mounts (5 – 9 mice/group, Fig. 1F).

Results:

An average of 546 ± 260 donor RGCs were detected in each neural retina with no statistical differences between groups. Treatment with SDF1 significantly increased donor RGC integration into the GCL from 16 ± 11% to 44 ± 11% (Fig. 1G). We confirmed the direct effect of SDF1 on donor cells by inhibiting in response to SDF1 by blocking SDF1-CXCR4 binding in donor cells with AMD3100 (17 ± 5.9%).

Further, our results show that inhibiting MP migration by roscovitine limits the percentage of donor RGCs that migrate to the GCL in response to SDF1 (No inhibition: 45 ± 15%; MP inhibition: 20 ± 3.7%), whereas inhibiting ST by CK666 does not affect donor RGC migration (ST inhibition: 47 ± 12%, Fig. 1G). The inhibition of migration did not affect survival, and each migration pattern was confirmed with an in vitro time-lapse study. Inhibiting ST with CK666 resulted in a significant decrease in RGC migration speed from 5.6 ± 2.2 to 4.7 ± 2.0 µm/s, whereas inhibiting MP migration with roscovitine decreased their speed to 3.2 ± 1.5 µm/s – demonstrating hESC-derived RGCs can migrate via both modalities. Together, these results indicate that MP migration is the primary mode of migration for surviving human donor RGCs in the retina.

Conclusion:

In conclusion, our study presents a novel strategy for guiding donor RGCs integration in the retina through chemokine-directed migration. By delivering donor RGC subretinally and engineering the microenvironment with chemokines, we overcame the ILM barrier and directed donor RGC migration in a developmentally relevant way. Specifically, an exogenous SDF1 gradient improved the structural integration of human stem cell-derived neurons 2.7-fold. Furthermore, our study provides valuable insights into the mechanisms of chemoattraction and the modes of donor RGC migration, with our results suggesting that MP migration is the primary mode of migration for surviving human donor RGCs in the retina.

Altogether, our findings have significant implications for restoring lost visual function in glaucoma and other retinal diseases and highlight the potential of chemokine-directed migration as a promising approach for future research and clinical applications. In addition, the established workflow using an “in silico – in vitro – in vivo” funnel to identify microenvironment modifiers can also be adapted to control other aspects of donor and newborn neuron behavior within the central and peripheral nervous system.

References:

1. Tham, Y.-C. et al. Global Prevalence of Glaucoma and Projections of Glaucoma Burden through 2040 A Systematic Review and Meta-Analysis. Ophthalmology 121, 2081–2090 (2014).

2. Erskine, L. & Herrera, E. Connecting the Retina to the Brain. Asn Neuro 6, 1759091414562107 (2014).

3. Storgaard, L., Tran, T. L., Freiberg, J. C., Hauser, A. S. & Kolko, M. Glaucoma Clinical Research: Trends in Treatment Strategies and Drug Development. Frontiers Medicine 8, 733080 (2021).

4. Oswald, J., Kegeles, E., Minelli, T., Volchkov, P. & Baranov, P. Transplantation of miPSC/mESC-derived retinal ganglion cells into healthy and glaucomatous retinas. Mol Ther - Methods Clin Dev 21, 180–198 (2021).

5. Zhang, K. Y., Aguzzi, E. A. & Johnson, T. V. Retinal Ganglion Cell Transplantation: Approaches for Overcoming Challenges to Functional Integration. Cells 10, 1426 (2021).

6. Venugopalan, P. et al. Transplanted neurons integrate into adult retinas and respond to light. Nat Commun 7, 10472 (2016).

7. Zhang, K. Y. et al. Role of the Internal Limiting Membrane in Structural Engraftment and Topographic Spacing of Transplanted Human Stem Cell-Derived Retinal Ganglion Cells. Stem Cell Rep 16, 149–167 (2021).

8. Kay, J. N. Radial migration: Retinal neurons hold on for the ride. The Journal of cell biology 215, 147–149 (2016).

9. Hayashi, K., Kubo, K., Kitazawa, A. & Nakajima, K. Cellular dynamics of neuronal migration in the hippocampus. Front Neurosci-switz 9, 135 (2015).

10. Buchsbaum, I. Y. & Cappello, S. Neuronal migration in the CNS during development and disease: insights from in vivo and in vitro models. Development 146, dev163766 (2019).

11. Wahlin, K. J. et al. CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids. Frontiers Cell Dev Biology 9, 764725 (2021).

12. Sluch, V. M. et al. Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells. Stem Cell Transl Med 6, 1972–1986 (2017).

Figure 1. Multipolar migration and the SDF1-CXCR4 axis direct donor RGC integration in mice. (A) Feature plots showing characteristic genes describing clusters of mature (RBPMS) and immature (Pou4f2) RGCs in the developing retina and a schematic demonstrating their capacity to migrate. (B) Dot plot showing the expression of select receptors involved in neural migration with arrows indicating receptors identified as potentially critical for RGC migration during development. (C) Schematic of cell recruitment transwell assay and representative immunofluorescent 3D reconstruction of RGCs on the apical and basal membrane surfaces following chemokine treatment. (D) Quantitative analysis of RGC recruitment, defined as the ratio of RGCs on the basal surface to the total number of RGCs, in response to chemokine treatment (E) Schematic representation of our subretinal transplantation strategy. (F) Depth-coded retinal flat mount displaying the positions of donor cells following transplantation. (G) Quantification of donor cells in response to small-molecule inhibitors for the CXCR4 receptor on RGCs – AMD3100, somal translocation – CK666, and multipolar migration – roscovitine. ** p < 0.01