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Background <p>Neurodegenerative conditions that affect the retina are currently the leading cause of incurable blindness in the developed world. Although gene and drug therapies are being developed to slow disease progression in some cases, restorative cell replacement approaches are needed for patients with significant vision impairment due to retinal degeneration. While a variety of different cell types have been evaluated in the context of retinal cell replacement, induced pluripotent stem cells (iPSCs), which can be generated and delivered as an autologous therapeutic, are in many ways the most attractive donor cell source currently available. Like embryonic stem cells, iPSCs must be differentiated into the target therapeutic cell type prior to transplantation. For instance, for patients with retinitis pigmentosa who have primary photoreceptor cell disease, photoreceptor cell derivation and enrichment are required prior to transplantation. Although other effective retinal differentiation protocols exist, they are often not fully compatible with clinical manufacturing.</p> Methods <p>Patient-derived iPSCs were generated via Sendai viral vector mediated reprogramming of dermal fibroblasts. Retinal organoids were generated using a stepwise 3D differentiation protocol testing different current good manufacturing practice (cGMP) compliant reagents and oxygen tension in a cGMP compliant Biospherix cell culture isolator. Organoids were dissociated with papain and photoreceptor precursor cells were transplanted into immune suppressed <i>Pde6b</i>-null rats. Human donor cell survival, cellular identity, and synaptic integration were assessed at 3- and 30-days post-injection.</p> Results <p>We developed of a xeno-free 3D retinal differentiation protocol based on the most robust adherent/non-adherent 3D differentiation strategies published to date. In addition, we demonstrate that while iPSC reprogramming efficiency is enhanced under reduced oxygen tension (i.e., 5%), efficient embryoid body and subsequent retinal organoid production require standard oxygen levels (i.e., 20%). Finally, we show that photoreceptor precursor cells obtained from 3D retinal organoids derived using the developed protocol under cGMP survive in the subretinal space of dystrophic <i>Pde6b</i>-null rats for 30 days post-transplantation and form new synaptic connections with host bipolar neurons. Importantly, synaptic connectivity between transplanted photoreceptor cells and host bipolar neurons appeared to have a positive trophic effect.</p> Conclusions <p>In this study, we report development of a xeno-free, cGMP compliant iPSC-3D retinal differentiation protocol for production of transplantable photoreceptor precursor cells.</p>

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Production of clinical grade patient iPSC-derived 3D retinal organoids containing transplantable photoreceptor cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月17日

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  • Laura R. Bohrer,
  • Luke A. Wiley,
  • Allison T. Wright,
  • Bradley Hittle,
  • Mallory J. Lang,
  • Louisa M. Affatigato,
  • Kimerly A. Powell,
  • Lorena M. Haefeli,
  • Ian C. Han,
  • Robert F. Mullins,
  • Edwin M. Stone,
  • Budd A. Tucker

摘要

Background

Neurodegenerative conditions that affect the retina are currently the leading cause of incurable blindness in the developed world. Although gene and drug therapies are being developed to slow disease progression in some cases, restorative cell replacement approaches are needed for patients with significant vision impairment due to retinal degeneration. While a variety of different cell types have been evaluated in the context of retinal cell replacement, induced pluripotent stem cells (iPSCs), which can be generated and delivered as an autologous therapeutic, are in many ways the most attractive donor cell source currently available. Like embryonic stem cells, iPSCs must be differentiated into the target therapeutic cell type prior to transplantation. For instance, for patients with retinitis pigmentosa who have primary photoreceptor cell disease, photoreceptor cell derivation and enrichment are required prior to transplantation. Although other effective retinal differentiation protocols exist, they are often not fully compatible with clinical manufacturing.

Methods

Patient-derived iPSCs were generated via Sendai viral vector mediated reprogramming of dermal fibroblasts. Retinal organoids were generated using a stepwise 3D differentiation protocol testing different current good manufacturing practice (cGMP) compliant reagents and oxygen tension in a cGMP compliant Biospherix cell culture isolator. Organoids were dissociated with papain and photoreceptor precursor cells were transplanted into immune suppressed Pde6b-null rats. Human donor cell survival, cellular identity, and synaptic integration were assessed at 3- and 30-days post-injection.

Results

We developed of a xeno-free 3D retinal differentiation protocol based on the most robust adherent/non-adherent 3D differentiation strategies published to date. In addition, we demonstrate that while iPSC reprogramming efficiency is enhanced under reduced oxygen tension (i.e., 5%), efficient embryoid body and subsequent retinal organoid production require standard oxygen levels (i.e., 20%). Finally, we show that photoreceptor precursor cells obtained from 3D retinal organoids derived using the developed protocol under cGMP survive in the subretinal space of dystrophic Pde6b-null rats for 30 days post-transplantation and form new synaptic connections with host bipolar neurons. Importantly, synaptic connectivity between transplanted photoreceptor cells and host bipolar neurons appeared to have a positive trophic effect.

Conclusions

In this study, we report development of a xeno-free, cGMP compliant iPSC-3D retinal differentiation protocol for production of transplantable photoreceptor precursor cells.