Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation. Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation

Intercellular cytoplasmic material transfer (MT) occurs between transplanted and developing photoreceptors and ambiguates cell origin identification in developmental, transdifferentiation, and transplantation experiments. Whether MT is a photoreceptor-specific phenomenon is unclear. Retinal ganglion cell (RGC) replacement, through transdifferentiation or transplantation, holds potential for restoring vision in optic neuropathies. During careful assessment for MT following human stem cell-derived RGC transplantation into mice, we identified RGC xenografts occasionally giving rise to labeling of donor-derived cytoplasmic, nuclear, and mitochondrial proteins within recipient Müller glia. Critically, nuclear organization is distinct between human and murine retinal neurons, which enables unequivocal discrimination of donor from host cells. MT was dependent on internal limiting membrane disruption, which is also required for retinal engraftment following transplantation. Our findings demonstrate that retinal MT is not unique to photoreceptors and challenge the isolated use of species-specific immunofluorescent markers for xenotransplant identification. Assessment for MT is critical when analyzing neuronal replacement interventions. Overall design: human ESC derived retinal ganglion cells were differentiated according to the small molecule-based method and purified on day 42 of differentiation. Purified RGCs then underwent single-cell RNA-seq according to the Drop-seq protocol.

细胞间胞质物质转移（intercellular cytoplasmic material transfer, MT）可发生于移植后与发育中的感光细胞之间，会导致发育、转分化及移植实验中的细胞来源鉴定出现混淆。目前尚不清楚MT是否为感光细胞特异性现象。
通过转分化或移植实现的视网膜神经节细胞（retinal ganglion cell, RGC）替代疗法，有望为视神经病变患者恢复视力。在将人干细胞来源的RGC移植至小鼠体内后，我们针对MT开展了严谨评估，期间发现RGC异种移植物偶尔会在受体米勒胶质细胞（Müller glia）内引发供体来源的胞质、细胞核及线粒体蛋白标记。
至关重要的是，人类与小鼠视网膜神经元的细胞核组织结构存在显著差异，这使得我们能够明确区分供体细胞与宿主细胞。MT的发生依赖于内界膜（internal limiting membrane）的破坏，而该过程也是移植后视网膜植入存活所必需的条件。
本研究结果表明，视网膜MT并非感光细胞所特有，同时对仅使用物种特异性免疫荧光标记物进行异种移植鉴定的做法提出了质疑。在分析神经元替代干预手段时，对MT进行评估至关重要。
整体实验设计：我们依据小分子诱导法分化得到人胚胎干细胞（human embryonic stem cell, ESC）来源的RGC，并在分化第42天完成纯化。随后，我们按照Drop-seq流程对纯化后的RGC进行单细胞RNA测序。