Optic nerve damage leads to degeneration and atrophy of retinal ganglion cells (RGCs), including various types such as glaucoma, optic neuritis, ischemic optic neuropathy, traumatic optic neuropathy, compressive optic neuropathy, and hereditary optic neuropathy. Since RGCs lack regenerative abilities, the majority of damaged cells tend to undergo apoptosis (So and Yip, 1998). In severe cases, this can result in blindness, significantly impacting the daily lives of individuals with optic nerve disorders. Therefore, finding an effective method to assist damaged RGCs, promote axon growth, and preserve or restore their function has long been a crucial focus in the field of neural regeneration.

In previous research on neuroprotective drugs for neurodegenerative diseases, although hundreds of drugs have shown protective effects in animal experiments, they often face significant challenges when progressing to clinical trials, and most fail to confirm their efficacy. Only a few drugs have received approval from the U.S. FDA for use in patients, such as Riluzole and Edaravone approved for amyotrophic lateral sclerosis (ALS) (Jaiswal, 2019; Neupane et al., 2023), and memantine and Aducanumab used for Alzheimer's disease (Beshir et al., 2022; Rahman et al., 2023). The current outcomes for the treatment of various optic neuropathies are limited, and the therapeutic effects of memantine for glaucoma are not as anticipated (Storgaard et al., 2021).

In recent years, the development of stem cell therapy has brought new hope to this field. One study found that mesenchymal stem cells could promote the survival and regeneration of RGCs in optic nerve crush rat models (da Silva-Junior et al., 2021). Injection of embryonic retinal progenitor cells (RPCs) into the vitreous of RGC-depleted mice also showed RGC differentiation and relevant gene expression (Cho et al., 2012). Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell differentiated from adult cells by specific transcription factors. iPSCs share similar morphology and regenerative abilities with embryonic stem cells and can be derived from the patient's cells, avoiding the risk of immune rejection associated with allogeneic transplantation. Previous studies have successfully differentiated human iPSCs into RGCs capable of expressing axonal transport and action potentials (Tanaka et al., 2015).

Pigment epithelium-derived factor (PEDF) is a 50-kDa glycoprotein composed of 418 amino acids. It belongs to the serine protease inhibitor (serpin) family. It was initially discovered in fetal retinal pigment epithelial cells but is also expressed in various ocular tissues, including the choroid, ciliary body, cornea, Müller cells, photoreceptors, and RGCs (Filleur et al., 2009; Behling et al., 2002; Karakousis et al., 2001). Previous studies have found that PEDF has anti-inflammatory, anti-tumor, anti-angiogenic, and neuroprotective activities (Filleur et al., 2009; Zhang et al., 2006; Belkacemi and Zhang, 2016; Bouck, 2002; Yabe et al., 2010). Regarding the protective effects of PEDF on RGCs, there were some relevant studies. PEDF is primarily secreted by Müller cells. In in vitro studies, co-culturing RGCs and Müller cells significantly reduced hypoxia-induced RGC damage. The protective effect was partially abolished when anti-PEDF antibodies were added (Unterlauft et al., 2012). The importance of the PEDF receptor in the survival of RGCs was further confirmed (Bürger et al., 2020). In the DBA/2J mouse model of glaucoma, transfection intravitreally with adeno-associated virus (AAV)-PEDF was observed to significantly reduce RGCs damage and visual function decline (Zhou et al., 2009). In another animal experiment, intravitreal injection of PEDF in rats after optic nerve crush also significantly increased the length and number of axons of RGCs (Vigneswara et al., 2013).

PEDF is a relatively large glycoprotein, and whether it can be transported to its site of action is an important issue, limiting its clinical applications. However, studies have found that PEDF 44-mer (amino acids 78–121) is a key structure in its neuroprotective ability (Yabe et al., 2010; Kawaguchi et al., 2010; Bilak et al., 2002). Previous research discovered that in a rat model of optic nerve injury, daily eye drops containing PEDF fragments were surprisingly more effective in protecting the survival of RGCs and promoting axon growth than weekly intravitreal injections (Vigneswara et al., 2015). A study on the protective effect of PEDF on photoreceptors found that the peptide P1 on the PEDF receptor has a high affinity for both 44-mer and another shorter peptide, 17-mer (amino acids 98–114) (Hernández-Pinto et al., 2019). Both 44-mer and 17-mer showed protective effects on cultured retina R28 cells, and intravitreal injection of these peptides in the rd1 mouse model of retinal degeneration reduced the death of photoreceptor cells (Hernández-Pinto et al., 2019). Smaller peptides may help address challenges related to the preparation, preservation, and permeability of the large molecular structure of PEDF.

The present study aimed to explore the effects of adding PEDF and associated peptides including 44-mer and 17-mer on the differentiation of human iPSCs into RGCs. We assessed the expression of RGC-related and neural markers and calculated the neurite length and density. The results of this study may provide a crucial new direction for the treatment of optic nerve degenerative diseases.