IGFBPs mediate IGF‐1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids

Development of the retina is regulated by growth factors, such as insulin‐like growth factors 1 and 2 (IGF‐1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF‐1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF‐1, by making it available or sequestering IGF‐1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage‐specific manner to mediate IGF‐1 functions. Our data show that IGF‐1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF‐1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF‐1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI‐31772) and IGF‐1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid‐stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF‐1's role in retinal lamination and photoreceptor development in a stage‐specific manner.

IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs 2 (IGF-1 and IGF-2) are multifunctional polypeptides, which are essential for regulating cell growth, development, and differentiation. 3,4 IGFs have been found in the retina of several species, such as Drosophila, chick, mice, and humans 1 including the human foetus older than 3 months. 5 In the eye, IGF-1 and its receptors are expressed in the sensory retina and retinal pigment epithelium during development and adulthood. 6 Work performed by our group has demonstrated an important role for the IGF-1 signaling pathway in the generation of laminated retinal tissue from human embryonic stem cells (hESCs). 7 Most of the IGF-1/2 is bound to one of the seven IGF-binding proteins (IGFBPs), which are structurally similar proteins shown to bind IGF-1 with an equal or greater affinity than IGF-1 receptor, acting as fine tuners of IGF-1 signaling globally and locally. 3,8,9 Binding of IGF-1 to IGFBPs increases its half-life in the circulation and blocks its potential binding to the insulin receptor. IGFBP-3 is the most abundant circulating IGFBP, carrying 75% to 80% IGF-1 and IGF-2 found in the serum. 8,10 The remaining 20% to 25% of IGFs are bound to one of the other IGFBPs. 8,10 The main function of IGFBPs is to bind IGFs and either transport them to their receptors, potentiating IGFs actions, or to sequester IGFs from their receptors (due to the greater affinity for IGF than its receptor) inhibiting their action. 3,8,10,11 It has been shown that proteolysis and fragmentation of IGFBPs or the attachment of the IGFBPs to cell surface proteoglycans and/or extracellular matrix components reduces their affinity to IGFs, resulting in an increase in the amount of IGFs available for IGF-1R binding, which enhances IGF-/2 activity. 8,11 However, several IGFBPs have cellular IGF-independent actions, which are mediated via binding to cell surface proteins or nuclear localization. 8,[11][12][13][14] The mechanisms underlying these IGF-independent actions are not fully understood, but may involve an interaction between the IGFBPs and different signaling pathways either at the cell surface or within the cell. 13,14 In neonatal mice, IGF-1 is expressed in all the retinal layers and neovascular vessels extending into the vitreous (tufts); however, IGF-1R is expressed predominantly in the photoreceptors. IGFBP2, IGFBP3, and IGFBP5 are expressed in all three retinal layers and expression of some of the IGFBPs (3, 5, and 7) shows large increases in the neovascular tufts. 15  In this article, we have undertaken a systematic expression study of all IGFBPs in the adult human retina, while assessing in parallel which of the IGFBPs is activated or inhibited in response to IGF-1 addition in pluripotent stem cell-derived retinal organoids. Importantly, we have used an IGBP inhibitor, NBI-31772 (1-(3,4-dihydroxybenzoyl)-3-hydroxycarbonyl-6, 7-dihydroxyisoquinoline) that binds to the IGFBPs and displaces biologically active IGF-1 18,19 to assess the IGFBPs mediated IGF-1 function in generation of hESC-derived retinal organoids.

| MATERIALS AND METHODS
A detailed description of all experimental procedures is presented in the Supporting Information.

| RESULTS AND DISCUSSION
To gain insights into the expression of the IGFBPs, we took advantage of a recent RNA-Seq analysis performed by our group 2 ( Figure S1A F I G U R E 1 Characterization of the IGFBP inhibitor's impact during retinal organoids formation. A, Diagram of the experimental design: hESCs with CRX-GFP were cultured in three different conditions: with IGF-1 individually or in combination with NBI-31772, and without IGF-1. These retinal organoids were harvested and collected at four time points (days 37, 60, 90, and 120) of differentiation. B, Examples of the resulting culture morphology under bright-field microscopy. Morphology of human retinal organoids differentiating with IGF-1 supplement (a-d), without IGF-1 (e-h) and with the addition of IGF-1 and NBI-31772 (i-l) at day 37 (a, e, i) day 60 (b, f, j), day 90 (c, g, k) and day 120 (d, h, l) of differentiation. Scale bars = 100 μm. C, Quantitative assessment of the total EBs area (μm 2 ) at day 37, day 60, day 90, and day 120 cultured in three different conditions: with or without IGF-1 and IGF-1 in combination with NBI-31772. Data are shown as mean ± SEM, n = 7 to 10 organoids per group. D,E, Effects of IGF-1 and combined IGF-1 and NBI-31722 addition on the percentage of embryoid bodies (EBs) displaying visible retinal structures. Quantitative assessment of retinal structures resembling the optic vesicle (OV) at early development (D) at day 37 and 60 and past optic vesicle at late development (E) at day 90 and 120 derived from hESCs cultured with or without IGF-1 and IGF-1 in combination with NBI-31772. All values are stated as the percentage of the total population of EBs with OVs. CRX, cone rod homeobox; GFP, green fluorescent protein; hESCs, human embryonic stem cells; IGF-1, insulin-like growth factor 1; IGFBP, insulin growth factor binding protein and IGFBP6 continued to be expressed at low levels ( Figure S2A). A recent study has shown a comparable rate of development between pluripotent stem cell-derived retinal organoids and human fetal retina. 20 We thus performed quantitative Real Time-PCR analysis of all IGFBPs in four hESC-derived retinal samples generated in the absence of IGF-1 and harvested at four differentiation time points (day 37, 60, 90, and 120), which fall within the human retinal development shown in Figure S2A. This analysis indicated that up to day 60 of differentiation, IGFBP2 and IGFBP5 were the highest and IGFBP1 and IGFBP6 were among the lowest of the IGFBPs expressed respectively within the retinal organoids ( Figure S2B). From day 90 to 120 of differentiation, IGFBP2, IGFBP5, and IGFBP7 were the highest and IGFBP1, IGBP3, and IGFBP6 were among the lowest of the IGFBPs expressed respectively within the retinal organoids ( Figure S2B).  Figure 3B). We think that this effect may be due to downregulation of IGFBP-7 in this group ( Figure S3). IGFBP-7 has a 100-fold lower affinity for IGF-1 and IGF-2, but high affinity for insulin (500-fold more elevated compared to other IGFBPs 25 ). This IGFBP7-IGF-1/2 independent activity may be the underlying reason for the increased number of Prox-1 + horizontal cells in the NBI-31772 + IGF-1 group; however, this needs further investigation.
At day 90 of differentiation, Ki-67 + cells were present as the neural retina was still developing ( Figure 3A[c,f,i]); however, no difference was observed between all the three groups ( Figure 3B). A few bipolar cells detected by the marker PKC-α (Figure S4A,D,G) were detected, forming a numerically small proportion of the retina's interneurons as confirmed by the quantification analysis ( Figure 3B). Müller glia cells were observed to span the whole retinal structure similar to the adult retina organization ( Figure S4C,F,I).
By day 120, IGF-1 and NBI-31772 + IGF-1 treated organoids were larger and with better lamination and stratification compared to retinal organoids grown without IGF-1 ( Figures 1C and 4A), which lost their stratification, corroborating the importance of IGF-1 for retinal organoid development. The highest number of photoreceptors was observed in the IGF-1 treated group and the lowest number was observed in the no IGF-1 group ( Figure 4B). Mature photoreceptors are observed in retinal organoids from day 150. 26,27 In accordance with this, only a few rods with typical morphology were present in the IGF-1 and NBI-31722 + IGF-1 groups ( Figure 4A[e,e 0 ,q,q 0 ]); however, the low numbers due to early differentiation stage precluded any firm statistical conclusions. Similarly Cone opsins (OPN1SW and OPN1LW/MW) were present only in IGF-1 group (Figure 4A[f,f 0 ,g,g 0 ]). Although the organoids grown without IGF-1 contained Müller glia cells, they were much shorter and situated mainly at the outer edge of the organoid and not dispersed throughout the structure ( Figure S5H).
In summary, our data show an important role for IGFBPs and IGF-1 in various stages of retinal organoids generation and maturation. The effects are specific to each stage of development and most likely mediated by different member of the IGFBP family. It is interesting to note that another widely used differentiation method, which involves embryoid body (EB) formation, followed by plating in 2D culture conditions, dislodging and culturing the organoids under 3D F I G U R E 4 The impact of IGFBP inhibition in the later stages of retinal organoid differentiation: day 120. A, Immunohistochemical analysis of human retinal organoids at day 120 of differentiation in three different culture conditions: with IGF-1 (a-g 0 ), without IGF-1 (k-l), or with IGF-1 and NBI-31772 (m-q 0 ). Antibodies used against HuC/D (red), RCVRN (red), Bassoon and Ribeye (red, white arrows), and mature photoreceptors: Rhodopsin (red) (e, e 0 , q, q 0 ), OPN1SW (red) (f, f 0 ), and OPN1LW MW (red) (g, g 0 ). Hoechst nuclear stain (blue) and endogenous GFP (green) also shown. B, Immunohistochemistry quantification analysis. Data are shown as mean ± SEM (n = 5). Scale bars = 50 μm (a, b, e, f, g, h, i, l, m, n, and q) and 10 μm (c, d, e 0 , f 0 , g 0 , j, k, o, p, and q 0 ). CRX, cone rod homeobox; GFP, green fluorescent protein; IGF-1, insulin-like growth factor 1; IGFBP, insulin growth factor binding protein; RCVRN, Recoverin and Rho, Rhodopsin conditions does not use IGF-1 supplementation in the media, but employs instead BMP-4 at the very early stages of differentiation, which raises interesting questions on the necessity of IGF-1 for generation of retinal organoids from pluripotent stem cells. [28][29][30] We cannot exclude that organoids generated with such method are not producing IGF-1 endogenously to sufficient levels. Neither can we comment on downstream pathways or target genes being activated by IGF-1 or BMP-4 unless both methods are studied in parallel in the same lab and with the same cell lines.