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. 2022 Aug 17;1(4):pgac162.
doi: 10.1093/pnasnexus/pgac162. eCollection 2022 Sep.

Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory

Yan-Ping Li  1 , Ya-Ting Wang  1 , Wen Wang  1 , Xiao Zhang  1 , Ren-Juan Shen  1 , Kangxin Jin  1 , Li-Wen Jin  2 , Zi-Bing Jin  1
Affiliations

Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory

Yan-Ping Li et al. PNAS Nexus. .

Abstract

Retinoblastoma (Rb) is a type of malignant tumor due to abnormal retinogenesis with biallelic mutations of the RB1 gene. Its pathogenesis has been proposed as a "two-mutation hypothesis" by Knudson since 1971; however, there remain some debates on disease onset sufficiency of the biallelic RB1 mutations. To obtain straightforward evidence for this hypothesis, we investigated whether two-hit mutations of the RB1 gene drive tumorigenesis in patient-induced pluripotent stem cell (hiPSC)-derived human retinal organoids (hROs) and whether single allelic mutation hiPSC-derived hROs exhibit molecular and cellular defects. We generated hiPSCs with a heterozygous germline mutation (RB1m1/ wt ) from a Rb patient. A second-allele RB1 gene mutation was knocked in to produce compound heterozygous mutations (RB1m1/m2 ) in the hiPSCs. These two hiPSC lines were independently developed into hROs through a stepwise differentiation. The hiPSC-RB1m1/m2 derived organoids demonstrated tumorigenesis in dishes, consistent with Rb profiles in spatiotemporal transcriptomes, in which developmentally photoreceptor fate-determining markers, CRX and OTX2, were highly expressed in hiPSC-RB1m1/m2 derived hROs. Additionally, ARR3+ maturing cone precursors were co-labeled with proliferative markers Ki67 or PCNA, in agreement with the consensus that human Rb is originated from maturing cone precursors. Finally, we demonstrated that retinal cells of hROs with monoallelic RB1 mutation were abnormal in molecular aspects due to its haploinsufficiency. In conclusion, this study provides straightforward supporting evidence in a way of reverse genetics for "two-hit hypothesis" in the Rb tumorigenesis and opens new avenues for development of early intervention and treatment of Rb.

Keywords: RB1; iPS cells; retinal organoids; retinoblastoma; two-mutation hypothesis.

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Figures

Fig. 1.
Fig. 1.
Generation, modification, and characterization of Rb patient-derived iPSC lines. (A) Scheme of hiPSC establishment from urine sample of the Rb patient. (B) Fundus appearances of the bilateral Rb after therapy. OD, right eye. OS, left eye. (C) Sanger sequencing of the RB1 gene in the Rb patient. A heterozygous 1-bp deletion in exon 7 results in a frameshift mutation (c.623delT, p.M208fs). (D) Representative bright filed image of urine cells. Scale bars, 200 µm. (E) Representative bright filed image of a hiPSC colony. Scale bars, 400 µm. (F) Sanger sequencing of cDNA sequence of targeted loci in iPSC-RB1m1/m2. (G) Representative bright filed images of iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2colonies. Scale bars, 400 µm. (H) Western blot analysis for RB1 protein expression in iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2 cells. (I) Relative percentage of cells within each cell cycle phase of iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2 lines. n = 3 for all samples.
Fig. 2.
Fig. 2.
Characterization of hROs derived from iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2. (A) Scheme of hROs differentiation derived from iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2. DD, differentiation day. (B) Retinal cell genesis at different timepoints in hROs. Each curve represented the FPKM of representative gene for each retinal cell over the time in hROs. AIPL1 for rod; ARR3 for cone; GRM6 for bipolar cell (BC); POU4F2 for RGC; and STX1A for horizontal cell and amacrine cell (HC and AC). n = 3. (C) Immunostaining of retinal progenitor cell markers RX and PAX6 in three groups of hROs at DD30. The top panel shows representative images of each group. Scale bars in top panel, 200 µm. The boxed regions in top panel were magnified in the middle and bottom panels accordingly. Scale bars in middle and bottom panels, 50 µm. Cell nucleus was counterstained with DAPI (blue). (D) Representative images of hROs (or hRBOs) at different timepoints. Right schematic diagrams showed the process of tumorigenesis in RB1m1/m2 hRBOs. Red dashed lines delineated the borders of tumors in RB1m1/m2 hRBOs. Scale bars, 400 µm. (E) The 2D virtual slice of micro-CT. The boxed regions were magnified in the right. (F) The micro-CT images of hROs and hRBOs at DD55. (i) The reconstructed 3D image of the RB1m1/wt hRO scanned with 1.5 µm vortex resolution. The bright-filed image of RB1m1/wt hRO was shown in the bottom left corner, Scale bars, 400 µm. (ii) The reconstructed 3D image of the RB1m1/m2 hRBO scanned with 1.2 µm vortex resolution. The bright-filed image of RB1m1/m2 hRBO was shown in the bottom left corner, Scale bars, 400 µm. White dashed lines in (i) and (ii) were used to enclose the regions for porosity calculation. (iii to vi) The 3D rendering images were obtained from Dragonfly software's thickness analysis of the organoid's surface and internal pores (indicated in blue).
Fig. 3.
Fig. 3.
Molecular profiling and characterization of hROs derived from iPSC-RB1wt/wt, -RB1m1/wt, and -RB1m1/m2. (A) Heatmap showed the DEGs between RB1m1/wt and RB1m1/m2 hROs at various differentiation timepoints. Hierarchical clustering of the significantly expressed genes identified two main gene clusters. Cluster 1: gene expression levels of 419 upregulated genes in RB1m1/wt hROs. Cluster 2: gene expression levels of 106 upregulated genes in RB1m1/m2 hRBOs. Light blue, RB1m1/wt hROs, n = 3. Red, RB1m1/m2 hRBOs, n = 3. DD, differentiation day. Control, RB1wt/wt hROs, n = 3. Y79, human Rb cell line, n = 4. (B) Significant gene sets from the Metascape enriched in clusters 1 and 2. (C) Immunostaining of cleaved caspase 3 in hROs and hRBOs. Representative images were shown for each group at DD75 and DD90. (D) Representative immunostaining images of Ki67 were shown for each group at DD75 and DD90. (E) Immunostaining of SYK, DEK, p16Ink4a and Ki67 in hROs and hRBOs at DD90. (i to iii) Representative images of the adjacent tissues in hRBOs; (iv to vi) Representative images of the solid tumors in hRBOs. White arrows pointed to cells with high cytoplasmic p16Ink4a in hRBOs. Scale bars, 50 µm. Cell nucleus was counterstained with DAPI (blue in C to E).
Fig. 4.
Fig. 4.
Rb tumor and adjacent tissues highly express cone and cone precursor markers. (A) Top image illustrates the locations of tumors and adjacent tissues. (B and C) Immunostaining of Ki67 and maturing cone marker ARR3 (in B) or cone precursor marker RXRγ (in C) in RB1m1/wt hROs and RB1m1/m2 hRBOs at DD90 and 105. Representative images were shown for the (i) RB1m1/wt hROs at DD90, (ii) adjacent tissue in RB1m1/m2hRBOs at DD90, (iii) tumor in RB1m1/m2 hRBOs at DD90, (iv) RB1m1/wt hROs at DD105, and (v) RB1m1/m2 hRBOs at DD105. White arrows pointed to cells with co-expression of Ki67 with ARR3 or RXRγ. Dashed circles enclosed the Flexner–Wintersteiner rosette-like structures. Scale bars, 50 µm. (D and E) Immunostaining of Ki67 and photoreceptor transcription factors CRX (in D) or OTX2 (in E) in RB1m1/wt hROs and RB1m1/m2 hRBOs at DD90 and DD105. Representative images were shown for the (i) RB1m1/wt hROs at DD90, (ii) adjacent tissue in RB1m1/m2hRBOs at DD90, (iii) tumor in RB1m1/m2hRBOs at DD90, (iv) RB1m1/wt hROs at DD105, (v) adjacent tissue in RB1m1/m2 hRBOs at DD105, and (vi) tumor in RB1m1/m2 hRBOs at DD105. Cell nucleus was counterstained with DAPI (blue). Scale bars, 50 µm.
Fig. 5.
Fig. 5.
RB1 heterozygous retinal cells in hROs have developmental defects and are inherently different from wildtype ones. (A) Representative images of immunostaining of photoreceptor markers CRX and RCVRN in RB1m1/m2 hRBOs, RB1m1/wt hROs, and RB1wt/wt hROs at DD75. The white-boxed region and yellow-boxed region in (i) were magnified in (ii) or (iii), respectively, which represented the tumor or the adjacent tissue in hRBOs, respectively. The white-boxed region in (iv) was magnified in (v), showing the region with typical retinal dysplasia in RB1m1/wt hROs but not in RB1wt/wt hROs. Cell nucleus was counterstained with DAPI (blue). Scale bars, 100 µm (i and iv), and 50 µm (ii, iii, v, and vi). (B) Representative images of immunostaining of photoreceptor markers CRX and RCVRN in RB1m1/m2 hRBOs, RB1m1/wt hROs, and RB1wt/wt hROs at DD90. Cell nucleus was counterstained with DAPI (blue). Scale bars, 50 µm. (C) Heatmap showed the DEGs in hROs between RB1m1/wt and RB1wt/wt at DD90. n = 3. (D) Enrichment of the significant gene sets in hROs between RB1m1/wt and RB1wt/wt by Metascape. Orange and light blue colors represented enriched gene sets in RB1wt/wt hROs and RB1m1/wt hROs, respectively.
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References

    1. Dimaras H, et al. 2015. Retinoblastoma. Nat Rev Dis Primers. 1:15021. - PMC - PubMed
    1. Knudson AG. 1971. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci. 68:820–823. - PMC - PubMed
    1. Knudson AG. 2001; Two genetic hits (more or less) to cancer. Nat Rev Cancer. 1:157–162. - PubMed
    1. Comings DE. 1973. A general theory of carcinogenesis. Proc Natl Acad Sci. 70:3324–3328. - PMC - PubMed
    1. Cavenee WK, et al. 1985; Genetic origin of mutations predisposing to retinoblastoma. Science. 228:501–503. - PubMed