Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient

Document Type : Original Article


1 Shenzhen Aier Eye Hospital, Shenzhen, China

2 Aier Eye Hospital, Jinan University, Shenzhen, China

3 Shenzhen Aier Ophthalmic Technology Institute, Shenzhen, China

4 Aier Eye Hospital Group, Changsha, China


We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE)
cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide
potential cell sources for both basic scientific research and clinical application.

Materials and Methods
In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old
female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka
factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR),
karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs
exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into
RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and
flow cytometry.

The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology
including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs
expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated
99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and
NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong
differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC-RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1,
Bestrophin, and PMEL17).

A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC
displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and
therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with
native RPE characteristics, which might provide potential regenerative treatments for AMD patients.


  1. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007; 448(7151): 313-317.
  2. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 2013; 499(7459): 481-484.
  3. Rowe RG, Daley GQ. Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet. 2019; 20(7): 377-388.
  4. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861-872.
  5. Wiegand C, Banerjee I. Recent advances in the applications of iPSC technology. Curr Opin Biotechnol. 2019; 60: 250-258.
  6. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010; 7(5): 618-630.
  7. Itakura G, Kawabata S, Ando M, Nishiyama Y, Sugai K, Ozaki M, et al. Fail-safe system against potential tumorigenicity after transplantation of iPSC derivatives. Stem Cell Reports. 2017; 8(3): 673-684.
  8. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Science. 2008; 322(5903): 945-949.
  9. Jakubosky D, D’Antonio M, Bonder MJ, Smail C, Donovan MKR, Young Greenwald WW, et al. Properties of structural variants and short tandem repeats associated with gene expression and complex traits. Nat Commun. 2020; 11(1): 2927.
  10. Zarbin M, Sugino I, Townes-Anderson E. Concise review: update on retinal pigment epithelium transplantation for age-related macular degeneration. Stem Cells Transl Med. 2019; 8(5): 466-477.
  11. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health. 2014; 2(2): e106-e116.
  12. Mettu PS, Allingham MJ, Cousins SW. Incomplete response to Anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities. Prog Retin Eye Res. 2021; 82: 100906.
  13. Cabral de Guimaraes TA, Daich Varela M, Georgiou M, Michaelides M. Treatments for dry age-related macular degeneration: therapeutic avenues, clinical trials and future directions. Br J Ophthalmol. 2022; 106(3): 297-304.
  14. Fisher CR, Ferrington DA. Perspective on AMD pathobiology: a bioenergetic crisis in the RPE. Invest Ophthalmol Vis Sci. 2018; 59(4): AMD41-AMD47.
  15. Boulton M, Dayhaw-Barker P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye (Lond). 2001; 15(Pt 3): 384-389.
  16. Simó R, Villarroel M, Corraliza L, Hernández C, Garcia-Ramírez M. The retinal pigment epithelium: something more than a constituent of the blood-retinal barrier--implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol. 2010; 2010: 190724.
  17. Buchholz DE, Hikita ST, Rowland TJ, Friedrich AM, Hinman CR, Johnson LV, et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 2009; 27(10): 2427-2434.
  18. Tucker BA, Mullins RF, Streb LM, Anfinson K, Eyestone ME, Kaalberg E, et al. Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. Elife. 2013; 2: e00824.
  19. Mandai M, Watanabe A, Kurimoto Y, Hirami Y, Morinaga C, Daimon T, et al. Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration. N Engl J Med. 2017; 376(11): 1038-1046.
  20. Smith EN, D’Antonio-Chronowska A, Greenwald WW, Borja V, Aguiar LR, Pogue R, et al. Human iPSC-derived retinal pigment epithelium: a model system for prioritizing and functionally characterizing causal variants at AMD risk loci. Stem Cell Reports. 2019; 12(6): 1342-1353.
  21. Sanchez LM, Montero-Sanchez A, Ponte-Zuñiga B, de la Cerda B, Bhattacharya SS, Diaz-Corrales FJ. Generation of a human iPS cell line (CABi003-A) from a patient with age-related macular degeneration carrying the CFH Y402H polymorphism. Stem Cell Res. 2019; 38: 101473.
  22. Brandl C, Zimmermann SJ, Milenkovic VM, Rosendahl SM, Grassmann F, Milenkovic A, et al. In-depth characterisation of retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC). Neuromolecular Med. 2014; 16(3): 551-564.
  23. D’Antonio-Chronowska A, D’Antonio M, Frazer KA. In vitro differentiation of human iPSC-derived retinal pigment epithelium cells (iPSC-RPE). Bio Protoc. 2019; 9(24): e3469.
  24. Schopperle WM, DeWolf WC. The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma. Stem Cells. 2007; 25(3): 723-730.
  25. Ruiz S, Diep D, Gore A, Panopoulos AD, Montserrat N, Plongthongkum N, et al. Identification of a specific reprogramming associated epigenetic signature in human induced pluripotent stem cells. Proc Natl Acad Sci USA. 2012; 109(40): 16196-16201.
  26. Glicksman MA. Induced pluripotent stem cells: the most versatile source for stem cell therapy. Clin Ther. 2018; 40(7): 1060-1065.
  27. Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coffey PJ, Clegg DO. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med. 2013; 2(5): 384-393.
  28. Lalu MM, McIntyre L, Pugliese C, Fergusson D, Winston BW, Marshall JC, et al. Safety of cell therapy with mesenchymal stromal cells (SafeCell): a systematic review and meta-analysis of clinical trials. PLoS One. 2012; 7(10): e47559.
  29. Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017; 16(2): 115-130.
  30. Zhang H, Su B, Jiao L, Xu ZH, Zhang CJ, Nie J, et al. Transplantation of GMP-grade human iPSC-derived retinal pigment epithelial cells in rodent model: the first pre-clinical study for safety and efficacy in China. Ann Transl Med. 2021; 9(3): 245.
  31. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci USA. 2011; 108(34): 14234-14239.
  32. Attwood SW, Edel MJ. iPS-cell technology and the problem of genetic instability-can it ever be safe for clinical use? J Clin Med. 2019; 8(3): 288.
  33. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol. 2015; 33(1): 58-63.
  34. Ye H, Wang Q. Efficient generation of non-integration and feeder-free induced pluripotent stem cells from human peripheral blood cells by sendai virus. Cell Physiol Biochem. 2018; 50(4): 1318-1331.
  35. Macarthur CC, Fontes A, Ravinder N, Kuninger D, Kaur J, Bailey M, et al. Generation of human-induced pluripotent stemcells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions. Stem Cells Int. 2012; 2012: 564612.
  36. Nakano T, Ando S, Takata N, Kawada M, Muguruma K, Sekiguchi K, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell. 2012; 10(6): 771-785.
  37. Meyer JS, Howden SE, Wallace KA, Verhoeven AD, Wright LS, Capowski EE, et al. Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells. 2011; 29(8): 1206-1218.
  38. Achberger K, Haderspeck JC, Kleger A, Liebau S. Stem cellbased retina models. Adv Drug Deliv Rev. 2019; 140: 33-50.
  39. Lowe A, Harris R, Bhansali P, Cvekl A, Liu W. Intercellular adhesion- dependent cell survival and ROCK-regulated actomyosindriven forces mediate self-formation of a retinal organoid. Stem Cell Reports. 2016; 6(5): 743-756.
  40. Capowski EE, Samimi K, Mayerl SJ, Phillips MJ, Pinilla I, Howden SE, et al. Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines. Development. 2019; 146(1): dev171686.