Page 23 - Read Online

Berber et al. J Transl Genet Genom 2021;5:292-303 https://dx.doi.org/10.20517/jtgg.2021.35 Page 294

METHODS
hiPSC reprogramming and maintenance
The isolation of adult human dermal fibroblasts and reprogramming to hiPSC (approved by the local ethics
committee reference No. 11-101-0228) were performed according to the method in with modifications as
[31]
[32]
per the work in with minor additional modifications. Briefly, hiPSCs were reprogrammed via
polycistronic lentiviral transduction utilizing the Human STEMCCA Cre-Excisable Constitutive
Polycistronic (OKS/L-Myc) Lentivirus Reprogramming Kit (Sigma-Aldrich, St. Louis, USA) according to
the manufacturer’s instructions. hiPSCs were cultured in mTeSR™ Plus medium (Stemcell Technologies,
Cologne, Germany) on plates coated with hESC-qualified Matrigel (Corning®, New York, USA) and
cryopreserved in CryoStor® CS10 medium (Stemcell Technologies, Cologne, Germany).

Retinal organoid differentiation
Method 1
Retinal organoids were differentiated according to Wahlin et al. with minor modifications. All cells were
[15]
cultured under normoxia. On day 0, hiPSCs were treated with StemPro™ Accutase™ (Life Technologies,
California, USA) for 9 min, and 2000 cells per well were plated on a Corning® Costar® Ultra-Low
Attachment 96 Well Plate (Sigma-Aldrich, St. Louis, USA) in mTeSR™ Plus medium (Stemcell
Technologies, Cologne, Germany) with 5 µm Blebbistatin (Cayman Chemical, Michigan, USA). Cells were
weaned onto BE6.2 medium [Supplementary Tables 1 and 2]. Then, 3 µM IWR-1e (Sigma-Aldrich, St.
Louis, USA) was added to the medium until day 7, and 1% Matrigel Growth Factor Reduced Basement
Membrane Matrix (Corning®, New York, USA) was added until day 8.

On day 10, the aggregates which formed retinal domains were counted and isolated with 27G cannulas (BD,
Franklin Lakes, USA) attached to 1 mL syringes (BD, Franklin Lakes, USA). Thereafter, the retinal
organoids were cultured in separate wells of Corning™ Costar™ Ultra-Low Attachment 24 Well Plates
(Thermo Fisher Scientific, Waltham, USA) following Hallam et al. . From day 10 to day 18, 100 nM SAG
[33]
(Cayman Chemical, Michigan, USA) was added to the medium. On day 12, the medium was switched to
LTR medium [Supplementary Table 3]. After day 20, 500 nM all-trans retinoic acid (Cayman Chemical,
Michigan, USA) and, from day 29 to day 42, 10 µM DAPT (Cayman Chemical, Michigan, USA) were added
to the medium.

Method 2
Retinal organoids were differentiated according to Zhong et al. with minor modifications. On day 0,
[14]
hiPSCs were treated with Dispase (Stemcell Technologies, Cologne, Germany) for 15 min and transferred to
Corning® Ultra-Low attachment cell culture flasks (Sigma-Aldrich, St. Louis, USA) in mTeSR™ Plus medium
(Stemcell Technologies, Cologne, Germany) with 10 µm Blebbistatin (Cayman Chemical, Michigan, USA)
to facilitate aggregate formation. Over the course of three days, the aggregates were weaned onto NIM
medium [Supplementary Table 4]. A medium exchange was performed on day 6, and, on day 7, the
aggregates were plated on Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning®, New
York, USA) coated CELLSTAR® Cell Culture 6 Well Plates (Greiner Bio-One, Kremsmuenster, Austria).
Half the medium was exchanged for fresh NIM every three days. On day 16, the medium was switched to
RDM medium [Supplementary Table 5]. On day 23, the retinal domains were counted. Then, 10 mM
HEPES (Thermo Fisher Scientific, Waltham, USA) was added to the medium, and the retinal organoids
were isolated as described in Method 1 and transferred to separate wells of a Corning® Costar® Ultra-Low
Attachment 96 Well Plate (Sigma-Aldrich, St. Louis, USA) following Hallam et al. . On day 43, the
[33]
medium was switched to RC2 medium [Supplementary Table 6]. From day 63 to day 90, 1 µM all-trans
retinoic acid (Cayman Chemical, Michigan, USA) was added to the medium five times weekly. On day 91,
the medium was switched to RC1 medium [Supplementary Table 7].