Figure 1.
Tumorigenicity testing (TPD50 log10) by subcutaneous transplantation of HeLa cells.
Log10TPD50 values (minimal cell doses for 50% of animals to form a tumor) for HeLa cells when transplanted subcutaneously in various immuno-deficient mouse strains (nude, SCID, NOD-SCID, NOG) with or without Matrigel as indicated. Abscissa, weeks after transplantation. Ordinate, Log10 TPD50 values, logarithmic scale.
Table 1.
Incidence of tumor formation after transplanting HeLa cells in various immunodeficient mice.
Table 2.
Tumorigenicity testing by subcutaneous transplantation of hiPSC-derived RPE into NOG mice.
Figure 2.
Characterization of hiPSC-derived RPE.
A: Phase contrast images of hiPSC-derived RPE cell lines 59-G3 RPE, K21-G18 RPE and 101–G25 RPE. B: Expression of pluripotency-related molecules POU5F1 (OCT3/4, upper panels) and RPE-related molecules BEST1 (lower panels) in lines 59-G3 RPE, K21-G18 RPE and 101–G25 RPE as detected by immunostaining with specific antibodies. Nuclei were stained with DAPI. Magnified photos of BEST1 staining are appended in left lower corners. C: Gene expression profiles of hiPSC-derived RPE cell lines 59-G3-RPE, K21-G18 RPE, 101-G25 RPE. Expression of pluripotent stem cell-related gene markers LIN28A and POU5F1, or RPE-related makers BEST1 (bestrophin), CRALBP, PAX6 and TYR (tyrosinase) in hiPSC cell lines 201B7 and 836B1, primary RPE (hRPE-1) and hiPSC-derived RPE cell lines 59-G3 RPE, K21-G18 RPE, 101-G25 RPE as determined by RT-PCR (left panel). GAPDH was used as an internal control. 50 ng RNA was used for one RT reaction. Gene expression of LIN28A (D) or POU5F1 (E) in hiPSC-derived RPE cell lines 59-G3 RPE, K21-G18 RPE, 101-G25 RPE, hiPSC 836B1 and primary RPE was quantified by qRT-PCR. UD: undetectable level (D). *, P<0.005 (E). P values for primary RPE, 101-G25 RPE, 59-G3 RPE, or K21-G18 RPE versus 836B1 are 0.000153, 0.000177, 0.000432 or 0.000489, respectively.
Figure 3.
Histological analyses of hiPSCs subcutaneously transplanted into NOG mice.
Tumor (teratoma) in NOG mouse was detected 5 weeks after transplanting 1.0×104 hiPSCs embedded with Matrigel (A, B). HE staining of sectioned hiPSC-derived teratoma consisted of three germ layers: cartilage-like tissue (mesoderm) (C), intestinal epithelium-like tissue (endoderm) (D) and neural rosette-like tissue (ectoderm) (E). Anti-Lamin A antibody (F) staining of rosette-like tissue. HE (G), Hoechst 33258 (H) and anti-Ki-67 antibody (I) staining of cartilage-like tissue.
Figure 4.
Histological analysis of hiPSC-derived RPE transplanted subcutaneously into NOG mice.
NOG mice were examined six months after transplantation of 1.0×106 hiPSC-derived RPE cells in Matrigel into subcutaneous tissue. No tumor was detected visually. Site of transplant (A), excised transplant (B), and excised Matrigel only transplant (Matrigel without RPE cells C). Transplants were sectioned and stained with HE (D) and anti-Ki67 antibody (E). Photomicrograph of unstained serial section (F), and section stained with Hoechst 33258 (G). Photomicrograph of unstained serial section (H) or stained with anti-Lamin A antibody (I). Photomicrograph of unstained serial section (J) or stained with anti-BEST1 antibody (K) and Hoechst 33258 (L) and merged (M). Ki-67 positive cells were not observed.
Figure 5.
Detection of human cells in host mouse tissue by Alu PCR.
DNA from hiPSC-derived RPEs (positive control, Lane 1), NOG mouse subcutaneous tissue just beneath the transplants (2), mouse liver (3), mouse heart (4), mouse spleen (5), mouse kidney (6) and mouse lung (7) were used as PCR templates. M: 1 kb marker (A). Alu PCR detects ≥0.1% human cells included in mouse cells determined by visual assessment of PCR products generated from various ratios of human: mouse DNA template mixtures. Percentage of human DNA in DNA mixture is shown in a respective lane number (1–8) (B). M: 1 kb marker.
Figure 6.
Histological analyses of hiPSCs or HeLa cells transplanted into the subretinal space of nude rats.
Eye balls were excised from a nude rat 7 weeks after subretinal transplantation of hiPSC. Non-transplanted right eye ball (NT) and left eye ball transplanted with 1×104 hiPSCs (hiPSC) (A). HE staining of cross section of NT eye ball (B) and hiPSC-transplanted eye ball (C). HE staining of hiPSC-derived teratoma with three germ layers: cartilage-like tissue (mesoderm, D), intestinal epithelium-like tissue (endoderm, E) and neuron-like tissue (ectoderm, F) in hiPSC-transplanted eye ball. (G – O) Eye balls were excised from a nude rat 5 weeks after subretinal transplantation of HeLa cells. Non-transplanted right eye ball (NT) and left eye ball transplanted with 1×105 HeLa cells (HeLa) (G). HE staining of cross section of HeLa cell-transplanted eye ball (H) and HeLa-derived tumor tissue (I). Anti-Ki-67-antibody (J), Hoechst 33258 (K) and HE staining (L) of serial sections of hiPSC-derived teratoma. Anti-Lamin A antibody (M), Hoechst 33258 (N) staining and microscopic image (O) of serial cross sections containing a boundary of hiPSC-derived teratoma and host rat tissue. Anti-Lamin A antibody specifically recognizes human cells in rat tissue.
Table 3.
Tumorigenicity testing by subretinal transplantation of hiPSC-derived RPE in nude rats.
Figure 7.
Histological analysis of hiPSC-derived RPE sheets transplanted into the subretinal space of nude rats.
(A) Eye balls of nude rat 9 months after subretinal transplantation of 0.8–1.4×104 hiPSC-derived RPE (in a 1 mm×1 mm cell sheet). Left eye ball transplanted with hiPSC-derived RPE (RPE) and non-transplanted right eye ball (NT). (B) HE staining of cross section of left eye ball following transplantation of hiPSC-derived RPE. (C) HE staining of section of eye ball following transplantation of hiPSC-derived RPE, high magnification. HE- (D), anti-Ki67 antibody- (E), and Hoechst 33258-staining (F) and merged (G) images of serial sections of nude rat retina after transplantation of hiPSC-derived RPE. Anti-Lamin A antibody (H), Hoechst 33258 staining (I), merged (J) and micrographic image (K) of serial sections of nude rat retina after transplantation of hiPSC-derived RPE. Anti-BEST1 antibody (L), Hoechst 33258 (M), merged (N) staining and micrographic image (O) of serial sections of nude rat retina following transplantation of hiPSC-derived RPE.