Table 1.
Summary of patient information.
Fig 1.
(A) Human cumulus-oocyte-complex (COC). (B) COCs treated with hyaluronidase for 1 minute. (C) Denudated oocyte. (D) Completely dissociated CCs. (E) Morphology of attached human CCs under phase contrast microscope. (F) Cultured CCs of spindle-like shape after passage. (G) Primarily formed iPSC colony after 25 days of induction. (H) hc-iPSCs after manually pick-up. Scale bar: 100 μm.
Fig 2.
In vitro characterizations of hc-iPSCs.
(A) The growth curve of hc-iPSCs (hc-iPSC-1, 2, 5, 6), and hESC H9 (3 replicates per line). (B) Population doubling time of hc-iPSC lines and hESC H9. (C) Alkaline phosphatase activity was detected in hc-iPSC-1, -2, -5 and -6 (Blue color, scale bar = 1 mm). Immunofluorescent with antibodies against OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81 were also detected in hc-iPSC lines. Scale bar = 100μm. (D) Flow cytometry confirmed the percentage of SSEA4 and TRA-1-60 positive cells in hc-iPSC lines (red color), as the numbers on graphs indicated, respectively. Isotype controls were shown in white color. (E) Semiquantitative PCR for expression of pluripotent genes. Exogeneous NANOG and POU5F1 (Exo-NANOG, Exo-POU5F1) genes were undetectable and endogeneous genes (Endo-NANOG, Endo-POU5F1) were expressed in hc-iPSC lines and hESC H9. cDNA of cumulus cells (CCs) at passage 3 was from mixture of patients. (+): template plasmids which containing POU5F1 or NANOG. An unexpected band was amplified by endogeneous NANOG primer in NANOG plasmid with incorrect size of PCR product, which may due to sequence similarity. (-): negative control without template. GAPDH was used for internal control. (F) X chromosome inactivating transcripts XIST were detected in mixture of CCs at passage 0 (P0 CCs), and passage 3 (P3 CCs), but nearly undetectable in hc-iPSCs. (G) hc-iPSCs showed normal female karyotypes without chromosome translocation, deletion or replication.
Fig 3.
In vitro differentiation and teratoma assay.
(A) The germlayer specific markers SOX17 (endoderm, red color), TUJ1 (ectoderm, green color), and the BRYCHURY (mesoderm, red color) were detected after in vitro differentiation in hc-iPSCs. DNA was stained by DAPI (blue). (B) Expression of germlayer specific genes (SOX17 for endoderm, PAX6 for ectoderm, and HAND1 for mesoderm) were all upregulated in day 10 EBs compared with hc-iPSCs. (C) Ciliated epithelium (endoderm), neuronal-like (ectoderm) and cartilage (mesoderm) cells were present in hc-iPSC derived teratomas (stained with H&E). Arrows indicate corresponding cell types.
Fig 4.
Immunofluorescent staining of (A) BLIMP1 (green color) and (B) OCT4 (green color) in 4-day-old EBs cultured with PGC induction medium on hc-iPSC-1, -2, -5 and -6 (third to fifth column), PBMC derived iPSC0102 and iPSC0207 (right two column). H9 hESCs (left column) adapted in 4i medium was also detected for BLIMP1 and OCT4 antibodies. Noted that there was no BLIMP1 signals detected in undifferentiated H9 in 4i condition. All samples were doubly stained with SOX17 antibody (red color) sequentially. DAPI was used for counterstain (blue). Scale bar = 50μm. (C) Quantification of immunofluoresent staining of PGCLCs. BLIMP1/SOX17 and OCT4/SOX17 double positive cells were counted for PGCLCs and normalized with the number of DAPI. Percentage of PGCLCs per EB of all lines was presented and significance was compared with control hESC H9. ** indicates P<0.005. *** indicates P<0.001.