PMC

Neural differentiation of LiPSC lines one passage post thaw. (A–C) Morphology of LiPSC lines was changing during the differentiation from passage 0 (P0) to P3. (D–E) Immunofluorescent staining of neural stem cells (NSCs) at the end of P3 revealed that they express NSCs-specific markers (Nestin and Pax6). (F) Folw cytometry analysis at the end of P3 demonstrated high expression of Pax6 by cells acquired NSCs morphology. Scale bars, 100 µm.

All three cGMP compliant LiPSCs lines were expanded in 3D suspension culture environment while maintaining their pluripotency. (A) Photomicrograph of the human PSCs (hPSCs) in 3D suspension culture during five passages in the Biott bioreactor. (B) Viable cell density during five pasages in Biott suspension bioreactors. (C) Three lines showed different cell-fold expansion during 3D culture. (D) All three lines demonstrated a high level of pluripotency specific markers: SSEA4, Tra-1-81, Tra-1-60, and Oct4 post expansion in the Biott bioreactor. Scale bars, 100 µm.

Differentiation of human iPSCs into definitive endoderm (DE) one passage post thaw. (A) Morphology of LiPSCs-derived cells was changing during the differentiation from day 1 to day 5. The cells were highly proliferative during the differentiated period and produced dense single-layered sheets. (B) Immunofluorescent staining of day 5 cells (all three LiPSC lines) revealed that they express DE-specific markers (FoxA2 and Sox17). (C) Flow cytometry analysis at the end of day 5 demonstrated that more than 80% of LiPSC 18R-derived cells express Sox17 and more than 99.0% express FoxA2. Scale bars, 100 µm.

All three current good manufacturing procedure (cGMP)-compliant LiPSCs lines differentiated into cardiomyocytes (CMs) in two-dimensional (2D) culture system. (A) Starting with good quality cells (around 100% confluency with very low differentiated areas), three LiPSC lines started morphological changes from day 1 onward until day 14. The first beating was observed for two lines (LiPSC-18R and Er2.2) around day 6–7 within several patches. A large beating area was shown for LiPSC-18R on day 10 and 14 (red circles). (B) All of the beating areas on day 14 of differentiation were positively stained for cardiac-specific troponin (cTnT) and Nkx2.5 markers for three LiPSC lines. (C) Three LiPSC lines showed positive population expressing NKX2.5 and cTnT. LiPSC-18R showed the highest population expressing NKX2.5 and cTnT among the three. Scale bars, 100 µm.

Thaw, expansion, and characterization of human LiPSC-18R post prolonged cryopreservation. (A) The induced pluripotent stem cells (iPSCs) attached and formed typical PSC colonies one day post thaw. The cells were passaged on day 6 and formed colonies one passage post thaw. Scale bars, 100 µm. (B) iPSCs stained positively with OCT4, TRA-1-60, SSEA4, NANOG, and AP. Scale bars, 100 µm. (C) iPSCs expressing the pluripotent stem cell internal and surface markers OCT4, SSEA4, TRA-1-60, and TRA-1-81 (dark pink). Light blue indicates the isotype control. (D) The iPSCs demonstrated a normal karyotype one passage post thaw. (E) iPSCs differentiated into embryoid bodies and readily expressing the markers for early ectoderm (TUJ1, beta-tubulin), endoderm (AFP, alpha feto protein), and mesoderm (SMA, smooth muscle actin). Scale bars, 100 µm.

Distribution of telomere lengths in fresh and passaged iPSCs. (A) Bars represent the relative frequency for every particular fluorescence intensity normalized (X axis). The 20th percentile (red bars) indicates the particular length below which 20% of the telomeres were observed. The median (MTL) and average telomere length (ATL) are also indicated in the histogram. The relative frequency distribution along the X axis allowed for the analysis of telomere length variability. (B) Telomere analysis technology (TAT) results (median telomere length, 20th percentile telomere length, and % short telomeres) for both samples. (C) Comparison of percentile curves for both samples. (D) Standard curve of Ct values of protein from cells against log of protein (ng). The cycle number at the threshold (Ct value) for each sample was interpolated in the curve in order to calculate relative telomerase activity (RTA). (E) Quantitative telomeric repeat amplification protocol (Q-TRAP) results (RTA) for two samples.