Human induced pluripotent stem cells (iPSC) have been generated by reprogramming somatic cells using a cocktail of stem cell transcription factors but the application has been limited in transplantation therapies. The pig represents an ideal model for human clinical research, in part because of its similarity to human physiology and immunology but also because of its use in assessing side effects in long-term preclinical studies. Porcine induced pluripotent stem cells (piPSC) have been established in many studies but their differentiation pattern has not been reported. The aim of this study was to estimate the efficiency and pattern of differentiated piPSC into all 3 germ layers using embryoid body (EB) formation. Two piPSC lines (VSMUi001-A and VSMUi001-D) were induced from porcine embryonic fibroblasts by retroviral overexpression of 5 human reprogramming transcription factors (OCT4, SOX2, KLF4, c-MYC, and LIN28). For EB formation, the piPSC were harvested by treating with TrypLE™ Select (Thermo Fisher Scientific, Waltham, MA, USA) and the cells were cultured in nonadherent 96-well plates in piPSC media without growth factors. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses were evaluated with Student t-tests for comparison between the 2 cell lines. Statistical significance was set at a P-value of < 0.05. The percentages of EB formation, which were calculated as the number of wells containing EB on Day 3 of differentiation, were 95.3 ± 3.42 and 89.1 ± 5.34 (VSMUi001-A and VSMUi001-D, respectively). However, there was no significant difference between the percentages of EB formation derived from the 2 cell lines. For EB size measurement, 20 EB per experiment were taken after incubation for 3, 7, 14, and 21 days. Both EB sizes increased over time (average diameter of 238.1 ± 6.18, 297.9 ± 4.10, 438.6 ± 13.33, and 728.8 ± 24.92 mm from VSMUi001-A, and 255.8 ± 5.12, 357.9 ± 3.94, 459.6 ± 11.88, and 439.4 ± 20.31 mm from VSMUi001-D). Moreover, both EB displayed homogeneity in size and shape (Day 3, 7), exhibited a cystic structure (Day 14), and a vesicular cavity was present (Day 21). For immunohistochemical analysis, both EB had lower levels of cleaved caspase 3, a marker of apoptotic cells, on Day 3 but higher levels of cleaved caspase 3 from Day 7 through 21. On the contrary, EB showed higher levels of Ki67, a marker of proliferating cells, on Day 3 but lower levels of Ki67 on Days 7, 14, and 21, respectively. In gene expression assessment, EB exhibited ectoderm gene (NeuroD1), mesoderm genes (TNNT2 and TNNI1), and endoderm genes (SOX17 and Endolase) at Day 7 and 21 by using RT-PCR. In conclusion, we report the successful in vitro formation of cystic EB from 2 piPSC lines, indicating that the piPSC could differentiate into 3 germ layers. This will allow researchers to unveil the roadmap of molecular cues needed for piPSC differentiation.
This research project is supported by grants from the Mahidol University, Thailand.
Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers
