Figure 4.

Human foreskin fibroblast supports self-renewal of mouse embryonic stem cells through the JAK-Stat3 pathway. (A) Alkaline phosphatase (AKP) staining of C57H1.2 mouse embryonic stem cells (ESCs) cultured on human foreskin fibroblast (HFF) treated with (a) 0 μM (dimethylsulfoxide (DMSO) only or (b) 10 μM and (c) 20 μM JAK inhibitor (JAKI). (B) Quantitative analysis of AKP-positive colonies of C57H1.2 mouse ESCs cultured on HFF with 0 μM (DMSO only), 10 μM and 20 μM JAKI. AKP-positive colonies in 10 (100×) microscopic fields were counted. The number of AKP-positive ESC colonies cultured with 0 μM JAKI is set as 1.0. Scale bars: 50 μm. **P < 0.01, n = 3. (C) Western blot analysis of the levels of phosphorylated Stat3 in C57H1.2 mouse ESCs grown on HFF treated with 0 μM (DMSO only), 10 μM and 20 μM JAKI, respectively. Experiments were conducted three times and the representative result is shown. (D) Quantitative RT-PCR analysis of the relative expression level of LIF in MEF and HFF. *P < 0.05, n = 3. The mRNA level of LIF in HFF is set as 1. (E) Comparison of the concentration of secreted IL-6 by ELISA. *P < 0.05, n = 3. (F) AKP staining of C57H1.2 mouse ESC colonies cultured on MEF alone, or MEF with 20 ng/ml human IL-6 (hIL-6) or HFF alone for one, two and three passages, respectively. Scale bars: 100 μm. (G) Quantitative analysis of results in (F). AKP-positive colonies in 10 (100×) microscopic fields were counted. The number of AKP-positive colonies of C57H1.2 mouse ESCs (mESCs) on HFF is set as 1.0. **P < 0.01, n = 3.