Figure 1.
Isolation of pAF-MSCs from the amniotic fluid of E35 porcine fetus.
(A) The E35 porcine fetus with whole amniotic membrane. The amniotic fluid was collected under sterile condition. (B) The morphology of pAF-MSCs after culturing for 3 passages in vitro. Most cells exhibited fibroblast-like shape. (C) ES cell-likes colony appeared occasionally in the primary pAF-MSCs culture, which was AP staining positive (inset). (D) The growth curve of the pAF-MSCs (at 6th passage). The doubling time was 34.6 h. (E) The cell cycle of pAF-MSCs was analyzed by FACS.
Figure 2.
Determination of specific gene markers in pAF-MSCs.
(A) The expression of cell surface antigens was examined by flow cytometry. The antibodies of CD34, CD117 and CD166 were labeled with PE, and CD45, CD44 and CD54 were labeled with FITC. (B) The mRNA expressions of CD90, CD117 and HLA-abc, but not CD45 were detectable in pAF-MSCs by semi-quantitative RT-PCR assay. The RNA samples were prepared from pAF-MSCs at 4th, 8th, 12th and 16th passages. The β-actin was used as internal control. (C) The pluripotent markers of ES cell were determined in pAF-MSCs (at 6th passage) by immunofluorescence assay. The antibodies against Oct4, Nanog, SSEA1, SSEA4, Tra-1-60 and Tra-1-81 were conducted and the positive staining showed green fluorescence (FITC). The nuclei were stained by Hoechst 33342 (blue fluorescence).
Figure 3.
The EBs and teratoma formation.
(A) The pAF-MSCs were cultured in suspension at the uncoated Petri dishes for EBs formation. After 24 h of culture EBs were formed and showed positive for alkaline phosphatase (AP) staining (inset). (B) When EBs cultured in the adherent dish for 24 h, the differentiated cells were observed. (C) The expression of three germ layers markers were detected by RT-PCR in the differentiated cells from EBs. M, DNA marker; Act, β-actin used as an internal control; FGF, Fibroblast growth factor 5 for the primitive ectoderm; AFP, Alpha fetoprotein for the endoderm; T-Bra, T-box gene brachyury for the mesoderm. (D) The pAF-MSCs (5×106 cells/injection) were subcutaneously injected into (BALB/c-Nu) the right axilla of three immunodeficient mice. One mouse injected with mES J1 cells (2×106 cells/injection) was as positive control. After six weeks post-injection, three mice injected with pAF-MSCs (on the left) were no teratomas, but mouse injected with mES J1 cells (on the right) showed the teratoma. (E–F) The TERT expression was detected by anti-TERT antibody in pig embryonic fibroblasts (E) and pAF-MSCs (F).
Figure 4.
The multi-lineage differentiation potential of pAF-MSCs in vitro.
The specific markers were detected by immunocytochemistry (ICC) and RT-PCR. (A) The pAF-MSCs differentiated into neurocytes. (1) ICC of Nestin; (2) ICC of NF-200; (3) RT-PCR assay for FGF5 and CD56. (B) The pAF-MSCs differentiated into adipocytes. (1) The lipocytes were induced for two weeks. The lipid droplets were round the nuclei (inset); (2) The cells were positive for Oil Red O staining; (3) RT-PCR assay for PPARγ and C/EBPα in adipocyte derived from pAF-MSCs. (C) The pAF-MSCs differentiated into cardiomyocytes. (1) ICC of α-actin; (2) ICC of ß-tublin; (3) RT-PCR assay for cardiomyocyte specific markers including Tbx5, Gata4, α-MHC, TNNi3, MYL and Nkx2.5. Act, β-actin was used as the internal control; M, DNA marker.
Figure 5.
The pAF-MSCs differentiated into beating cardiomyocytes in vitro.
(A) The EBs cultured in suspension with cardiac differentiation medium for 17 day expressed cardiomyocyte special markers, including α-actin, β-tublin and Nkx2.5. (B) The pAF-MSCs continually grew in cardiomyogenic medium for 17 days in the tissue culture plate to induce the differentiation into cardiomyocytes. The immunofluorescence analysis was conducted to determine α-actin, ß-tublin and Nkx2.5 expression in the uninduced pAF-MSCs (0 d) and the induced pAF-MSCs (17 d). (C) The percentage of beating cell masses induced by the different media was counted. * indicates difference at p<0.05; ** indicates significant difference at p<0.01. (D) Transmission electron microscope analysis of the beating cell masses. 1, pig heart tissue was used as the control. The arrows indicate the Z lines (×25 k); 2, the cardiac-muscle like filament from the beating cell masses derived from pAF-MSCs. The arrows indicated the glycogen pool and glycogen granules (×30 k). 3, the cardiac-muscle like structure was observed in the beating cell masses (×12 k). 4, The enlarged photo showed the cell connections (arrows) (×50 k).
Table 1.
Primer sequences for RT-PCR.