Abstract:
Previous study demonstrated that feeders are important for growth and differentiation of rhesus monkey embryonic stem cells (rES)(Thomson JA et al., 1995 PNAS \bf 92, 7844-7848). In this study, we developed 5 rhesus monkey feeder cell lines for rES culture: MESF (ear skin fibroblasts from 2-week-old neonatal monkeys), MAF (adult Fallopian tube cells), MGF (adult follicular granulose fibroblast-like cells), MGK (adult follicular granulose kidney-like cells), and MSC (single-cell cloning from MESF), all of which were fibroblast-like cells. Two experiments were designed to investigate rES cell growth and differentiation potentials. In experiment 1, rES (R366.4) was cultured on MESF, MAF, MGF, MGK, and MSC, with MEF (mouse embryonic fibroblasts) as control. In experiment 2, rES was cultured on the mixed feeders: MSC: MGK at 1�:�1, 7�:�3, 8�:�2 and 9�:�1, depending on the results of experiment 1. Results of experiment 1 showed that the rES underwent undifferentiated growth on MESF, MAF, MGF and MSC (cultured for 15 passages), but not on MGK, with morphology typical of rES on MEFs, and exhibiting positive alkaline phosphatase staining, positive expression of Oct-4 and GAPDH, and negative expression of PAX6, AFP, BMP4 and hCG of rES as well as normal karyotypes. Embryonic bodies (EBs) were formed on Day 7 to 8 from the rES cultured on MESF, MAF, MGF and MSC. The EBs showed positive expression of hCG, AFP and BMP4. Differentiation of neuron, epithelium and muscle cells was observed after 21 days of continuous culture of the rES. The colonies of rES cultured on MESF, MAF, MGF and MSC were higher than on MEF (1.5 to 3 times). Interestingly, expansion speeds and differentiation rates on MSC were less than on MESF. These results also indicated that fibroblast cells, on the one hand, were alone sufficient for supporting prolonged undifferentiated growth of rES, and on the other hand, other mixed cells except fibroblast cells had the ability to promote the growth and differentiation of R366.4 rES. Results of experiment 2 showed that the mixed feeders (MSC�:�MGK) at 8�:�2 or 9�:�1 supported undifferentiated growth of rES but not feeders at 7�:�3 or 1�:�1. Our work demonstrated that the rhesus monkey feeders were better able to support undifferentiated growth and maintain differentiation potentials of rES than were MEFs feeders. The rES could grow if the ratio of mixed other cells serving as the feeders exceeded 20%.