This result suggested that the cells require some other factors to maintain their immature properties. In our study, a number of different culture conditions were tested on SP cells for optimization. SP cells grew better and retained the SP characteristics on MEF feeder cells or in conditioned medium from MEF feeder cells, just like the case with mouse TS cells. Several different growth factors were also tested on SP cells. Because FGF2 gave the best result, FGF2 was chosen for a supplement with our SP medium, HSM. Although vCTB cells can be isolated from villi at any stage of pregnancy for primary culture, they quickly cease proliferating and differentiate within about 5 days. Previous studies reported that proliferation in 1st trimester villous explant could be increased at 4�C5 weeks by supplying EGF or IGF. FGF4 was also reported to inhibit differentiation of the 1st trimester explant and to prolong cell proliferation. Our SP cells from primary vCTB maintained the SP morphology in HSM containing FGF2 for at least 2 weeks after SP isolation. We did not check the later stage, but FGF2 is also a candidate mitogen for vCTB primary culture. We also discovered that IL7R and IL1R2 are novel markers of SP cells derived from both HTR-8/SVneo and primary vCTB. Most of the SP cells expressed IL7R and IL1R2, but in contrast, NSP cells failed to express IL7R and IL1R2. It is known that IL7R is expressed on Chloroquine Phosphate dendritic cells and monocytes, and activates multiple pathways that regulate lymphocyte survival, glucose uptake, proliferation and differentiation. IL7R signal in particular plays an essential role in T and B cell development and homeostasis. Although a previous study reported that IL7R was expressed in both vCTB and STB at an early stage of pregnancy its expression was rather weak. The function of IL7R in trophoblast differentiation remains unknown. The other marker, IL1R2, was reported to antagonize IL1 activity by acting as a decoy target for IL1 in polymorphonuclear cells, a specific type of leukocyte. IL1R2 expression has not been reported in placenta. Our in vitro data suggested that only a small proportion of vCTB cells expressed IL7R and IL1R2 and that they lost IL7R and IL1R2 expression as they differentiated into STB or EVT cells. Further investigation should reveal the function of IL7R and IL1R2 in the mechanism of human trophoblast differentiation. Isolation of human TS cells is necessary to investigate the early trophoblast cell lineages with self-renewing properties and the capability to differentiate into all trophoblast cell types of the mature placenta. The pathology of pregnancy-associated complication is believed to be based on abnormal trophoblast differentiation, defects in trophoblast invasion and spiral artery remodeling. To Tulathromycin B understand the pathology, in vitro model using human TS cells will provide tremendous benefits. Furthermore, human TS cells may lead us to a new approach for treating patients with placental dysfunction with TS cell transfer. Our study provides new insights into the characteristics of human TS/ progenitor cells. This study also reveals several key factors that are practical and available markers for TS/ progenitor cell isolation, and which might be essential for the maintenance of TS/ progenitor cells. These new insights should help us to understand human TS cell biology and develop novel therapeutic technologies for placental disorders. This offers a unique opportunity to investigate structure/function shifts during evolution and, by comparison with data from the two other bilaterian clades, to help define the basic assortment of genes required to manage reproduction.