In turn, SP cells could be obtained from HTR-8/SVneo, and they exhibited features of stem cells/ progenitor cells, which include differentiation into multiple trophoblast cell lineages, Catharanthine sulfate colony formation, long-term proliferation and self-renewal. Numbers of adult stem cell/ progenitor cell subpopulations have been defined using the fluorescent dye Hoechst 33342 in various human tissues. ABCG2 expression was described to efflux Hoechst 33342 from SP cells. High expression of ABCG2 was also observed in HTR-8/SVneo-SP cells. Further more, our SP cells exclusively expressed ID2, which was reported to be a marker of vCTB stem cells. These data suggested that our trophoblast SP fraction included a stem cell/progenitor cell population. Markedly abundant expression of BMP4 was observed in SP but not in NSP cells. BMP4 is known to induce human embryonic stem cell differentiation into trophoblast. There are no studies about BMP4 expression in placenta. Considering previous reports, our results suggested that BMP4 secreted by trophoblast stem cells/ progenitor cells could be important for trophoblast commitment at an early stage or for maintenance of trophoblast cell lineages. We also demonstrated that SP cells expressed some trophoblast differentiation markers, CSH1, CGB and HLA2G as a result of spontaneous differentiation in HBM. CK7 has been recognized as a key marker identifying all different trophoblast subtypes. Surprisingly, SP cells and IL7R/IL1R2 double-positive cells from both HTR-8/SVneo and primary vCTB cells expressed an undetectable level of CK7. NSP cells or spontaneously differentiated cells from SP or IL7R/IL1R2 double-positive cells gained abundant CK7 expression. Our result may be compatible with a previous report that CK7 was absent in HTR-8/SVneo cells and was re-expressed by converting the culture condition. Some groups that successfully obtained cytotrophoblast progenitor cells from human embryonic stem cells could not demonstrate upregulation of CK7 in the progenitor cells. The work of these groups and our data suggest that CK7 expression was upregulated in the process of trophoblast differentiation from the progenitor cell stage. In murine placenta, Cdx2, Eomes and Errb are known to be critical for the maintenance of TS cells. In human, CDX2 was reported to be Mepiroxol differentially expressed in trophectoderm and not in inner cell mass. Furthermore, villous tissues from human early pregnancy were reported to show a higher level of CDX2 expression than those from term placentas. On the other hand, although many trials have been undertaken to obtain human TS cells from human ES cells, most of the studies failed to show induction of CDX2, EOMES or ERRB in their cells. Although EOMES expression was observed in IL7R/ IL1R2 double-positive cells from primary trophoblast culture, HTR-8/SVneo SP cells did not show upregulation of CDX2, EOMES or ERRB compared with NSP 
cells. Careful examination is needed to elucidate the requirements of CDX2, EOMES or ERRB expression for maintenance of human trophoblast stem cells. In addition to HTR-8/SVneo cells, some of these results were also confirmed using human primary vCTB. We could also obtain SP cells and IL7R/IL1R2 double-positive cells from human primary vCTB and found that they expressed EOMES, a TS marker. The cells also remained immature in HSM medium and could differentiate into multiple trophoblast cell lineages including STB and EVT. Although most of the SP and IL7R/ IL1R2 double-positive cells maintained the ability to proliferate and retained the SP morphology in HSM without differentiation.