The therapeutic potential of ESC-derived stem cells has been hindered by the formation of teratomas. Purifying ESC-derived stem cells is a potential approach to overcome this barrier. SP cells are identified and isolated in many different tissues, tumors, and cell lines, and are generally accepted as a unique character for stem cells . The percentages of SP cells derived from ESCs range from 1% to 16% of total viable cells, depending on the stage of ESC development . In the current study, SP cells were identified and sorted in the induction stage cells from mouse ESCs cultured in EGF, SF, and FCS medium, and the SP percentage in EGF group was significantly higher than that in control groups. Consisted with our previous findings showing that EGF can induce the differentiation of ESCs into Msi1high cells and increase the percentage of Msi1 positive cells, 5-day EGF administration enhanced the percentage of SP cells (Figure 2C), suggesting that the SP fraction probably contains a large proportion of stem or progenitor cells derived from ESCs, including the Msi1 positive cells .
Until recently, it was impossible to isolate IESCs and NSCs based on identifying any single marker expressed on the cell surface. As a protein in the cytoplasm and nucleus required for asymmetric cell division, Msi1 is expressed in NSCs as well as in IESCs . In our previous study, the Msi1-positive cells sorted from ESC-derived cells after a pMsi1-GFP vector transfection had the potential to differentiate into neural and intestinal epithelial cells in vivo . However, the isolation process is complicated and completely depends on cell transfection, which restricts its application. It was reported by Dekaney et al that the SP fraction sorted from mouse jejunum had the stem-like characters and highly expressed Msi1 . SP cells sorted from colon also expressed Msi1, β-integrin, and CD133. Consist with these previous studies, our results demonstrated that SP fraction contained almost all Msi1-positive cells (Figure 3B, D), indicating that sorting SP fraction by FACS, which is regarded as an effective and convenient method to enrich for stem or progenitor cells, could be a reliable method to enrich for Msi1-positive cells from the differentiated cell population derived from ESCs.
Zhou et al found that ABCG2, a subtype member of the ATP binding cassette (ABC) transporter, is a molecular determinant of the SP phenotype in mouse bone marrow [52, 53]. Furthermore, ABCG2 expression was also identified in SP cells sorted from other tissues, such as skeletal muscle, liver, mammary gland, lung, and skin [27, 30, 32, 33, 41]. These studies demonstrated that ABCG2 plays an important role in the SP phenotype. However, it had become clear that the expression of ABCG2 was not detected in the all SP cells. The NSP cells sorted from mouse ESCs expressed Bcrp1 at a level equivalent to that from the SP fraction . It was reported by Alt et al that ABCG2 expression was not detected in the SP cells from human umbilical cord blood . This discrepancy was also reported in mammary gland cells and haemopoietic cells [55, 56]. In our study, the SP phenotype is partially caused by ABCG2 activity, as evidenced by the marked reduction in SP cells with the administration of verapamil, an inhibitor of the ABCG2, which can block the formation of the SP fraction. The results showed that a similar expression of ABCG2 between SP and NSP fraction sorted from Msi1high cells was detected, indicating that ABCG2 expression is not sufficient to confer the SP phenotype (Figure 5A, B). Because there is a significant overlap in the substrate specificity of ABC transporters, with each of the commonly studied family members ABCG2, MDR1, and MRP1 being capable of effluxing Hoechst 33342 dye, the mechanism contributing to SP phenotype sorted from Msilhigh cells should be further investigated [55, 56].
Msi1, a marker for NSC and IESC, plays key roles in the maintenance of the stem cell state and its differentiation, which had been shown by several scholars and us [20–22, 57]. The Msi1 protein can also be found in tissues from patients with endometriosis and endometrial carcinoma, photoreceptor cells, retinal stem cells, and the hair follicle stem cell nich [58, 59]. To confirm that the SP cells sorted from Msilhigh cells had the phenotype of NSC and IESC, the expressions of other markers for IESCs (Lgr5, Hes1, Bmi1, and Ascl2) and NSCs (Sox2 and Nestin) in SP and NSP fractions were detected, respectively (Figure 4) [42–46]. The results revealed that sorting SP fraction could enrich for Lgr5, Hes1, Bmi1, and Nestin high expression cells, further indicating that SP cells could have the developed potentials of IESCs and NSCs. Recent studies showed that IESCs could be grouped into two different phenotypes . One group presented with Msi1 and Hes1 expression resides in intestinal crypts near the transit-amplifying cells. Another marked with Lgr5 and Ascl2 resides between Paneth cells at the small intestinal crypt base. However, Ascl2 expression in SP fraction was similar with NSP fraction and not consistent with the expression of Lgr5 (Figure 4A, D). The reason for the inconsistent expression between Lgr5 and Ascl2 is not clear and should be further investigated.
As mentioned above, the strong expression of Msi1 and other markers for IESCs and NSCs were detected in SP fraction. SP cells sorted from Msi1high cells were engrafted into the NOD/SCID mice to illuminate their developed profiles compared with NSP cells in vivo. The results showed that the SP grafts tended to differentiate into AD-like, NT-like, and sack-like structures (Figure 6A). This pathological profile suggested that SP graft contained more tissues and structures developed from NSCs and IESCs. Consistent with this observation, the expression of neural tissue markers (Nestin and Tubulin β III) and intestinal epithelial cells markers (Tubulin β III, SI, Fabp2, Tff3, and Lyz1) were significantly higher in SP grafts compared with NSP grafts (Figure 6B-6D). In addition, immunochemistry showed that more Tubulin β III positive cells partially constructed with a nest-like profile and more Fabp2-positive cells were observed in the SP grafts (Figure 6E). These results provided strong evidence that SP cells sorted from Msi1high cells had the similar potential of Msi1 positive cells, which could develop into mature neural and intestinal epithelial tissues in vivo. Because forming grafts in NOD/SCID mice is a method for investigating the developed potential of the Msi1-positive cells in vivo, we have not yet performed the experiments necessary to prove that the cells isolated from Msi1high cells will not form teratomas in the mice with normal immune function.