Adoption of dynamic suspension culture system for generating SEs
We have developed a highly efficient two-step protocol to produce NK cells from physically reprogrammed somatic cells (Fig. 1a). In order to generate SEs, LO2, HOSEC, and WPMY-1 cells were harvested and transferred to a dynamic suspension culture system without integration, feeder layer, and serum. The morphological changes of cells after dynamic suspension culture were shown in Fig. 1b. Cell aggregates were observed in LO2 and HOSEC cells on day 1, and SEs with irregular shapes appeared on day 3~5. After 5~7 days, SEs with consistent sphere morphology can be formed in both LO2 and HOSEC cells, and the size continued to increase. After 9 days, SEs derived from LO2 (L-SEs) and HOSEC (H-SEs) were in the form of homogeneous spheres wrapped by membrane. SEs derived from WPMY-1 cells (W-SEs) were formed on day 7, which was shorter than L-SEs and H-SEs. After 2~3 days, the sphere model of W-SEs was formed rapidly, and the size continued to increase. After 7 days, W-SEs were in the form of homogeneous spheres wrapped by membrane. These results indicate that somatic cells can be efficiently reprogrammed into SEs by dynamic suspension culture system. SEs were compact spheres with clear borders.
During the dynamic suspension culture process, the diameters of L-SEs, H-SEs, and W-SEs increased from 36.68 2.49 μm, 34.63 1.98 μm, and 49.41 4.00 μm to 97.20 0.89 μm, 95.43 1.56 μm, and 98.20 9.21 μm, respectively (Fig. 1c). At the beginning and end of the dynamic suspension culture, the average diameters of SEs derived from somatic cells were 40.24 4.62 μm and 96.94 0.81 μm, respectively (see Additional file 1). During the dynamic suspension culture process, the diameters of L-SEs, H-SEs, and W-SEs increased 2.67 0.18, 2.77 0.16, and 1.99 0.05-fold, respectively, and the average diameter of somatic cell-derived SEs increased 2.48 0.25-fold (see Additional file 1). These data show that at the end of the culture, the diameters of the three somatic cell-derived SEs are similar and significantly increased compared to the beginning of the dynamic suspension culture (Fig. 1c).
From 1.22 0.05106 LO2, 0.98 0.06106 HOSEC, and 1.58 0.06106 WPMY-1 cells, we obtained 0.54 0.12106 L-SEs, 0.23 0.06106 H-SEs, and 0.26 0.06106 W-SEs, respectively (see Additional file 2). On average, we obtained 0.34 0.10106 SEs from 1.26 0.17106 somatic cells (see Additional file 2). These data indicate that the total number of cells changes during the formation of SEs.
SEs express pluripotency-associated markers
The pluripotency-associated markers of SEs were identified by classical assays. Immunofluorescence staining showed that L-SEs, H-SEs, and W-SEs expressed pluripotency-associated markers, such as nuclear markers NANOG, SOX2, OCT4, and surface markers SSEA3 and SSEA4 (Fig. 2a-c).
Two-color flow cytometry analysis demonstrated that L-SE, H-SEs, and W-SEs were positive for NANOG, SOX2, OCT4, SSEA-3, and SSEA-4 (Fig. 3a). Although the expression levels of NANOG, SOX2, OCT4, SSEA-3, and SSEA4 were higher than those of the control group (see Additional file 3), their expression levels in L-SE, H-SE, and W-SE were comparable (Fig. 3b). The percentage of cells expressing pluripotency-related markers was similar in L-SEs, H-SEs, and W-SEs (33.28 5.57%, 35.21 3.42% and 32.66 8.41%), and the average percentage of cells expressing pluripotency-related markers was 33.72 0.77% in SEs derived from somatic cells (see Additional file 3). Generation of iPSCs from somatic cells is affected by many factors, such as the initial cell type, reprogramming methods, and culture conditions. Generally, the reprogramming efficiency varies in the range of 10%, and it takes about 12-30 days . In our study, the reprogramming efficiency was comparable for LO2, HOSEC, and WPMY-1(14.93 5.06%, 7.90 1.03% and 5.48 1.84%). We obtained an average reprogramming efficiency of 9.44 2.83% when our integration-free, feeder-free, and serum-free dynamic suspension culture system was adopted for the reprogramming of somatic cells (Fig. 3c).
The western blot experiments indicated that NANOG, SOX2, and OCT3/4 were expressed in L-SEs, H-SEs, and W-SEs cells, and the expression of OCT3/4 was higher in the three somatic cell-derived SEs (Fig. 4a and see Additional file 4). W-SEs expressed NANOG protein at higher levels compared to L-SEs and H-SEs. L-SEs expressed SOX2 protein at higher levels compared to H-SEs and W-SEs. There was no significant difference in the protein level of OCT3/4 among the three somatic cell-derived SEs (Fig. 4b). Taken together, the results of immunofluorescence staining, flow cytometry analysis, and western blot analysis indicate that SEs generated in the dynamic suspension culture system express pluripotent-associated markers.
Teratoma formation from SEs with normal karyotype
To test pluripotency in vivo, we transplanted SEs subcutaneously into the groin of immunodeficient (SCID) mice. Ten weeks after injection, we observed tumor formation. Histological examination showed that the tumor contained various tissues (Fig. 5a), including thyroid tissues (endoderm), chondrocyte (mesoderm), and neural tissues (ectoderm). Finally, L-SEs, H-SEs, and W-SEs displayed normal karyotypes (Fig. 5b).
In summary, our dynamic suspension culture system allows us to efficiently generate integration-free iPSCs (in the form of SEs) from somatic cells. SEs are characterized by classic assays, including contribution to all three germ layers in vivo. More importantly, all classic assays were performed after 7~9 days of dynamic suspension culture suggesting that our protocol can provide high-quality iPSCs in a short time.
Directed differentiation of SEs into NK cells
We next examined whether SEs could be induced to directly differentiate into NK cells without employing CD34+ cell enrichment and feeder. The protocol of NK cell induction is summarized in Fig. 6a. We transferred L-SEs, H-SEs, and W-SEs to 24-well plates and maintained them under differentiation conditions for 4~5 weeks. Cells spread after 3 days, and a large number of suspension cells were observed after 30 days (Fig. 6b). Morphologically, L-SEs, H-SEs, and W-SEs could differentiate into small, round, suspended, and resembled lymphoid cells under cytokines conditions.
From 0.27 0.06106 L-SEs, 0.11 0.03106 H-SEs, and 0.13 0.03106 W-SEs, we obtained 0.34 0.06106 L-SEs-NK, 0.22 0.11106 H-SEs-NK, and 1.10 0.21106 W-SEs-NK cells, respectively (see Additional file 5). On average, we obtained 0.55 0.27106 SEs-NK cells from 0.17 0.05106 SEs (see Additional file 5). The total number of L-SEs-NK, H-SEs-NK, and W-SEs-NK cells increased about 1.40 0.25, 2.12 1.08, and 9.28 1.78-fold, and the average total number of SEs-NK cells increased about 4.26 2.52-fold (see Additional file 5). These data indicate that the total number of cells changes during the formation of SEs-NK cells.
Surface marker analysis of SEs-NK cells
The expression of CD57 on NK cells has been demonstrated by a large number of studies [19-24]. More recently, it has been established that CD57 expression defines functionally mature sub-populations of NK cells [25-27]. Immunofluorescence staining showed that CD57 was expressed on L-SEs-NK, H-SEs-NK, and W-SEs-NK cells, but not on LO2, HOSEC, and WPMY-1 cells (Fig. 7a).
Flow cytometry showed that the expression level of CD57 on L-SEs-NK, H-SEs-NK, and W-SEs-NK cells was higher than that on LO2, HOSEC, and WPMY-1 cells (Fig. 7b and see Additional file 6). For SEs-NK cells, we found that a majority of L-SEs-NK and H-SEs-NK cells expressed CD57 (see Additional file 6). Compared to somatic cells, SEs-NK cells included a significantly higher proportion of NK cells expressing CD57 (see Additional file 6). These results indicate that SEs-NK cells generated in this study are CD57+ NK cells, indicating that they are highly mature and terminally differentiated NK cells.
Cytotoxic activity of SEs-NK cells
Cytotoxicity is an important functional feature of NK cells. The cytotoxicity of NK cells can be determined by measuring the percentage of K562 cells lysed by NK cells after incubation. The cytotoxic activities of L-SEs-NK cells against K562 at E: T ratios of 20:1, 50:1, 80:1, and 100:1 were 61.22 6.42%, 59.40 4.82%, 63.40 4.42%, and 61.83 5.10%, respectively, H-SEs-NK cells were 49.72 0.89%, 50.76 3.82%, 49.99 4.00%, and 51.97 3.19%, respectively, and W-SEs-NK cells were 22.54 3.53%, 33.11 1.01%, 30.08 2.14%, and 28.94 0.95%, respectively (Fig. 8a). Similar to the expression of CD57 in SEs-NK cells, L-SEs-NK cells and H-SEs-NK cells had higher ability of K562 cytotoxicity than W-SEs-NK (Fig. 8b). Therefore, through a combination of cytokines, SEs can be directly induced to differentiate into mature NK cells with cytotoxic function, without the need for CD34+ cell enrichment and feeder culture system.