Figure 3

(a) Western blot analysis. After 55 days of in vitro differentiation treatment of eGFP expressing hiPSdNP the levels of antigens related to cellular proliferation and neural and/or glial differentiation change. The proliferation related Ki67 antigen decreases sharply in DhiPSdNP compared to hiPSdNP. Similarly, in DhiPSdNP Nestin and SOX2 both labeling neuro-glial precursors decrease, while MAP2 and β-III-tubulin that are present in postmitotic neurons, and GFAP, that is mainly present in astrocytes, increase. A housekeeping protein like β-Actin, remains the same. Numbers indicate the apparent molecular weight in kilodaltons. An uncropped image of the westerns used to assemble Fig. 3a is provided in Fig. S1. (b) Splenocytes from CD1 mice immunized with hiPSdNP or DhiPSdNP, or not immunized (Naive), were stimulated with PMA/ionomycin, in the presence of brefeldin A, and tested for IFNγ production by intracellular staining and flow cytometry. The histograms report the quantification of IFNγ production by CD4⁺ T lymphocytes; bars: average. Values are subtracted of background, i.e. spontaneous IFNγ release by unstimulated CD4⁺ or CD8⁺ T cells. N = 3 mice per group. One-way ANOVA followed by Tukey’s test: *p < 0.05. (c) Same as in B but the histogram shows the quantification of IFNγ production by CD8⁺ T lymphocytes. (d) hiPSdNP and DhiPSdNP were tested in vitro for suppressive activity against T cells. Responder CFSE-labeled splenocytes were stimulated with anti CD3 and anti CD28 antibodies, and left alone (+) or in the presence of hiPSdNP/DhiPSdNP, and tested after 5 days by flow cytometry. hiPSdNP/DhiPSdNP: responder ratio 1:1, 1:1.5 or 1:2 as indicated. Histogram reports percentage of proliferation for CD4⁺ T lymphocytes. Negative (−) controls were unstimulated splenocytes. The experiment was repeated three times. One-way ANOVA followed by Tukey’s test: **p < 0.005. (e) Same as in D but we measured proliferation of CD8⁺ T lymphocytes. (f) Kaplan–Meier analysis of survival of hiPSdNP transplanted in immunocompetent CD1 mice (CD1) and Wistar rats (WR) or immunodeficient mice (NOD-SCID) alone or as a mixture with unlabeled DhiPSdNP (hiPSdNP + N) or with adult rat cerebellar cells (hiPSdNP + Cb). (g) Mouse colliculum P45. Mice were transplanted at P3 with a mixture of EGFP labelled hiPSdNP and unlabelled DhiPSdNP. Dapi staining shows that the EGFP positive cells are mono-nucleated and their chromatin condensation pattern is very different (arrow) from that of the host cells (arrows). Scale bar: 10 µm. (h) Mouse cerebellum and brainstem 90 after transplant. The animal was transplanted in utero with a mixture of eGFP positive hiPSdNP and a suspension of cells derived by mechanical dissociation of adult rat cerebellum. Sagittal section eGFP and Dapi counterstained. The main cluster of transplanted cells is localized in the superior colliculus (SC) but eGFP positive cells are present also in the inferior colliculus (IC) and the brainstem. GL: granular layer, ML: molecular layer. Scale bar: 100 µm. On the right an enlargement of the area of the transplant. eGFP positive cells show different degrees of morphological differentiation and some display glial and neuronal morphologies. Scale bar: 25 µm. (i) Same as in h. Arrows indicate eGFP cells showing hNCAM immunopositivity. Scale bar: 15 µm.