Volume 12, Issue 4, Pages 407-412 (April 2013) Lack of Immune Response to Differentiated Cells Derived from Syngeneic Induced Pluripotent Stem Cells  Prajna Guha, John W. Morgan, Gustavo Mostoslavsky, Neil P. Rodrigues, Ashleigh S. Boyd  Cell Stem Cell  Volume 12, Issue 4, Pages 407-412 (April 2013) DOI: 10.1016/j.stem.2013.01.006 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Differentiated Cells Derived from Syngeneic iPSCs Lack Immunogenicity In Vitro (A) Micrographs are representative of endothelial cells, hepatocytes, and neuronal cells differentiated from either the B6 embryonic stem cell (ES) line, STEMCCA-generated induced pluripotent stem cell lines (iPS), or the episomal iPSC line (Epi). (B) RNA prepared from each of the differentiated cell types (endothelial cells, hepatocytes, and neuronal cells) was subjected to reverse transcriptase reaction and qPCR was performed for markers of endothelial cells (top panel), hepatocytes (middle panel), and neuronal cells (bottom panel), respectively (n = 3 separate experiments per cell line). Results were normalized to GAPDH. Untreated cells were used as a control. Triplicates were used for each PCR. Error bars represent ± SEM. ∗p < 0.05. (C) Representative immunofluorescence staining with respective tissue-specific markers. Top two rows: endothelial cell grafts were stained for VE-Cadherin (green) and PECAM (red). Middle two rows: hepatocyte grafts were stained for CD24 (green) and α-fetoprotein (AFP) (red). Bottom two rows: neuronal cell grafts were stained for Nestin (green) and γ Tubulin (red). Data are representative of five independent experiments. Scale bar = 10 μm for endothelial cell and hepatocyte grafts and 50 μm for neuronal cell grafts. (D) Gene expression of putative immunogenic genes Hormad, Zg16, and Retn was assessed in pluripotent stem cells (PSC), embryoid bodies (EB), endothelial cells (Endo), hepatocytes (Hep), and neuronal cells (Neu) derived from ES, iPS1, iPS2, and Epi cell lines, respectively. Data are represented as ± SEM of triplicates of three independent experiments. ∗p < 0.05. (E) Experimental design for the CFSE-T cell proliferation assay using CD4+ and CD8+ T cells isolated from spleens of B6 (syngeneic) or CBA (allogeneic) mice. The fluorescent dye CFSE can be used to track a cell’s division history because it is equally distributed between daughter cells upon mitosis, resulting in loss of signal as detected by flow cytometry. CFSElo/− cells have undergone proliferation while maintenance of CFSE signal denotes nondividing cells. Only viable (Hoescht−) CD4+ or CD8+ T cells were used to evaluate the loss of CFSE dye signal. (F) CFSE-T cell proliferation assay comparing the proliferation of CFSE-labeled CD4+ and CD8+ T cells (responders, R) isolated from spleens of B6 (syngeneic) or CBA (allogeneic) mice cocultured with PSCs, EBs, or tissue specific cells (TSCs) (stimulators, S) at a 1:2 (R:S) ratio for 72 hr. The percentage of proliferating cells (CFSElo) was quantified and shown as ± SEM of triplicates of two independent experiments. See also Figure S1. Cell Stem Cell 2013 12, 407-412DOI: (10.1016/j.stem.2013.01.006) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Differentiated Cells Derived from Syngeneic iPSCs Are Not Rejected In Vivo, nor Do They Induce Secondary T Cell Responses (A) Representative H&E staining of endothelial cells (Endo), hepatocytes (Hep), and neuronal cells (Neu) engrafted into syngeneic B6 recipient mice. K and G indicate kidney and graft, respectively. (B) Representative immunostaining of endothelial cells, hepatocytes, and neuronal cells engrafted into syngeneic B6 recipient mice stained for tissue-specific cell markers. Top three rows: endothelial cell grafts stained with VE-Cadherin (green) and PECAM (red). Middle three rows: hepatocyte grafts stained with CD24 (green) and α-fetoprotein (AFP) (red). Bottom three rows: neuronal grafts stained with Nestin (green) and γ Tubulin (red). Scale bar = 50 μm. (C) Syngeneic endothelial, hepatocyte, and neuronal cell grafts were stained for CD4 (green, top panel) or CD8 (green, bottom panel) to analyze T cell graft infiltration. To label the engrafted cells, endothelial cells were stained with VE-Cadherin (VCAD), hepatocytes with CD24, and neuronal cells with CD29 (red). Scale bar = 20 μm. (D) CFSE-labeled CD4+ and CD8+ T cells (responders) isolated from spleens of B6 (syngeneic) or CBA (allogeneic) mice engrafted 30 days previously with PSCs, EBs, or TSCs were cocultured with freshly isolated PSCs, EBs, or TSCs (stimulators) at a 1:2 (R:S) ratio for 72 hr. The percentage of CFSElo cells, having been stimulated to proliferate, was quantified and shown as ± SEM of triplicates of two experiments. (E) Early and late-stage apoptosis of PSCs, EBs, and TSCs cocultured for 72 hr with ex vivo isolated CD4+ or CD8+ T cells from spleens of B6 (syngeneic) or CBA (allogeneic) mice engrafted 30 days previously with PSCs, EBs, or TSCs was examined. After 72 hrs, PSCs, EBs, or TSCs were harvested and stained with the vital dye Hoechst and apoptotic marker Annexin V to determine whether antigen-experienced T cells would directly kill syngeneic iPSC-derived cells. Cells undergoing early apoptosis are Annexin-V+ Hoescht− and those undergoing late apoptosis are Annexin-V+ Hoescht+. Data are represented as mean value ± SEM of triplicates of two experiments. (F) Q-PCR for putative immunogenic genes Hormad, Zg16, and Retn was performed with RNA isolated from grafts of PSCs, EBs, or tissue-specific cells (n = 3 separate experiments per group). Background expression of genes from sham-transplanted kidneys was subtracted from PCR results from those recipients receiving a graft. Results were normalized to GAPDH. Triplicates were used for each PCR. Error bars represent ± SEM. See also Figures S1 and S2 and Table S1. Cell Stem Cell 2013 12, 407-412DOI: (10.1016/j.stem.2013.01.006) Copyright © 2013 Elsevier Inc. Terms and Conditions