hiPSCs were infected for 24 h and sorted three days later for GFP+ and NGFR-PE+ expression for the decoy and over-expression functional assays, respectively

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hiPSCs were infected for 24 h and sorted three days later for GFP+ and NGFR-PE+ expression for the decoy and over-expression functional assays, respectively. pathway (Yoshida et al., 2012). A minority of cases arises in young children with Noonan syndrome (NS; OMIM163950), a genetic disorder with increased RAS/MAPK signaling (Tartaglia and Gelb, 2008). Fifty percent of NS patients and 35% of JMML cases carry gain-of-function (GOF) mutations altering SHP-2. However, the molecular mechanisms through which mutations result in deranged myelopoiesis are not well understood. SHP-2 is usually a member of the tyrosine phosphatase family and regulates several biological processes, particularly embryogenesis and hematopoietic cell development (Tartaglia and Gelb, 2008). For NS, particular germ-line mutations in underlie JMML, which has clinical features similar to those in children with JMML arising from somatic mutations in although with generally better outcomes. Mutations in genes, and are mutually unique in JMML, suggesting that one hit in this pathway is sufficient for leukemogenesis (Tartaglia and Gelb, 2008; Perez et al., 2010; Yoshida et al., 2012). The ability to induce hiPSCs from terminally differentiated cells such as skin fibroblasts (Takahashi et al., 2007) provides an opportunity to study disease pathogenesis. For hereditary disorders associated with cancer such as NS/JMML, AM 114 hiPSCs derived from non-cancerous cells permit investigation of the role of AM 114 the inherited mutations mutations. Results Generation of hiPSC lines and clinical manifestations in NS/JMML-derived hematopoietic cells Using somatic cell reprogramming (Takahashi et al., 2007), we obtained hiPSC lines from skin fibroblasts harboring mutations in from two subjects with NS/JMML. As controls, we used five AM 114 hiPSC lines derived from skin fibroblasts of unrelated healthy individuals and two subjects with NS, the latter to clarify which perturbations were attributable to the gain-of-function mutations generally those specific to JMML pathogenesis (Tables S1 and S2). All mutations resided in the N-SH2 domain name, destabilizing SHP-2s inactive conformation. Pluripotency was established based on characteristics (Figures S1ACB), and teratoma formation (Physique S1C). All hiPSC lines had a AM 114 normal karyotype (Physique S1C). NS and NS/JMML hiPSCs retained heterozygosity for their mutation (Physique S1F). Transgenes were integrated at 1C4 sites and were silenced (Physique S1DCE). After EGF stimulation, ERK activation was increased and sustained in the NS and NS/JMML hiPSC lines (Physique S1G), consistent with mutation GOF effects. Following an established protocol using a cocktail of cytokines (IGF-1, VEGF, EPO, IL-11, IL-3, IL-6, bFGF, SCF, and TPO) (Grigoriadis et al., 2010; Kennedy et al., 2007) (Figures S1H, S2A and Supplemental Experimental Procedure), hiPSC lines were differentiated into hematopoietic progenitor lineages, showing common morphology for the various cell types (Physique S1H). Flow cytometry analysis showed that this differentiating lines expressed the pan-hematopoietic marker CD45 at least until Day 28 (Physique S2B). Neither the B-lymphocyte antigen CD19 nor the T-cell surface glycoprotein CD3 gamma chain (hybridization with a dual probe showed an absence of the fusion (Physique S3A). Open in a separate window Physique 1 Clinical Features of NS/JMML hiPSC-Derived Hematopoietic Cells(A) Hematopoietic growth factor dose-response curves to GM-CSF. Values are the mean standard error of the mean for three impartial experiments. At 0.01 ng/ml GM-CSF, ** is p 0.01 in NS/JMML Patient #4 CTRL. At 0.1 ng/ml GM-CSF, ** is p 0.01 in NS/JMML Patients #3 and #4 controls. JAK-STAT and MAPK signaling pathways, chronic myelocytic leukemia, acute myelocytic leukemia and cell cycle (red, up-regulated; blue, down-regulated). (G and H) Representative immunoblots show increased pSTAT5 and total STAT5 (G), and pERK (H) protein expression levels in NS/JMML CD33+ myeloid cells. Lanes were run on the same gel but were noncontiguous. (I) and expression levels in CD33+ myeloid cells. Bar graphs represent mean Icam1 standard error of the mean. (J) Annexin V and (K) Ki-67 staining of CD33+ myeloid cells show apoptosis and proliferation, respectively. Bar graphs represent mean standard error of the mean. The CTRL group consisted of iPSC lines, each derived from different healthy individuals. Data represent a composite of three individual experiments. See also Figure S4. Genotype-specific dysregulation of genes related to biological processes such as metabolism, development, and proliferation was observed in CD33+ myeloid cells using.