In-vitro differentiation of iPS cells towards mesodermal and haematopoietic lineages.

<p>(A) Schematic representation of protocol for haematopoietic differentiation of X-CGD mouse derived iPS cells. EB, embryoid bodies; ColIV, collagen type IV coated plate; Flk-1+ cells, FMS-like tyrosine kinase I positive cells; OP9, M-CSF deficient OP9 stromal cell line; LSK, Lineage negative, Sca-1 positive, c-kit positive cells. (B) Characterization of day six EB cultured under low-attachment conditions. Semi-quantitative RT-PCR analyses showing comparative expression of pluripotency and differentiation markers in wild-type (wt-IPSC) and diseased iPS cells (cgd-IPSC). Left panel shows expression levels of various markers in day six EBs, while those in the right panel shows comparative levels of expression in cells after culturing in collagen IV coated flasks in haematopoietic differentiation medium. Middle panel shows in-situ immunophenotypic staining of six-day EBs with antibodies targeting characteristic antigens expressed in the three germinal layers. (C) FACS analysis showing generation of Flk-1 positive cells from EBs upon collagen IV culture.(D) FACS plot showing derivation of lineage negative, Sca-1 positive and c-kit positive (LSK) population post OP9 co-culture of Flk-1 positive cells.</p

Reprogramming of X-CGD mouse fibroblasts to induced pluripotent stem cells.

<p>(A) Images of iPSC showing ES cell like morphology (high nucleus to cytoplasm ratio), high levels of alkaline phosphatase (AP) activity, and expression of pluripotency markers Sox2, Oct4, Klf4, Nanog, SSEA-1, and c-Myc. Bright-field images were acquired with a standard Olympus microscope (20X objective). Fluorescent images were acquired with a Zeiss LSM 710 confocal microscope (25X objective). (B) Haematoxylin & Eosin staining of teratoma sections showing derivatives from three germinal layers (Ecto, ectoderm; Endo, endoderm; Meso, mesoderm). Structures shown include neural tube (ectodermal), striated muscles and cartilaginous structures (mesodermal), and gut-like epithelium (endodermal). (C) Immunofluorescent confocal images of teratoma sections showing antibody staining targeting tissue derivatives present in three germinal layers.Tuj1: neuronal class III β-tubulin; ∝-FP: alpha-fetoprotein; ∝-act: alpha-actinin; DAPI: 4′-6-Diamidino-2-phenylindole.(D) Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses showing silencing of exogenously introduced transgenes as shown by their presence in fibroblasts (three days post transduction, D3), and absence in cgd-IPSC clone (passage five). Specific primers were designed to target regions of retroviral transgenes and endogenous sequences of reprogramming factors. GAPDH, glyceraldehdye 3-phosphate dehydrogenase.</p

Myeloid differentiation of iPS cell derived LSK cells.

<p>Colony Forming Unit (CFU) assay was performed to test the ability of LSK cells to undergo terminal differentiation in semi-solid methylcellulose media containing suitable cytokine cocktail. (A) Graph showing the relative distribution of CFU-GM (granulocyte/macrophage), CFU-G (granulocyte), and CFU-M (macrophage) colonies scored on the basis of their morphology from three independent cultures. Error bars denote standard error of mean (SEM). (B) Representative cytospin preparation of Diff-Quik stained cells isolated from characteristic colonies showing presence of granulocytes and macrophages including and not restricted to mature and functional neutrophils. (C) FACS plot showing presence of double positive Gr-1 (granulocyte differentiation marker) and CD11b (myeloid and NK cells marker) cells isolated from colonies growing in methycellulose differentiation medium.</p

Efficacy of novel lentiviral vector for generation of disease-free haematopoietic cells.

<p>Embryoid bodies were transduced with lentiviral vector encoding codon optimized <i>gp91^phox</i> from an internal chimeric promoter (LentiChimgp91). (A) Graph showing positive correlation between the multiplicity of infection (m.o.i) and generation of NBT positive colonies. Error bars denote standard error of mean (SEM). Phase contrast images of CFU-GM colonies showing results from NBT assay. (B) FACS derived histograms showing results of dihydrorhodamine123 (DHR) test upon phorbol myristate acetate (PMA) stimulation. DHR loaded neutrophils upon PMA stimulation show a shift in fluorescence due to reduction of DHR123 by respiratory burst generated by functional neutrophils.</p

Lin- primary progenitor cells expressing BCR-JAK2 show increased proliferation capacity compared to wild-type (wt) and mock progenitor cells.

<p>Lin- progenitor cells obtained from Balb/c mice were transduced with pLZR (mock cells) or pLZR-BCR-JAK2 retroviral particles and seeded for proliferation assay during 30 days.</p

BCR-JAK2 is constitutively phosphorylated, triggering STAT5 activation which translocates to the nucleus and induces STAT5 target gene expression on transduced Ba/F3 cells.

<p>(<b>a</b>) Western blot analysis of Ba/F3 cells infected with BCR-JAK2, TEL-JAK2 or vector alone (mock). Cellular lysates were immunoprecipitated with anti-pTyr Ab and immunoblotted with anti-JAK2 Ab to see pJAK2 (upper panel: endogenous JAK2: 130 kDa, BCR-JAK2: 90 kDa, TEL-JAK2: 72 kDa). TEL-JAK2 protein expression is higher than BCR-JAK2 in transduced Ba/F3 cells. Whole cell lysates were probed with anti-pSTAT5 and anti-STAT5 Ab's (bottom). The expression levels of Tubulin were used as a loading control. (<b>b</b>) Enriched cytoplasmic (C) and nuclear (N) extracts from Ba/F3-mock,Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 cells were prepared from total cell lysates and blotted with anti-pSTAT5 Ab. TATA-Binding protein (TBP) and IkBα were used as loading controls for enriched nuclear and cytoplasmic fractions, respectively. (<b>c</b>) Expression of <i>Bcl-xL, Osm and Socs2</i> in Ba/F3-mock, Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 cells by qPCR. For comparative purposes, mRNA levels in untreated cells were normalized to 1. Bars represent fold changes of each gene normalized using <i>GADPH</i> levels. Results are given as mean ± SEM (n = 3).</p

Nude mice injected with Ba/F3-BCR-JAK2 cells develop tumors.

<p>Mice were subcutaneously injected with 10⁷ Ba/F3-mock cells (left flank) and Ba/F3-BCR-JAK2 cells (right flank). (<b>a</b>) Left panel: white light photograph showing mice bearing a tumor only on the right side, where Ba/F3-BCR-JAK2 cells were injected (black arrows). Right panel: EGFP⁺ tumor (white arrows) captured on a digital photostation showing the reflected image of the mice (right side). No EGFP expression was detected when Ba/F3-mock cells were injected (left side). (<b>b</b>) Flow cytometry analysis of EGFP-expressing tumor cells. (<b>c</b>) RT-PCR analysis to detect BCR-JAK2 expression using BCR-B and JAK2-3 primers. RNA from three different tumors was analyzed compared with RNA from Ba/F3-mock and Ba/F3-BCR-JAK2 cells used as negative and positive controls, respectively. pLZR-BCR-JAK2: plasmid bearing <i>BCR-JAK2</i> was used as positive control. MW, marker.</p

DNA methylation pattern on tumour suppressor genes in peripheral blood mononuclear cells of FA patients.

<p>PBMC from FA patients and healthy donor were isolated as in A, DNA was isolated and methylation profile was identified in 2 FA samples and 2 healthy donors samples using EpiTect Methyl II Complete PCR array (SABiosciences). Samples CTR1 and CTR2 represent control and FA1 and FA2 represent FA patients. Each row represents a tumour-supressor genes and each column represent a single DNA sample. The methylation degree are represented by the level of intensity of the square, red representing greater than 10% promoter hypermethylation, and green representing less than 10% promoter methylation (unmethylated) alleles for the tumour-suppressor gene.</p

TG101209, a JAK2 inhibitor, down-regulates JAK2, BCR-JAK2, and STAT5 tyrosine-phosphorylation, as well as target gene expression of Ba/F3-BCR-JAK2 transduced cells.

<p>(<b>a</b>) Sigmoidal dose-response curve showing viability of Ba/F3-mock growing with IL-3 (IC₅₀ = 3180 nM) and Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 growing in absence of IL-3 (IC₅₀ = 246 and 369 nM, respectively). The percentage of growth, relative to that of cells in the absence of drug, is plotted for increasing concentrations of TG101209 (where X axis is the logarithm of concentration). (<b>b</b>) Western blot analysis of transduced Ba/F3 cells treated for 12 h with 1 µM TG101209 (TG) or the vehicle (DMSO). Cellular lysates were immunoprecipitated with anti-pTyr Ab and immunoblotted with anti-JAK2 (upper panel). Whole cell lysates were probed with anti-pSTAT5, anti-STAT5 and anti-Bcl-xL Ab's (bottom). The expression levels of Tubulin were used as a loading control. STAT5 levels were used as a loading control for pSTAT5. (<b>c</b>) qPCR for <i>Bcl-xL, Osm and Socs2</i> expression on Ba/F3-mock, Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 cells treated for 12 h with 1 µM TG101209. For comparative purposes, mRNA levels in untreated cells were normalized to 1. Bars represent fold changes of each gene after normalization with <i>GADPH</i> levels. Samples from three independent experiments were measured. Results are given as mean ± SEM (n = 3).</p

TG101209 induces apoptosis of Ba/F3-BCR-JAK2 cells.

<p>Ba/F3-mock growing with IL-3, Ba/F3-BCR-JAK2 and Ba/F3-TEL-JAK2 cells (10⁵/ml) were grown in 24-well plates and treated either with DMSO or 1 µM TG101209 (TG) for 24 h. Ba/F3-mock cells growing for 24 h without IL-3 were dead (data not shown). Apoptosis was measured using flow cytometry analysis as the percentage of cells expressing annexin-V/7-AAD on the cell surface. (a) One representative experiment out of three is shown. (b) Quantification of apoptotic cells. Results are given as mean ± SEM (n = 3).</p