Partial restoration of TKI-sensitivity of CD34<i>⁺</i> hematopoietic progenitors derived from CML-iPSCs.

<p>Partial restoration of sensitivity to TKI of CD34⁺ hematopoietic progenitors derived from CML-iPSCs. Apoptosis in untreated versus imatinib cultures (5 µM, 24 h) was evaluated after annexin-V staining by FACS analysis, in CD34⁺ cells derived from CB-iPSC #11, CML-iPSCs #1.24 and #1.31.</p

Effect of shRNA against BCR-ABL1 on CML-iPSC #1.31 clone proliferation.

<p>(<b>A</b>) Western blot analysis of BCR-ABL1 and ABL expression in CML-iPSC #1.31 with shRNA control (shC) and with shRNA against BCR-ABL1 (shBCR). (<b>B</b>) Left panel: Proliferation of CML-iPSC (#1.31) with shC or shBCR. iPSCs counts at day 6 expressed as percentages relative to same iPSC (CML-iPSC #1.31) with shC. Mean +/− SD, n = 3. Right panel: Dose-effect of imatinib exposure for 6 days on iPSCs (CML-iPSC #1.31, CML-iPSC #1.31 with shC or with sh BCR). iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC without TKI. Mean ± SD, n = 3.</p

BCR-ABL1 independent proliferation.

<p>(<b>A</b>) Dose-effect of imatinib exposure (0–5 µM) for 6 days on CML-iPSC clones #1.22 and #1.31. Colony frequency is evaluated by alkaline phosphatase staining conducted at day 6. (<b>B</b>) Dose-effect of imatinib exposure for 6 days on iPSCs survival. iPSCs counts were conducted at day 6 and are expressed as percentages relative to same iPSC . Mean +/− SD n = 3, *: p<0.05 versus clone #1.22 with the same exposure. (<b>C</b>) Dose-effect of ponatinib exposure for 6 days on CML-iPSC clones (#1.22 Ph-, #1.24 and #1. 31 Ph+) survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC without TKI. Mean +/- SD, n = 3. * p <0.05 vs iPSC #1.22 (internal control Ph-) at the same TKI exposure. (<b>D</b>) Western-blot analysis of ABL, phosphotyr (p-Tyr) pattern, CRKL and phosphoCRKL (p-CRKL) in CML-iPSCs in absence (−) or presence (+) of imatinib (20 µM) for 48 h.</p

Transgene independence of CML-iPSCs survival in presence of TKI.

<p>(<b>A</b>) PCR for the integrated vectors OSK 1 and MshP53 in 11 subclones of CML-iPSC #1.31 pretreated with CRE adenovirus. Generation of transgene-free subclone CML-iPSC #1.31i: excision of the 2 vectors. (<b>B</b>) Immunohistochemistry of pluripotency markers: OCT4, SOX2, KLF4, NANOG, SSEA-4 and TRA1-60 in human transgene-free iPSC subclones (after excision) derived from CD34⁺ from CML patient (#1.22 exc and #1.31 exc) (<b>C</b>) Dose-effect of TKI exposure (with imatinib (left panel) or ponatinib (right panel)) for 6 days on human excised CML-iPSCs (# 1.22, #1.31) and CB-iPSC (#11) subclones survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC clone without TKI. Mean ± SD of triplicate.</p

Characterization of iPSC clones.

<p>(<b>A</b>) Representative immunofluorescence of pluripotency markers in human iPSC clones derived from CD34⁺ CB cells (CB-iPSC #11) and CD34⁺ from CML first patient (CML-iPSCs #1.22, #1.24 and #1.31) and from CML second patient (#2.1 and #2.2), staining with anti-OCT4, anti-SOX2, anti-KLF4, anti-NANOG, anti-SSEA-4 and anti-TRA1-60. MEFs surrounding human iPSCs served as a negative control for immunofluorescence (magnification x100 or x200). (<b>B</b>) Representative alcian blue staining of histological sections of teratoma derived from human CB-iPSC #11 and CML-iPSC #1.31 encompassing tissues with all three germ layers (magnification x25 and x200).</p

PCR analysis of genomic DNA encoding the glycoprotein Ia gene surrounding the 807, 837 and 873 polymorphisms.

<p><i>(A)</i> Amplified products (1332 bp) were resolved by 1% agarose gel electrophoresis and stained with ethidium bromide. Lane 1: molecular weight marker; lane 2: blank; lanes 3 to 11: different genotyped individuals. <i>(B)</i> Analysis of <i>ITGA2^{807C/T, 837C/T, 873 G/A}</i> polymorphisms by PCR-RFLP using <i>Bgl II</i> and <i>Asn I</i> endonucleases on 1.5% agarose gel. Lane 7: molecular weight marker; other lanes: different genotypes according to reference 29 (lanes 1, 4, 5: 2/2, lanes 2, 3, 6: 1/2, lane 7: 1/3, lane 8: 2/3, lane 9: 1/1).</p

Cardiovascular Risk Factors in the Study Sample.

<p> <b>Results as mean ± SD; ns: no significant different when p>0.05.</b></p>*<p>THC: tetrahydrocannabinol, † Lp(a): lipoprotein(a), ‡ hsCRP: ultrasensible C-reactive protein.</p><p>(PPAD, premature peripheral arterial diseases; PAOD, peripheral arterial occlusive disease; TAO, thromboangiitis obliterans).</p

Clinical Characteristics of the Study Sample.

<p>Results as mean ± SD; ns: no significant different when p>0.05.</p><p>(PPAD, premature peripheral arterial diseases; PAOD, peripheral arterial occlusive disease; TAO, thromboangiitis obliterans).</p

Risk Factors for PAOD Patients and TAO.

*<p>p<0.001, †p<0.01, ‡ p<0.05, ns: no significant different when p>0.05.</p><p>(PAOD, peripheral arterial occlusive disease; TAO, thromboangiitis obliterans).</p