qMRLab: Quantitative MRI analysis, under one umbrella

ABSTRACT: Magnetic resonance imaging (MRI) has revolutionized the way we look at the human body. However, conventional MR scanners are not measurement devices. They produce digital images represented by “shades of grey”, and the intensity of the shades depends on the way the images are acquired. This is why it is difficult to compare images acquired at different clinical sites, limiting the diagnostic, prognostic, and scientific potential of the technology. Quantitative MRI (qMRI) aims to overcome this problem by assigning units to MR images, ensuring that the values represent a measurable quantity that can be reproduced within and across sites. While the vision for quantitative MRI is to overcome site-dependent variations, this is still a challenge due to variability in the hardware and software used by MR vendors to produce quantitative MRI maps. Although qMRI has yet to enter mainstream clinical use, imaging scientists see great promise in the technique’s potential to characterize tissue microstructure. However, most qMRI tools for fundamental research are developed in-house and are difficult to port across sites, which in turn hampers their standardization, reproducibility, and widespread adoption. To tackle this problem, we developed qMRLab, an open-source software package that provides a wide selection of qMRI methods for data fitting, simulation and protocol optimization Figure 1. It not only brings qMRI under one umbrella, but also facilitates its use through documentation that features online executable notebooks, a user friendly graphical user interface (GUI), interactive tutorials and blog posts

Variable behavior of iPSCs derived from CML patients for response to TKI and hematopoietic differentiation.

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents

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

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

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

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