Journal: PLoS ONE

Article Title: Heterozygous and Homozygous JAK2 V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients

doi: 10.1371/journal.pone.0074257

Figure Lengend Snippet: (A) Cytological analysis showing EryP (red arrows), MK (black arrows), monocytes (green arrows) and granulocytes (blue arrows). (B) iPS cells from P1(H), P2(h) or control were induced for hematopoietic differentiation with EPO (1 U/mL), TPO (20 ng/mL), SCF (25 ng/mL) and IL-3 (10 ng/mL) till day 13. 1x10 5 cells were plated in semi-solid conditions (both methylcellulose and plasma clots) and hematopoietic progenitors were counted in both conditions at day 12 and 10, respectively. (C) Proportions of each progenitor (CFU-MK, CFU-GM and EryP) was calculated for each iPS cell line. (D) Percentage of alpha and beta locus globins was calculated after qRT-PCR in iPS and ES-derived GPA + cells or adult CD34 + -derived erythroblasts. (E) Fold changes represent the amplification of CD41 + GPA + cells at day 12 into CD41 + cells or GPA + cells at day 18. Alternatively, fold changes represent the amplification of CD14 + cells between days 15 and 21. (F) Hematopoietic differentiation potential of iPS cells was quantified by plating one TRA-1-81 + cell per well in a 96-well plate coated with OP9 stromal cells. The absolute number of CD41 + , GPA + and CD14 + cells in each clone was measured by flow cytometry at day 18 (mean ± SEM, n>10; 2 independent experiments) (Mann Whitney test, two-tailed, * P<0.05, ***P<0.001).

Article Snippet: On day 7, EPO (1 U/mL) (Amgen, Thousand Oaks, CA), TPO (20 ng/mL) (Kirin, Tokyo, Japan), SCF (25 ng/mL) (Biovitrum AB, Stockholm, Sweden) and IL-3 (10 ng/mL) (Miltenyi Biotec, Paris, France) were added and on day 11-12, cells were enzymatically dissociated.

Techniques: Quantitative RT-PCR, Derivative Assay, Amplification, Flow Cytometry, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Heterozygous and Homozygous JAK2 V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients

doi: 10.1371/journal.pone.0074257

Figure Lengend Snippet: (A) GPA + CD41 + cells from P1(H), P2(h) or control were plated in methylcellulose in the presence of SCF (25 ng/mL) and increasing concentrations of EPO. EryP colonies were counted 12 days later. (B) The percentage of large EryP (>50 cells per colony) in total EryP was also calculated. Results are the mean ± SEM of 3 independent experiments. (C) Cloning efficiency of GPA + CD41 + cells for each genotype was calculated (D) GPA + cells were cytokine-deprived overnight in serum-free medium and then seeded in IMDM alone for 4 hours. Cells were then stimulated with 10 U/mL EPO or not. (E) CD41 + cells from P1(H), P2(h) or control were plated in plasma clots without or with increasing concentrations of TPO. CFU-MK colonies were counted at day 10 after CD41a indirect staining. Results are the mean ± SEM of 3 independent experiments (*P<0.05). (F) Primary cells from one control and 4 patients (P1(H), P2(h), P3(H) and P4(h)) were grown either with SCF (25 ng/mL) ±EPO (1 U/mL) or with SCF (25 ng/mL) ± TPO (20 ng/mL), and cloned at one progenitor cell/well. The percentage of endogenous erythroid colonies (EEC) or endogenous CFU-MK was calculated for each condition.

Article Snippet: On day 7, EPO (1 U/mL) (Amgen, Thousand Oaks, CA), TPO (20 ng/mL) (Kirin, Tokyo, Japan), SCF (25 ng/mL) (Biovitrum AB, Stockholm, Sweden) and IL-3 (10 ng/mL) (Miltenyi Biotec, Paris, France) were added and on day 11-12, cells were enzymatically dissociated.

Techniques: Clone Assay, Staining

Journal: Blood

Article Title: Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms

doi: 10.1182/blood.2022016846

Figure Lengend Snippet: Induction of differentiation of MK progenitors by CALR-del52. (A) Stability study of rhCALR-del52 colony-forming unit MK culture medium. Six samples maintained at 37°C for various lengths of time were measured in duplicate by ELISA and analyzed using a 1-phase decay model (Prism6) to determine the averaged half-life and R 2 . Error bars represent SDs. CD34 + CD41 + progenitors from 4 to 6 patients with mutated CALR (B), 3 patients with JAK2 V617F (B), and 3 normal controls (C) were sorted and cloned at 1 cell per well in 96-well plates in serum-free medium containing SCF and treated with a single, large dose of CALR-del52. Percentages of MK colonies were calculated compared with the Tpo condition. Results are shown as mean ± SEM. ∗∗∗∗ P < .0001, ∗∗∗ P < .001. One-way analysis of variance, Holm-Sidak multiple comparisons test. (D) Effect of daily treatment of CALR-del52 (0.1, 1, or 5 μg/mL) on MK colony formation. CD34 + CD41 + progenitors from 4 patients with mutated CALR were tested and the percentages of MK colonies were calculated compared with the Tpo condition. Results are shown as mean ± SEM. ∗∗ P < .01. One-way analysis of variance, Holm-Sidak multiple comparisons test.

Article Snippet: CD34 + cells were purified from patient peripheral blood and cultured for 3 days in serum-free medium in the presence of thrombopoietin (Tpo) (20 ng/mL) (Kirin Brewery, Tokyo, Japan) and stem cell factor (SCF) (25 ng/mL) (Biovitrum AB, Stockholm, Sweden).

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay