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. 2024 Mar 1;109(3):968-973.
doi: 10.3324/haematol.2023.283858.

Engineering a humanized animal model of polycythemia vera with minimal JAK2 V617F mutant allelic burden

Tyler M Parsons  1 , Aishwarya Krishnan  1 , Wangisa M B Dunuwille  1 , Andrew L Young  1 , Jason Arand  1 , Wentao Han  1 , Grant A Challen  2
Affiliations

Engineering a humanized animal model of polycythemia vera with minimal JAK2 V617F mutant allelic burden

Tyler M Parsons et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Engineering a humanized model of polycythemia vera. (A) Knock-in efficiency of VF and VV mutations in CD34+ cells of indicated source determined by next-generation sequencing. (B) Engraftment of human cells in the peripheral blood of NSGS mice determined by flow cytometry. (C) Variant allele frequency (VAF) of engineered mutations in the peripheral blood (PB) of NSGS mice determined by digital droplet plolymerase chain reaction. (D) Lineage distribution of engrafted human CD45+ cells in the PB of NSGS mice. (E) Blood counts of indicated recipient groups across the experimental time course. (F) Spleen weights of mice receiving indicated human cells. (G) Engraftment of human cells in the spleens of NSGS mice. N=5-7 mice per group, data are compiled from 2 independent experiments. *P<0.05, **P<0.01, ***P<0.001. Mean ± standard error of the mean values are shown. NSGS: age-matched irradiated non-transplanted mice; WBC: white blood cells; BM: bone marrow; CB: cord blood; VF: JAK2V617F; VV: JAK2V617V.
Figure 2.
Figure 2.
Manifestation of polycythemia vera pathologies from a minimal JAK2V617F mutant allele burden. (A) Engraftment of human cells in the bone marrow (BM) of NSGS mice. (B) Lineage distribution of engrafted human CD45+ cells in the BM of NSGS mice. (C) Absolute number of human hematopoietic stem cells (HSC) in BM of NSGS recipient mice for each donor group. (D) Western blot analysis showing activation of the JAK/STAT pathway in VF targeted cells. (E) Variant allele frequency (VAF) of engineered mutations in BM of NSGS mice determined by digital droplet polymerase chain reaction. (F) VAF of engineered mutations specifically within human cell fractions. N=5-7 mice per group, data are compiled from 2 independent experiments. *P<0.05, ***P<0.001. Mean ± standard error of the mean values are shown. NSGS: age-matched irradiated non-transplanted mice; CB: cord blood; VF: JAK2V617F; VV: JAK2V617V.
Figure 3.
Figure 3.
Histopathology of xenografted mice. (A) Representative bone marrow (BM) sections of NSGS mice from indicated groups showing reticulin staining. (B) Quantification of the degree of reticulin fibrosis in BM of recipient mice from indicated groups. (C) Representative histological images of BM sections of NSGS mice from indicated recipient groups. (D) Histopathology showing multinucleated megakaryocytes (MK) in BM of a mouse receiving BM-derived JAK2V617F targeted cells. (E) Quantification of the percentage of dysmorphic and multinucleated MK in the BM of recipient mice from indicated groups. (F) Correlations of BM reticulin fibrosis grade with pathological parameters (non-linear regression) for recipients transplanted with BM-derived JAK2V617F-targeted cells. Line of best fit (red) and 95% confidence intervals are shown. N=5-7 mice per group, data are compiled from 2 independent experiments. *P<0.05, ***P<0.001. Mean ± standard error of the mean values are shown. NSGS: age-matched irradiated non-transplanted mice; CB: cord blood; HCT: hematocrit; WBC: white blood cells; Hb: hemoglobin; Plt: platelets; MK: megakaryocytes; VF: JAK2V617F; VV: JAK2V617V.
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