Correction of metachromatic leukodystrophies (MLD) in the mouse model by transplantation of genetically modified hematopoietic stem cells

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

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466. Modeling and Immune Monitoring of Human Adaptive T and B Antigen-Specific Responses in Humanized Mice Upon Immunizations With Lentivirus-Induced Dendritic Cells: Potential Application for Functional Evaluation of Genetically Modified Human Hematopoietic Stem Cells

Molecular Therapy ◽

10.1016/s1525-0016(16)35479-x ◽

2014 ◽

Vol 22 ◽

pp. S179

Keyword(s):

Stem Cells ◽

Dendritic Cells ◽

Hematopoietic Stem Cells ◽

Potential Application ◽

Genetically Modified ◽

Immune Monitoring ◽

Humanized Mice ◽

Functional Evaluation ◽

Hematopoietic Stem

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Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity

Blood ◽

10.1182/blood-2008-02-138214 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2713-2721 ◽

Cited By ~ 70

Author(s):

Qizhen Shi ◽

Scot A. Fahs ◽

David A. Wilcox ◽

Erin L. Kuether ◽

Patricia A. Morateck ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Factor Viii ◽

Hemophilia A ◽

Genetically Modified ◽

Hematopoietic Stem ◽

Inhibitory Antibodies ◽

Sublethal Irradiation

Abstract Although genetic induction of factor VIII (FVIII) expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of preexisting FVIII inhibitory antibodies to determine whether such antibodies would affect therapeutic engraftment. We generated a line of transgenic mice (2bF8) that express FVIII only in platelets using the platelet-specific αIIb promoter and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from heterozygous 2bF8 transgenic (2bF8tg+/−) mice was transplanted into immunized FVIIInull mice after lethal or sublethal irradiation. After BM reconstitution, 85% of recipients survived tail clipping when the 1100-cGy (myeloablative) regimen was used, 85.7% of recipients survived when 660-cGy (nonmyeloablative) regimens were used, and 60% of recipients survived when the recipients were conditioned with 440 cGy. Our further studies showed that transplantation with 1% to 5% 2bF8tg+/− BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not negate engraftment of 2bF8 genetically modified hematopoietic stem cells, and transplantation of these hematopoietic stem cells can efficiently restore hemostasis to hemophilic mice with preexisting inhibitory antibodies under either myeloablative or nonmyeloablative regimens.

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Copy Number Determination of Genetically-Modified Hematopoietic Stem Cells

Genetic Modification of Hematopoietic Stem Cells - Methods in Molecular Biology ◽

10.1007/978-1-59745-409-4_19 ◽

2009 ◽

pp. 281-298 ◽

Cited By ~ 3

Author(s):

Todd Schuesler ◽

Lilith Reeves ◽

Christof von Kalle ◽

Elke Grassman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Copy Number ◽

Genetically Modified ◽

Hematopoietic Stem ◽

Copy Number Determination

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Abstract 12653: The Therapy-related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-ischemic Heart Failure in a Murine Model

Circulation ◽

10.1161/circ.142.suppl_3.12653 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Yoshimitsu Yura ◽

Emiri Miura-Yura ◽

Kenneth Walsh

Keyword(s):

Stem Cells ◽

Angiotensin Ii ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Cancer Patients ◽

Nlrp3 Inflammasome ◽

Cardiac Dysfunction ◽

Hematopoietic Stem ◽

Cardiac Stress ◽

Clonal Hematopoiesis

Background: Therapy-related clonal hematopoiesis in cancer patients is typically associated with somatic mutations in hematopoietic cell genes that encode regulators of the DNA-damage response (DDR) pathway. The Protein Phosphatase Mg2+/Mn2+ Dependent 1D ( PPM1D ) gene is the most frequently mutated DDR gene associated with therapy-related clonal hematopoiesis. While epidemiological evidence suggests an association between therapy-related clonal hematopoiesis and cardiovascular disease in cancer patients, causal and mechanistic relationships have never been evaluated in an experimental system. Methods: To test whether hematopoietic cell mutations in PPM1D can increase the susceptibility to cardiac stress, we evaluated cardiac dysfunction in response to angiotensin II infusion in a mouse model where clonal-hematopoiesis-associated mutations in Ppm1d were produced by CRISPR-Cas9 technology. Results: Mice transplanted with hematopoietic stem cells containing clinically relevant mutations in exon 6 of Ppm1d exhibited augmented cardiac remodeling following the continuous infusion of angiotensin II. Ppm1d -mutated macrophages showed impairments in the DDR pathway and had an augmented proinflammatory profile. Mice transplanted with Ppm1d mutated cells exhibited elevated IL-1β in the stressed myocardium, and bone marrow derived macrophages produced more IL-1β in response to LPS stimulation. The administration of an NLRP3 inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d -mutated hematopoietic stem cells under conditions of Angiotensin II-induced stress. Conclusions: A mouse model of Ppm1d -mediated clonal hematopoiesis was more susceptible to cardiac stress following of angiotensin II infusion. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that therapy-related clonal hematopoiesis involving mutations in PPM1D could contribute to the cardiac dysfunction observed in cancer survivors.

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