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Goat Model for CML

ABSTRACT: To evaluate the long-term growth potential of BCR-ABL-transduced primitive human hematopoietic cells, lin- cord blood cells containing an MSCV-BCR-ABL-IRES-GFP (BCR-ABL) or control-GFP transgene (MIG) were injected IP into fetal goats at 45-55 days of gestation. Six transplant goats were born alive. One was examined three weeks after birth and showed GFP+ cells in the blood, bone marrow (BM), liver, kidney, lung, heart, and both skeletal and smooth muscle. FISH analysis also showed the liver of this goat contained BCR-ABL-GFP transgenic cells. The remaining five goats appear normal although, in some, the WBC count is elevated 3- to 5-fold. GFP+ cells, including cells identifiable by FACS as human CD34+ cells, have been detected in the blood of all these goats. The presence of BCR-ABL-GFP transgenic cells in the BM and liver was confirmed by FISH analysis, and quantitative real-time PCR analysis of genomic DNA isolated from unpurified BM cells obtained from three of the transplant goats demonstrated 3-5×104 copies of the transgene per microgram of DNA. Microarray transcript profiling was performed on blood and liver tissues of normal goats, BCR-ABL chimeric goats, MIG chimeric goats, and normal human samples. RNA for human genes was detected in goats transplanted with cord blood cells but not in normal goats, and the RNA abundance of some genes in BCR-ABL chimeric goat blood was similar to or greater than levels observed in MIG goat blood or normal human samples. Quantitative RT-PCR confirmed the differential expression of several genes in goats carrying the BCR-ABL vs. control transgene. These results demonstrate long-term engraftment but slow expansion in a large animal model of primitive human hematopoietic cells transduced with a BCR-ABL fusion gene and transplanted in utero. This novel xenotransplant goat model should be useful for analyzing the initial phases of development of human CML and for assessing new therapies with potential long-term benefits.

ORGANISM(S): Capra hircus Homo sapiens

PROVIDER: GSE17588 | GEO | 2009/08/12

SECONDARY ACCESSION(S): PRJNA118521

REPOSITORIES: GEO

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Project description:To evaluate the long-term growth potential of BCR-ABL-transduced primitive human hematopoietic cells, lin- cord blood cells containing an MSCV-BCR-ABL-IRES-GFP (BCR-ABL) or control-GFP transgene (MIG) were injected IP into fetal goats at 45-55 days of gestation. Six transplant goats were born alive. One was examined three weeks after birth and showed GFP+ cells in the blood, bone marrow (BM), liver, kidney, lung, heart, and both skeletal and smooth muscle. FISH analysis also showed the liver of this goat contained BCR-ABL-GFP transgenic cells. The remaining five goats appear normal although, in some, the WBC count is elevated 3- to 5-fold. GFP+ cells, including cells identifiable by FACS as human CD34+ cells, have been detected in the blood of all these goats. The presence of BCR-ABL-GFP transgenic cells in the BM and liver was confirmed by FISH analysis, and quantitative real-time PCR analysis of genomic DNA isolated from unpurified BM cells obtained from three of the transplant goats demonstrated 3-5Ã104 copies of the transgene per microgram of DNA. Microarray transcript profiling was performed on blood and liver tissues of normal goats, BCR-ABL chimeric goats, MIG chimeric goats, and normal human samples. RNA for human genes was detected in goats transplanted with cord blood cells but not in normal goats, and the RNA abundance of some genes in BCR-ABL chimeric goat blood was similar to or greater than levels observed in MIG goat blood or normal human samples. Quantitative RT-PCR confirmed the differential expression of several genes in goats carrying the BCR-ABL vs. control transgene. These results demonstrate long-term engraftment but slow expansion in a large animal model of primitive human hematopoietic cells transduced with a BCR-ABL fusion gene and transplanted in utero. This novel xenotransplant goat model should be useful for analyzing the initial phases of development of human CML and for assessing new therapies with potential long-term benefits. Experiment Overall Design: Total RNA was extracted from liver (L) and blood (B) samples of normal goats (ng), humans (hu), chimeric goats engrafted with human cord blood stem cells containing control (mig) vector, and chimeric goats engrafted with CML (bcrabl) vector. RNA samples were profiled on Affymetrix human U133A GeneChips and examined for differentially expressed genes in CML vs control goats, filtering for signals significantly above background levels observed in normal goat to select for specific human gene expression.

Project description:MiR-142 is dynamically expressed and plays a regulatory role in hematopoiesis. Based on the simple observation that miR-142 levels are significantly lower in CD34+CD38- cells from blast crisis (BC) chronic myeloid leukemia (CML). CML patients compared with chronic phase (CP) CML patients (p=0.002), we hypothesized that miR-142 deficit plays a role in BC transformation. To test this hypothesis, we generated a miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mouse by crossing a miR-142−/− mouse with a miR-142+/+BCR-ABL mouse. While the miR-142+/+BCR-ABL mice developed and died of CP CML, the miR-142−/−BCR-ABL mice developed a BC-like phenotype in the absence of any other acquired gene mutations and died significantly sooner than miR-142+/+BCR-ABL CP controls (p=0.001). Leukemic stem cell (LSC)-enriched Lineage-Sca-1+c-Kit+ cells (LSKs) from diseased miR-142−/−BCR-ABL mice transplanted into congenic recipients, recapitulated the BC features thereby suggesting stable transformation of CP-LSCs into BC-LSCs in the miR-142 KO CML mouse. Single cell (sc) RNA-seq profiling showed that miR-142 deficit changed the cellular landscape of the miR-142−/−BCR-ABL LSKs compared with miR-142+/+BCR-ABL LSKs with expansion of myeloid-primed and loss of lymphoid-primed factions. Bulk RNA-seq analyses along with unbiased metabolomic profiling and functional metabolic assays demonstrated enhanced fatty acid β-oxidation (FAO) and oxidative phosphorylation (OxPhos) in miR-142−/−BCR-ABL LSKs vs miR-142+/+BCR-ABL LSKs. MiR-142 deficit enhanced FAO in miR-142−/−BCR-ABL LSKs by increasing the expression of CPT1A and CPT1B, that controls the cytosol-to-mitochondrial acyl-carnitine transport, a critical step in FAO. MiR-142 deficit also enhanced OxPhos in miR-142−/−BCR-ABL LSKs by increasing mitochondrial fusion and activity. As the homeostasis and activity of LSCs depend on higher levels of these oxidative metabolism processes, we then postulate that miR-142 deficit is a potentially druggable target for BC-LSCs. To this end, we developed a novel CpG-miR-142 mimic oligonucleotide (ODN; i.e., CpG-M-miR-142) that corrected the miR-142 deficit and alone or in combination with a tyrosine kinase inhibitor (TKI) significantly reduced LSC burden and prolonged survival of miR-142−/−BCR-ABL mice. The results from murine models were validated in BC CD34+CD38- primary blasts and patient-derived xenografts (PDXs). In conclusion, an acquired miR-142 deficit sufficed in transforming CP-LSCs into BC-LSCs, via enhancement of bioenergetic oxidative metabolism in absence of any additional gene mutations, and likely represent a novel therapeutic target in BC CML.

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Goat Model for CML

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