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: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.

Project description:BACKGROUND: BCR-ABL1+ chronic myeloid leukemia (CML) is characterized by abnormal production of leukemic stem (LSC) and progenitor cells and their spread from the bone marrow into the blood resulting in extramedullary myeloproliferation. So far, little is known about specific markers and functions of LSC in CML. METHODS: We examined the phenotype and function of CD34+/CD38─/Lin─ CML LSC by a multi-parameter screen approach employing antibody-phenotyping, mRNA expression profiling, and functional studies, including LSC repopulation experiments in irradiated NOD-SCID-IL-2Rgamma-/- (NSG) mice, followed by marker-validation using diverse control-cohorts and follow-up samples of CML patients treated with imatinib. RESULTS: Of all LSC markers examined, dipeptidylpeptidase IV (DPPIV=CD26) was identified as specific and functionally relevant surface marker-enzyme on CD34+/CD38─ CML LSC. CD26 was not detected on normal CD34+/CD38─ stem cells or LSC in other hematopoietic malignancies. The percentage of CD26+ CML LSC decreased to undetectable levels during successful treatment with imatinib in all patients (p<0.001). Whereas the sorted CD26─ stem cells obtained from CML patients engrafted irradiated NSG mice with multilineage BCR-ABL1-negative hematopoiesis, CD26+ LSC engrafted NSG mice with BCR-ABL1+ cells. Functionally, CD26 was identified as target-enzyme disrupting the SDF-1alpha-CXCR4-axis by cleaving SDF-1alpha a chemotaxin for CXCR4+ stem cells. Whereas CD26 was found to inhibit SDF-1alpha-induced migration, CD26-targeting gliptins reverted this effect and blocked the mobilization of CML LSC in a stroma co-culture assay. CONCLUSIONS: CD26 is a robust biomarker of LSC and a useful tool for their quantification and isolation in patients with BCR/ABL1+ CML. Moreover, CD26 expression may explain the extramedullary spread of LSC in CML. To define specific mRNA expression patterns and to identify specific LSC markers in CML LSC, gene array analyses were performed. RNA was isolated from sorted CD34+/CD45+/CD38─ CML LSC, CD34+/CD45+/CD38+ CML progenitor cells, CML MNC, sorted CD34+/CD38─ cord blood (CB) SC, CB-derived CD34+/CD38+ progenitor cells, and CB MNC. Total RNA was extracted from sorted cells using RNeasy Micro-Kit (Qiagen) and used (100 ng total RNA) for Gene Chip analyses. Preparation of terminal-labeled cRNA, hybridization to genome-wide human PrimeView GeneChips (Affymetrix, Santa Clara, CA, USA) and scanning of arrays were carried out according to the manufacturer's protocols (https://www.affymetrix.com). Robust Multichip Average (RMA) signal extraction and normalization were performed according to http://www.bioconductor.org/ as described.18 Differences in mRNA expression levels (from multiple paired samples) were calculated as mRNA ratio of i) CML LSC versus CB SC, ii) CML LSC versus CD34+/CD38+ CML progenitors, and normal cord blood SC versus cord blood progenitors. To calculate differential gene expression between individual sample groups where appropriate, we performed a statistical comparison using the LIMMA package as described previously. Briefly, LIMMA estimates the fold change between predefined sample groups by fitting a linear model and using an empirical Bayes method to moderate the standard errors of the estimated log-fold changes for each probe set.

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Goat Model for CML

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