scholarly journals SPARC: a method to genetically manipulate precise proportions of cells

2019 ◽  
Author(s):  
Jesse Isaacman-Beck ◽  
Kristine C. Paik ◽  
Carl F. R. Wienecke ◽  
Helen H. Yang ◽  
Yvette E. Fisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Calcium Imaging ◽  
Transgene Expression ◽  
Cell Populations ◽  
Individual Animal ◽  
Cell Type ◽  
Targeted Expression ◽  
Experimental Approaches ◽  
Mitotic Cells

AbstractMany experimental approaches rely on controlling gene expression in select subsets of cells within an individual animal. However, reproducibly targeting transgene expression to specific fractions of a genetically-defined cell-type is challenging. We developed Sparse Predictive Activity through Recombinase Competition (SPARC), a generalizable toolkit that can express any effector in precise proportions of post-mitotic cells in Drosophila. Using this approach, we demonstrate targeted expression of many effectors and apply these tools to calcium imaging of individual neurons and optogenetic manipulation of sparse cell populations in vivo.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

scholarly journals Genetic engineering for in vivo optical interrogation of neuronal responses to cell type-specific silencing

2021 ◽  
Author(s):  
Firat Terzi ◽  
Johannes Knabbe ◽  
Sidney B. Cambridge
Keyword(s):  
High Resolution ◽  
Genetic Engineering ◽  
Transgene Expression ◽  
Neuronal Morphology ◽  
Dependent Manner ◽  
Cell Type ◽  
Fluorescent Marker ◽  
Genetic Perturbations ◽  
Cell Type Specific

SummaryGenetic engineering of quintuple transgenic brain tissue was used to establish a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo. Co-expression of a constitutive, Cre-dependent fluorescent marker selectively allowed single cell analyses before and after inducible, tet-dependent transgene expression. Here, we used this method for acute, high-resolution manipulation of neuronal activity in the living brain. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least three days. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, ‘HighFive’) method thus allows visualizing temporally precise, genetic perturbations of defined cells.

Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Liang Du ◽  
Jingwan Zhang ◽  
Alexander Clowes ◽  
David Dichek
Keyword(s):  
Gene Expression ◽  
Blood Flow ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Arterial Blood Flow ◽  
Vein Grafts ◽  
Arterial Blood ◽  
En Face

Background Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins. Hypothesis We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer. Methods Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area. Results B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results. Conclusions Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals Cell-Type Specific Oxytocin Gene Expression from AAV Delivered Promoter Deletion Constructs into the Rat Supraoptic Nucleus in vivo

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e32085 ◽  
Author(s):  
Raymond L. Fields ◽  
Todd A. Ponzio ◽  
Makoto Kawasaki ◽  
Harold Gainer
Keyword(s):  
Gene Expression ◽  
Supraoptic Nucleus ◽  
Cell Type ◽  
Promoter Deletion ◽  
Cell Type Specific

Investigation of cell physiology in the animal using transgenic technology

1992 ◽  
Vol 262 (2) ◽  
pp. C261-C275 ◽  
Author(s):  
A. P. Koretsky
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Regulation Of Gene Expression ◽  
Cell Physiology ◽  
Transgenic Technology ◽  
Cell Type ◽  
Mutant Proteins ◽  
The Past ◽  
Targeted Expression ◽  
Made In

Over the past 10 years significant progress has been made in techniques for manipulating the genome of the animal. Production of transgenic mice has led to important insights into the regulation of gene expression, the molecular basis of cancer, immunology, and developmental biology. The tools necessary to generate transgenic mice are becoming widely available, making it possible to study a variety of problems. In this review a description of the strategies being used to address problems of interest in cell physiology using transgenic mice is given. Elucidation of the rules governing the regulation of gene expression now permits the targeted expression of a protein to a particular organ or cell type within an organ. Overexpression of proteins, expression of foreign or mutant proteins, mislocalization of proteins, and directed elimination of proteins are all procedures that can now be used to generate interesting animal models for physiological studies. The applications of these techniques to a variety of problems in normal and abnormal physiology are discussed in this review.

CD146 Expression in Primary Bone Marrow MSC Progenitor/Stem Cells Is Dependent On Their In Vivo Location.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 251-251 ◽  
Author(s):  
Ariane Tormin ◽  
Ou Li ◽  
Stuart Walsh ◽  
Mats Ehinger ◽  
Jan Claas Brune ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Surface Markers ◽  
Double Positive ◽  
Marrow Space ◽  
Single Positive ◽  
Cd146 Expression

Abstract Abstract 251 Primary MSC progenitor/stem cells (MSC-PC/SC) represent only a minute fraction of the bone marrow cells and they give rise to the well-known mesenchymal stromal cells (MSC) in culture. In vivo, MSC-PC/SC are probable constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Enrichment of primary MSC progenitors, i.e. CFU-F, has been recently reported based on expression of surface markers such as CD271, CD146, GD2, SSEA4, etc. Based on the expression of CD271 and CD146 on primary MSC (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007), we have recently identified two BM subpopulations (CD271+/CD146+/ CD45− and CD271+/CD146−/low/ CD45−) that highly enrich for primary MSC-PC/SC (Tormin et al, Blood 2008, 112[11]:843). These two populations contained all assayable CFU-F and both gave rise to typical cultured MSC (expression of standard surface markers, differentiation capacity into adipocytes, osteoblasts, chondrocytes). Interestingly, MSC derived from CD146−/low cells acquired CD146 expression in culture, and we therefore aimed to further investigate whether CD146 expression correlates to functional differences, e.g. stemness, or possibly differences in localization. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC. Single cell sorting of CD271/CD146/CD45− cells (n=6) confirmed the results of our prior CFU-F experiments, i.e. high enrichment of CFU-F and multipotency in the two putative stem cell populations. Six-color FACS analysis of primary BM cells showed that both populations coexpressed typical MSC markers (CD90, CD105, PDGFR-β, STRO-1), but not GD2, SSEA4, and endothelial markers. Single cell multiplex PCR on sorted primary MSC-PC/SC showed that both cell populations were negative for CD45, but did express “early” genes (Oct4, Sox2, Nanog), marker genes for the adipogenic lineage (CEBPA, LPIN1) and osteogenesis-related genes (ALPL, Runx2). Gene expression of CD146 correlated to its surface expression with some CD146 bands also detected among the CD146−/low sorted population. Next, we investigated possible differences in localization utilizing confocal microscopy of normal human BM sections. Reticular CD271/CD146 double positive and reticular CD271 single positive cells were identified. Double positive cells were mainly located adjacent to larger vessels and sinusoids but were also found in the marrow space. In contrast, CD271 single positive cells were primarily found in the endosteal space. These cells were furthermore negative for CD45 in contrast to CD45 coexpressing CD271+/low cells in the marrow space. As expected, CD146 single positive endothelial cells were found surrounding larger vessels. Thus, expression of CD146 in CD271+/CD45− cells correlated with localization (primarily endosteal versus primarily perivascular) and we therefore hypothesized that CD146 expression might be regulated by hypoxia levels. To test this, MSC were cultured under normoxic versus hypoxic conditions using deferoxamine mesylate (DFO) to mimic hypoxia. When sorted CD271+/CD146−/low/CD45− cells were cultured in normoxia, CD146 expression was lower compared with cultures initiated with CD271+/CD146+/CD45− cells in the beginning and up to the 1st passage. Thereafter, CD146 expression was comparable. However, when established MSC (CD146+) were cultured in the presence of DFO, CD146 expression was clearly downregulated, and after 7 days about 25% of cells became CD146 negative compared to 3% in normoxic controls. No changes were observed for CD90 and CD271 expression. Taken together, CD271+/CD146+/CD45– and CD271+/CD146–/low/CD45– bone marrow cells are putative primary MSC stem/progenitor populations. Both are highly enriched for primary MSC progenitors, they have comparable functional characteristics as well as comparable surface marker and gene expression profiles. Differences in CD146 expression correlated to localization and are likely to be caused by differences in oxygen levels. We therefore conclude that CD146 expression allows to distinguish the vary rare (0.19 ± 0.09%) primary endosteal niche MSC (CD271+/CD146−/low/CD45−) from vascular niche MSC (CD271+/CD146+/CD45−, 0.31 ± 0.13% of BM cells). Disclosures: No relevant conflicts of interest to declare.

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3296-3296
Author(s):  
Raul Teruel Montoya ◽  
Xianguo Kong ◽  
Shaji Abraham ◽  
Lin Ma ◽  
Leonard C. Edelstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Binding Sites ◽  
Transgene Expression ◽  
Cell Types ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematological Disorders

Abstract Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures: No relevant conflicts of interest to declare.

Leukemia Stem Cell Potential of Different Progenitor Subpopulations in Myeloid Blast Phase CML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3489-3489
Author(s):  
Ross Kinstrie ◽  
Dimitris Karamitros ◽  
Nicolas Goardon ◽  
Heather Morrison ◽  
Richard E Clark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Research Funding ◽  
Cell Populations ◽  
Novel Therapies ◽  
Blast Phase ◽  
Self Renewal ◽  
Stem Cell Populations

Abstract Blast phase (BP)-CML remains the most critical area of unmet clinical need in the management of CML and novel, targeted therapeutic strategies are urgently needed. In the tyrosine kinase inhibitor (TKI) era, the rate of progression to BP is 1 to 1.5% per annum in the first few years after diagnosis, falling sharply when major molecular response is obtained. Around 10% of patients present with de novo BP-CML and despite the use of TKIs, median survival after the diagnosis of BP-CML is between 6.5 and 11 months.Therefore, improved understanding of the biology of BP-CML and novel therapies to prolong therapeutic responses are urgently sought. Studies of myeloid malignancies show that acquisition of tumor-associated mutations occurs principally in a step-wise manner. Initiating mutations usually originate in an hematopoietic stem cell (HSC) to give rise to preleukemic stem cell populations that expand through clonal advantage. Further mutation acquisition and/or epigenetic changes then lead to blast transformation and disruption of the normal immunophenotypic and functional hematopoietic hierarchy. At this stage, multiple leukemic stem cell (LSC) populations (also termed leukemia initiating cell populations) can be identified. We previously showed, in AML, that the CD34+ LSC populations were most closely related to normal progenitor populations, rather than stem cell populations, but had co-opted elements of a normal stem cell expression signature to acquire abnormal self-renewal potential (Goardon et al, Cancer Cell, 2011). CD34+CD38- LSCs were most commonly similar to an early multi-potent progenitor population with lympho-myeloid potential (the lymphoid-primed multi-potential progenitor [LMPP]). In contrast, the CD34+CD38+ LSCs were most closely related to the more restricted granulocyte-macrophage progenitor (GMP). In chronic phase CML, the leukemia-propagating population is the HSC, and the progenitor subpopulations do not have stem cell characteristics. To date, studies to isolate LSC populations in BP-CML have been limited, identifying the GMP subpopulation only as a possible LSC source (Jamieson et al, NEJM, 2004). Furthermore, in vivo LSC activity has not been assessed. We therefore set out to assess the LSC characteristics of different primitive progenitor subpopulations in myeloid BP-CML both in vitro and in vivo. We isolated different stem and progenitor cell subpopulations using FACS; HSC (Lin-CD34+CD38-CD90+ CD45RA-), multipotent progenitor (MPP; Lin-CD34+CD38-CD90-CD45RA-), LMPP (Lin-CD34+CD38-CD90-CD45RA+), common myeloid progenitor (CMP; Lin-CD34+CD38+CD45RA-CD123+), GMP (Lin-CD34+CD38+CD45RA+CD123+) and megakaryocyte erythroid progenitor (MEP; Lin-CD34+CD38+CD45RA-CD123-). The functional potential of these purified populations was examined in 13 patients by: (i) serial CFC replating assays to study progenitor self-renewal (n=10); (ii) In vivo xenograft studies using NSG mice with serial transplantation to identify populations with LSC potential (n=6). Our data conclusively demonstrate that functional LSCs are present in multiple immunophenotypic stem/progenitor subpopulations in myeloid BP-CML, including HSC, MPP, LMPP, CMP and GMP subpopulations. There was inter-patient variability in terms of both in vitro and in vivo functional properties. Fluorescence in situ hybridisation (FISH) was used to assess clonality in the different progenitor subpopulations and identify which populations contained cells with additional cytogenetic abnormalities (ACAs) with a view to improving our understanding of the clonal hierarchy. Interestingly, there were no significant differences in ACAs in the different progenitor subpopulations in the majority of samples studied, suggesting that clonal evolution tends to occur in the HSC compartment in myeloid BP-CML. Preliminary gene expression profiling studies of the different progenitor subpopulations, using Affymetrix Human Gene 1.0 ST Arrays, demonstrated highly variable gene expression, supporting the functional heterogeneity seen. Taken together, our results demonstrate that myeloid BP-CML is a very heterogeneous disorder with variable LSC populations. Further interrogation of these populations will likely identify novel therapies which will specifically target the LSC. Disclosures Copland: Bristol-Myers Squibb: Consultancy, Honoraria, Other, Research Funding; Novartis: Consultancy, Honoraria, Other; Ariad: Consultancy, Honoraria, Research Funding.

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