[Show abstract][Hide abstract] ABSTRACT: The dog is recognized as a highly predictive model for pre-clinical research. Its size, life span, physiology and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model hasn't has not been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents, and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for pre-clinical models of genetic diseases. Specifically, we 1) established markers for identification and tracking canine spermatogonial cells; 2) established methods for enrichment and genetic manipulation of these cells; 3) described canine SSC behavior in culture; and 4) demonstrated engraftment of genetically manipulated SSC, and production of transgenic sperm. These findings help set the stage for generation of transgenic canine models via SSC transplantation.
[Show abstract][Hide abstract] ABSTRACT: The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs
remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture
times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor
receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic
cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive
selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher
copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon
insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR
of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion
of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing
Preview · Article · Mar 2012 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained <or=1.7 x 10(7) TNC/kg. Recipients were given 9.2 Gy total-body irradiation (TBI) and DLA-matched unrelated cord blood with postgrafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with 1 dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (P = .03, log-rank test); neutrophil and platelet recovery was delayed (both P = .005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the nonengrafted unit facilitates engraftment of the dominant unit.
Full-text · Article · Mar 2010 · Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation
[Show abstract][Hide abstract] ABSTRACT: Dogs given nonmyeloablative conditioning and marrow grafts from 2 dog leukocyte antigen (DLA)-identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. In this study, we used trichimeras to evaluate strategies for adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from 2 DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy of total body irradiation (TBI) and given a short course of postgraft immunosuppression. After stable hematopoietic engraftment was confirmed, a kidney was transplanted from 1 of the 2 marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes from each kidney donor were then used to sensitize the alternate marrow donor. The trichimeric recipients were given donor lymphocyte infusions (DLIs) from the sensitized dogs and monitored for chimerism, graft-versus-host disease (GVHD), and kidney rejection. After DLI, we observed both prompt rejection of the transplanted marrow and donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably due to shared minor histocompatibility antigens, host chimerism also disappeared, and GVHD in skin, gut, and liver developed. The native kidneys, although exhibiting lymphocytic infiltration, remained functionally normal. This study demonstrates that under certain experimental conditions, the kidney--an organ ordinarily not involved in graft-versus-host reactions--can be targeted by sensitized donor lymphocytes.
Full-text · Article · Dec 2008 · Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation
[Show abstract][Hide abstract] ABSTRACT: Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.
[Show abstract][Hide abstract] ABSTRACT: Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.
[Show abstract][Hide abstract] ABSTRACT: Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix. Using artificial mixtures of retrovirally marked, single-cell-derived clones, we demonstrate that LAM-PCR fails to detect 30-40% of the clones, even after exhaustive analysis. Furthermore, the relative abundance of specific clones within a mix is not accurately represented, deviating by as much as 60-fold from their true abundance. We describe an optimized, multiarm, high-throughput modification of LAM-PCR that improves the global detection capacity to greater than 90% with exhaustive sampling, facilitates accurate estimates of the total pool size from smaller samplings, and provides a rapid, cost-effective approach to the generation of large insertion-site data bases required for evaluation of vector integration preferences. The inability to estimate the abundance of individual clones within mixtures remains a serious limitation. Thus, although LAM-PCR is a powerful tool for identification of integration sites and for estimations of clonal complexity, it fails to provide the semiquantitative information necessary for direct, reliable tracking of individual clones in a chimeric background.
Full-text · Article · Jul 2007 · Stem Cells and Development
[Show abstract][Hide abstract] ABSTRACT: The human osteosarcoma-derived cell line, SAOS-2, exhibits many of the phenotypic characteristics of osteoblasts including the deposition of types I and V collagens in an extracellular matrix. Lesser amounts of collagen XI chains were also detected. The cell layer collagen contains hydroxylysyl pyridinoline cross-links but without the accompanying lysyl pyridinoline typical of human bone collagen. This indicates that the lysine residues at the two helical cross-linking loci are fully hydroxylated. The isoform of lysyl hydroxylase, LH1, known to be required for full hydroxylation at these sites, was shown to be highly expressed by SAOS-2 cells. Our findings provide insight on the mechanism of post-translational overmodification of lysine residues in collagen made by osteosarcoma tumors, and may be relevant for understanding a similar overmodification observed in osteoporotic bone.
[Show abstract][Hide abstract] ABSTRACT: Nearly 15 years have elapsed since the US Food and Drug Administration last approved a major new hematopoietic cytokine. Promiscuous binding to multiple receptors, or to receptors expressed by multiple tissues, reduces growth factor specificity and promotes side effects. Here we show that hematopoiesis can be differentially regulated using receptors rather than ligands. Conditional derivatives of both fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) induced a sustained expansion of mouse marrow cells ex vivo, and erythroid cells in vivo. Only F36VFGFR1 could support the ex vivo expansion of short-term repopulating hematopoietic stem cells (HSCs), the ex vivo survival of long-term repopulating HSCs, and the prolonged in vivo expansion of granulocytes, monocytes, and platelets. Only F36VMpl induced a response sufficiently rapid to accelerate recovery from radiation-induced anemia. These results establish receptors as a new class of hematopoietic regulators possessing activities unobtainable with growth factors.
[Show abstract][Hide abstract] ABSTRACT: Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate. Only differentiating erythroid precursors and multipotential and erythroid progenitors (colony-forming unit [CFU]-mix and burst forming unitserythroid [BFUe]) responded; however, the nature of the response differed markedly between bone marrow and spleen. In the marrow, F36VMpl induced a 12- to 17-fold expansion of differentiated erythroid precursors and a loss of CFU-mix and BFUe. In the spleen, F36VMpl induced a marked rise in BFUe and CFU-mix and, relative to marrow, a much less prominent rise in more mature red cells. Clonal analysis was most consistent with the interpretation that the spleen and bone marrow differentially regulate the response of human progenitors to a mitotic signal, possibly influencing progenitor expansion versus differentiation. These findings establish CIDs as in vivo growth factors for human hematopoietic cells.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific
gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the β-globin locus control
region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated
virus (AAV) inverted terminal repeats (Ad.LCR-β-GFP). This vector possesses the adenovirus type 35 fiber knob that allows
efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth
factor-dependent CD34+ erythroleukemic cell line, and in cord blood-derived human CD34+ cells. Stable transduction of MO7e cells with Ad.LCR-β-GFP was more efficient and less subject to position effects and silencing
than transduction with a vector that did not contain the β-globin LCR. Analysis of integration sites indicated that Ad.LCR-β-GFP
integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed
integration sites were within the chromosomal β-globin LCR. None of the Ad.LCR-β-GFP integrations occurred in exons. The integration
pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the β-globin LCR-containing
vector. Infection of primary CD34+ cells with Ad.LCR-β-GFP did not affect the clonogenic capacity of CD34+ cells. Transduction of CD34+ cells with Ad.LCR-β-GFP resulted in vector integration and erythroid lineage-specific GFP expression.
Full-text · Article · Oct 2005 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Methods for regulating the growth of transplanted cells may have many applications in gene and cell therapy. One such method uses conditional signaling molecules that can be activated in response to artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CID-triggered growth signal, in vivo. CD34+ cells transduced with a lentivirus vector encoding a derivative of the thrombopoietin receptor (F36VMpl) and green fluorescent protein (GFP) were transplanted into immune deficient mice. CID treatment was associated with a >12 – fold expansion of GFP+ CD71+ erythroid cells that were localized mainly in the marrow, and a unique subset of multipotential and erythroid progenitors that were confined to the spleen. Both the CD71+ cells and the progenitor colonies were intensely GFP+. The CID response was oligoclonal, with most colonies containing a single transgene copy. Despite expressing F36Vmpl, GFP+ B lymphoid and myeloid cells showed no response. CID-responsive progenitors and CD71+ cells were detectable for up to 5 weeks following the end of CID; however these effects were not sustained in secondary transplant recipients. These findings establish CIDs as growth factors for genetically modified human hematopoietic cells.The University of Washington has submitted a patent application for this application. Any royalties accruing from this application may lead to income for Dr. Blau.
Full-text · Article · Aug 2005 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy. The only known route of clearance of busulfan is by GST-mediated conjugation. Using a fibroblast cell line as a model, we have tested the effects of overexpression of three human GST (GSTA1, GSTP1, and MGSTII) on cell survival under a busulfan or melphalan challenge. In two separate assay formats using chronic exposure to busulfan, MGSTII conferred a reproducible twofold selective advantage. GSTA1 and GSTP1 had no effect on busulfan resistance, and melphalan resistance was not affected by expression of any of the GSTs in these assays. In an acute (24-hour) melphalan exposure assay, MGSTII conferred about a twofold selective advantage. Busulfan was not toxic in this assay. RTPCR analysis of human bone marrow CD34+ cells showed that MGSTII is not highly expressed in this stem/early progenitor population. These data indicate that MGSTII may be a useful selective agent in hematopoietic gene therapy.
No preview · Article · Feb 2005 · Cancer Investigation
[Show abstract][Hide abstract] ABSTRACT: A multivariate analysis of 121 dogs conditioned with 200, 100, or 50 cGy of total body irradiation (TBI) followed by hematopoietic stem cell transplantation from matched littermates showed that TBI dose was the only factor examined that was statistically significantly associated with the percentage of donor myeloid engraftment in stable long-term chimeras ( P = .008). To understand the direct effects of low-dose irradiation on hematopoietic stem/progenitor cells, nonirradiated and irradiated human CD34 + cells were evaluated for competitive repopulating ability in nonobese diabetic/severe combined immunodeficiency beta2m -/- mice. As expected, the results showed a radiation dose-dependent loss of competitive repopulating ability. Flow cytometric analysis indicated that, within a viable cell gate, there was reduced expression of P-selectin glycoprotein ligand-1 and L selectin on irradiated compared with nonirradiated CD34 + cells; this suggests that irradiated stem/progenitor cells may be compromised in their ability to home to or interact with the marrow microenvironment. However, the CD34 + /P-selectin glycoprotein ligand-1 dim cells also showed activation of caspase-3, indicating that they were destined to die. These results suggest that the TBI dose determines the degree of myeloid engraftment by compromising the resident stem/progenitor cell compartment.
No preview · Article · Jan 2005 · Biology of Blood and Marrow Transplantation
[Show abstract][Hide abstract] ABSTRACT: HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.
[Show abstract][Hide abstract] ABSTRACT: Leukemias arising in 2 patients treated with gene therapy for X-SCID may be viewed to result from a collaboration between a transcription factor, abberantly expressed due to insertional mutagenesis, and a constitutively active, receptor-initiated growth signal. We are undertaking a systematic comparison of retrovirus integration sites in transduced hemopoietic cells prior to and following in vivo selection. The system for selection uses a conditional derivative of the thrombopoietin receptor, F36Vmpl, that transmits a growth signal in the presence of an artificial ligand called a chemical inducer of dimerization (CID). Our initial studies have focused on comparing insertion sites identified by Southern analysis with those identified by LAM-PCR. A cohort of 11 mice was transplanted with marrow cells transduced with a bicistronic vector encoding green fluorescent protein (GFP) and F36Vmpl. Marrow aspirates were obtained at baseline and following administration of 3 monthly cycles of CID (AP20187 10mg/kg IP daily for 3 days). 10 of 11 mice exhibited strong responses to CID treatment, with the percentage of GFP positive red cells rising from
Full-text · Article · Jan 2004 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side "population" (SP) of bone marrow. This SP from bone marrow, as well as other tissues, is reported to contain immature stem cells with considerable plasticity. Some cell lines also efflux Hoechst and generate SP profiles. Reverse transcription-polymerase chain reaction (RT-PCR) and efflux inhibition studies with the lung carcinoma cell line, A549, implicated the ABCG2 transporter as a Hoechst efflux pump. Furthermore, it is shown that transient expression of ABCG2 generates a robust SP phenotype in human embryonic kidney (HEK293) cells. The results allow the conclusion that ABCG2 is a potent Hoechst efflux pump. Semiquantitative RT-PCR was used to characterize the developmental pattern of expression of ABCG2 in hematopoiesis. It is expressed at relatively high levels in putative hematopoietic stem cells (isolated as SP, 34+/38- or 34+/KDR+ populations) and drops sharply in committed progenitors (34+/38+, 34+/33+, or 34+/10+). Expression remains low in most maturing populations, but rises again in natural killer cells and erythroblasts. Comparison of messenger RNA (mRNA) levels for the 3 major multidrug-resistant efflux pumps, MDR1, MRP1, and ABCG2, in bone marrow SP cells reveals that ABCG2 is the predominant form in these cells. These data suggest that ABCG2 contributes significantly to the generation of the SP phenotype in hematopoietic stem cells. Furthermore, the sharp down-regulation of ABCG2 at the stage of lineage commitment suggests that this gene may play an important role in the unique physiology of the pluripotent stem cell.
[Show abstract][Hide abstract] ABSTRACT: Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.
[Show abstract][Hide abstract] ABSTRACT: The Swarm rat chondrosarcoma cell line, RCS-LTC, deposits an extracellular matrix that contains the typical type II, IX, and XI collagen phenotype of hyaline cartilage, but the fibrils appear abnormally thin. By N-terminal sequence analysis, the type II collagen from the matrix was shown to have retained its N-propeptides with no evidence of normal processing to type II collagen. Amplification and sequencing of cDNA prepared from the pro alpha1(II) mRNA of these cells showed a normal N-propeptide cleavage site. Furthermore, the type II N-procollagen could be processed to type II collagen by incubation with culture medium from normal chondrocytes. The findings indicate that the RCS-LTC cell line fails to express an active type II procollagen N-proteinase and, therefore, offers a useful culture system in which to study the role of N-propeptide removal in fibrillogenesis.
Preview · Article · Aug 1997 · European Journal of Biochemistry