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C Anthony Blau

Publications and source records attributed to C Anthony Blau.

At least 19 recordsLinked to original sources
PubMed2006

A comparison of NIH-approved human ESC lines.

In October 2003, the NIH established three extramural "Exploratory Centers for Human Embryonic Stem Cell Research." Our center acquired 15 of the 22 NIH-approved cell lines. Lines were tested for: (a) freedom from mycoplasma contamination; (b) appropriate pattern of gene expression during self-renewal and differentiation; (c) ability to adapt to uniform culture conditions; (d) ability to grow at clonal densities; (e) karyotype; (f) growth efficiency; and (g) efficiency of stable transfection following electroporation. One line harbored mycoplasma. Ten lines were converted to uniform conditions. Nine lines were fully characterized. Human ESC (hESC) lines varied markedly with respect to growth efficiency as measured by the amount of time it took to plate and double (31-57 hours), cloning efficiency (0.8%-9.2%), and stable transfection rates following electroporation (0%-53% relative to a standard mouse ESC line). One hESC line had an unstable karyotype at an early passage. Modifications of the proposed Material Transfer Agreements with hESC suppliers were required to improve accessibility to hESC lines by local researchers. The NIH-approved hESC lines vary in their behavior in culture. Many hESC lines can be maintained using culture conditions less onerous than those recommended by their suppliers. Intellectual property issues pose a significant obstacle to research using NIH-approved hESC lines.

Animals↗
PubMed2006

Growth factor receptors as regulators of hematopoiesis.

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.

Amino Acid Substitution↗
PubMed2006

Establishment of cell lines that exhibit correct ontogenic stage-specific gene expression profiles from tissues of yeast artificial chromosome transgenic mice using chemically induced growth signals.

Transgenic mice produced with human yeast artificial chromosomes (YACs) generally display transgene expression patterns that reflect those of the normal human host. Because mice are expensive and time-consuming to generate and maintain, extensive mutation-phenotype correlation studies cannot be readily carried out. Cell lines are better suited for analysis of a plethora of mutations. However, these types of gene regulatory studies have been complicated by the lack of suitable cell lines, most of which do not exactly replicate the gene expression patterns observed in vivo. We reasoned that cells established from tissues of YAC transgenic mice might express the transgenes in the correct tissue and developmental stage-specific pattern from which they were derived because YAC transgenic mice display correct regulation of gene expression during ontogeny. We used our human beta-globin locus YAC (beta-YAC) transgenic mice to demonstrate this approach. All existing erythroid cell lines coexpress beta-like globins from different developmental stages or express them inappropriately based on the developmental stage from which they were obtained. Cell populations were established from the adult bone marrow (BM) of beta-YAC transgenic mice, which express exclusively adult beta-globin, using dimerizer technology. A derivative of the thrombopoietin receptor (mpl) was used to bring the proliferative status of primary BM marrow cells under the control of a small molecule drug called a chemical inducer of dimerization (CID). Cells generated in this manner can be expanded to extremely large numbers, remain strictly CID-dependent, and retain megakaryocytic, erythroid, and granulocytic potential. Marrow cells transduced with a retrovirus vector encoding the mpl derivative proliferated extensively in the presence of the CID, AP20187. RNAse protection assays demonstrated that the transcripts for human beta-globin and mouse alpha-globin were present, while gamma-globin transcripts were absent, thus, these cells had the predicted expression phenotype. Exposure to 5-azacytidine or introduction of a hereditary persistence of fetal hemoglobin mutation activated gamma-globin, which was expressed in addition to beta-globin, again consistent with the predicted expression profile of these cells. This approach extends the usefulness of YAC transgenic mice for the generation of cell lines amenable to more detailed studies regarding gene regulation.

Animals↗
PubMed2005

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal.

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.

Animals↗
PubMed2005

{gamma}-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human {beta}-globin locus yeast artificial chromosome ({beta}-YAC) transgenic mice.

Identification of trans-acting factors or drugs capable of reactivating gamma-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express epsilon- and gamma-globin but not beta-globin; transgenic mouse erythroleukemia 585 cells express predominantly human beta-globin but also gamma-globin; and transgenic murine GM979 cells co-express human gamma-and beta-globin. Human beta-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of beta-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively beta-globin and therefore could be employed to select or screen inducers of gamma-globin expression. A thrombopoietin receptor derivative that brings the proliferative status of primary mouse bone marrow cells under control of a chemical inducer of dimerization was employed to institute and maintain these cell populations. Human beta-globin was expressed, but gamma-globin was not; a similar expression pattern was observed in cells derived from fetal liver. gamma-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from -117 Greek hereditary persistence of fetal hemoglobin human beta-globin locus yeast artificial chromosome (beta-YAC) mice, showing that the hereditary persistence of fetal hemoglobin (HPFH) phenotype was maintained in these cells or was reactivated by an artificial zinc finger-gamma-globin transcription factor and the previously identified fetal globin transactivators fetal Krüppel-like factor (FKLF) and fetal globin-increasing factor (FGIF). These cells may be useful for identifying transcription factors that reactivate gamma-globin synthesis or screening gamma-globin inducers for the treatment of sickle cell disease or beta-thalassemia.

Animals↗
PubMed2005

Collection of blood stem cells from patients with sickle cell anemia.

Prior to initiating gene therapy trials for sickle cell disease (SCD), methods to collect sufficient numbers of hematopoietic stem and progenitor cells will need to be developed. Bone marrow harvest entails significant morbidity that could be severe in patients with SCD. In addition, an ability to perform repeated stem cell collections so that several transfers of genetically modified cells could be attempted would be advantageous. In other settings, apheresis collection of mobilized blood stem cells has become the preferred source of stem cells for transplantation. Unfortunately, patients with SCD do not tolerate granulocyte-colony stimulating factor and therefore cannot be mobilized using these conventional methods. In this pilot study, we investigated whether withdrawal of hydroxyurea therapy results in an increase in circulating numbers of CD34+ cells and hematopoietic progenitors. In addition, we performed leukapheresis in one patient with severe SCD in an attempt to determine whether blood stem cell collection can be performed safely in patients who would be candidates for SC gene therapy trials. Our results highlight some of the potential difficulties in initiating gene therapy clinical trials for sickle cell disease.

Adult↗
PubMed2005

Congenital amegakaryocytic thrombocytopenia in three siblings: molecular analysis of atypical clinical presentation.

OBJECTIVE: An 11-year-old girl, presenting with fatigue and bruising, was found to be profoundly pancytopenic. Bone marrow exam and clinical evaluation were consistent with aplastic anemia. Family members were studied as potential stem cell donors, revealing that both younger siblings displayed significant thrombocytopenia, whereas both parents had normal blood counts. We evaluated this pedigree to understand the unusually late presentation of congenital amegakaryocytic thrombocytopenia (CAMT). MATERIALS AND METHODS: The coding region and the intron/exon junctions of MPL were sequenced from each family member. Vectors representing each of the mutations were constructed and tested for the ability to support growth of Baf3/Mpl(mutant) cells. RESULTS: All three siblings had elevated thrombopoietin levels. Analysis of genomic DNA demonstrated that each parent had mutations/polymorphisms in a single MPL allele and that each child was a compound heterozygote, having inherited both abnormal alleles. The maternal allele encoded a mutation of the donor splice-junction at the exon-3/intron-3 boundary. A mini-gene construct encoding normal vs mutant versions of the intron-3 donor-site demonstrated that physiologic splicing was significantly reduced in the mutant construct. CONCLUSIONS: Mutations that incompletely eliminate Mpl expression/function may result in delayed diagnosis of CAMT and confusion with aplastic anemia.

Alleles↗
PubMed2005

Controlled-rate freezing of human ES cells.

A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.

Cell Survival↗
PubMed2004

Modulating erythrocyte chimerism in a mouse model of pyruvate kinase deficiency.

In vivo selection may provide a means to increase the relative number of cells of donor origin in recipients with hemopoietic chimerism. We have tested whether in vivo selection using chemical inducers of dimerization (CIDs) can direct the expansion of transduced normal donor erythrocytes in recipients with chimerism using a mouse model of pyruvate kinase deficiency. Marrow cells from normal CBA/N mice were transduced with a vector (F36Vmpl(GFP)) that promotes cell growth in the presence of CIDs. Transduced cells were then transplanted into minimally conditioned, pyruvate kinase-deficient recipients (CBA-Pk-1(slc)/Pk-1(slc)) to establish stable chimerism. CID administration resulted in expansion of normal donor erythrocytes and improvement of the anemia. The preferential expansion of normal erythrocytes also resulted in a decrease in erythropoietin levels, reducing the drive for production of pyruvate kinase-deficient red blood cells. CID-mediated expansion of genetically modified erythrocytes could prove a useful adjunct to transplantation methods that achieve erythroid chimerism.

Anemia↗
PubMed2004

Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia.

There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1-7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

Animals↗
PubMed2004

In vivo selection of genetically modified erythroid cells using a jak2-based cell growth switch.

Cell-based therapies have potential widespread applications in clinical medicine, and methods for controlling the fate of transplanted cells are needed. We have previously described a means for directing the growth of genetically modified cells in vivo using a derivative of the thrombopoietin receptor, mpl, that is reversibly activated by a drug called a chemical inducer of dimerization (CID). Since Jak2 participates in signaling from a number of different cytokine receptors (including mpl), we tested whether direct activation of the JH1 domain of Jak2 would broaden the repertoire of hematopoietic lineages responsive to the CID. While the engineered Jak2 induced a significant rise in genetically modified red cells, as we have observed previously with mpl, it lacked mpl's ability to expand genetically modified platelets and failed to expand genetically modified granulocytes, B cells, or T cells. These findings identify a signaling molecule other than mpl that can function as a cell growth switch in vivo and demonstrate that signaling molecules used for in vivo selection need not be confined to receptors. The erythroid-restricted growth response suggests that CID-activated Jak2 may be well suited to gene therapy applications in sickle cell anemia or beta-thalassemia.

Animals↗
PubMed2004

Differences in F36VMpl-based in vivo selection among large animal models.

Animal models are indispensable tools for understanding physiological and pathological processes, as well as for developing new therapies. Ultimately, the results of animal experimentation must provide information that can guide the development of therapeutic approaches in humans. Significant differences have been reported comparing a gene therapy approach between different animal models. However, little information exists describing differences among the available large animal models. Here we evaluated, in the hemopoietic cells of baboons, a system of selection that has previously demonstrated activity in mice, in dogs, and in human cells ex vivo. This system employs a derivative of the murine thrombopoietin receptor (F36Vmpl), which is conditionally activated in the presence of a small-molecule drug called a chemical inducer of dimerization (CID). Whereas cultured mouse, human, and, to a lesser extent, dog hemopoietic cells all proliferate in response to the F36Vmpl signal, we observed only a minor and variable response to the F36Vmpl signal in the cultured cells of baboons. Similarly, we have noted significant rises in the frequency of transduced hemopoietic cells in mice and in dogs upon CID administration in vivo; however, here we show that responses to CID administration in three baboons were modest and variable. These findings have general implications for the evaluation and development of new strategies for gene therapy.

Animals↗
PubMed2004

[Long term regulated expansion and committed differentiation of JAK2 gene transfected hematopoietic stem/progenitor cells in vitro].

OBJECTIVE: To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro. METHODS: A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated. RESULTS: A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S. CONCLUSIONS: AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.

Animals↗
PubMed2003

Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate model.

Conditional suicide genes derived from pathogens have been developed to confer drug sensitivity and enhance safety of cell therapy, but this approach is limited by immune responses to the transgene product. We examined a strategy to regulate survival of transferred cells based on induction of apoptosis through oligomerization of a modified human Fas receptor by a bivalent drug (AP1903). Three macaques (Macaca nemestrina) received autologous T cells retrovirally engineered to express a Fas suicide-construct (LV'VFas). High levels of transduced cells were present in blood following cell transfer, but LV'VFas(+) cells declined rapidly after AP1903 administration. A small fraction of LV'VFas(+) cells resisted elimination by AP1903, in part due to insufficient levels of transgene expression in resting T cells, because reactivation of these cells in vitro enhanced sensitivity to AP1903. An immune response to the transgene product was observed, but epitope mapping indicated the response was directed to discrete components of human LV'VFas that were variant with the corresponding macaque sequences. These data demonstrate that chemically induced dimerization can be used to regulate survival of adoptively transferred T cells in vivo.

Adoptive Transfer↗
PubMed2003

Small-molecule-directed mpl signaling can complement growth factors to selectively expand genetically modified cord blood cells.

Efforts toward achieving gene therapy for blood disorders are plagued by low rates of gene transfer into hemopoietic stem cells. Recent studies suggest that this obstacle can be circumvented using selection. One way to achieve selection employs genes that encode receptor-bearing fusion proteins capable of inducing cell growth in response to drugs called chemical inducers of dimerization (CIDs). We have previously shown that genetically modified marrow cells from mice can proliferate for up to a year in culture in response to CID-initiated signals arising from the thrombopoietin receptor (mpl). The sustained growth observed in mouse hemopoietic cells results from an mpl-induced self-renewal of multipotential hemopoietic progenitor cells. In contrast, human hemopoietic cells proliferate only transiently in response to the mpl signal (from differentiation of transduced erythroid and megakaryocytic progenitors), while human myeloid progenitors fail to respond. Here, we show that myeloid progenitors from human cord blood can be induced to proliferate and/or differentiate in response to the mpl signal by providing additional signals via a combination of growth factors. These findings are relevant for the eventual clinical application of CID-regulated cell therapy.

Antigens, CD34↗
PubMed2002

Pharmacologically regulated in vivo selection in a large animal.

The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.

Animals↗
PubMed2002

CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy.

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded products. Here we have developed a transduction and selection strategy for generating large numbers of polyclonal T cells transduced with a retroviral vector encoding the human low-affinity nerve growth factor receptor (LNGFR) for selection and a Fas-based suicide construct (LV'VFas). Ligation of CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to the generation of gene-modified T cells that retained clonal diversity, functional properties, and a homing receptor profile similar to untransduced peripheral blood lymphocytes. Microbeads conjugated directly to antibody specific to LNGFR significantly improved the immunomagnetic selection of LV'VFas-modified T cells and assisted in scaling of the selection procedure to therapeutic cell numbers. Thus, these studies identified a strategy that requires only a brief ex vivo culture and does not use drug selection to obtain large numbers of functional gene-modified polyclonal T cells that can be used for adoptive immunotherapy.

Animals↗