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B Murdoch

Publications and source records attributed to B Murdoch.

At least 37 records · Page 2Linked to original sources
PubMed1999

Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells.

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34(+)CD38(-)Lin- stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor beta, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34(+)CD38(-) phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34(+) CD38(-)Lin- cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.

Activin Receptors↗
PubMed1998

Treatment of non-obese diabetic (NOD)/Severe-combined immunodeficient mice (SCID) with flt3 ligand and interleukin-7 impairs the B-lineage commitment of repopulating cells after transplantation of human hematopoietic cells.

Until recently, the identification of cellular factors that govern the developmental program of human stem cells has been difficult due to the absence of repopulation assays that detect human stem cells. The transplantation of human bone marrow (BM) or cord blood (CB) into non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice has enabled identification of primitive human cells capable of multilineage repopulation of NOD/SCID mice (termed the SCID-repopulating cell [SRC]). Here, we examined the effect of long-term in vivo treatment with various combinations of human cytokines on the developmental program of SRC. Detailed flow cytometric analysis of engrafted mice indicated that the vast majority of the human graft of untreated mice was comprised of B lymphocytes at various stages of development as well as myeloid and primitive cells; T cells were not reproducibly detected. Many studies, including murine in vitro and in vivo data and human in vitro experiments, have suggested that flt3 ligand (FL) and/or Interleukin-7 (IL-7) promotes T- and B-cell development. Unexpectedly, we found that treatment of engrafted mice with the FL/IL-7 combination did not induce human T- or B-cell development, but instead markedly reduced B-cell development with a concomitant shift in the lineage distribution towards the myeloid lineage. Effects on lineage distribution were similar in engrafted mice transplanted with highly purified cells indicating that the action of the cytokines was not via cotransplanted mature cells from CB or BM cells. These data show that the lineage development of the human graft in NOD/SCID mice can be modulated by administration of human cytokines providing a valuable tool to evaluate the in vivo action of human cytokines on human repopulating cells.

Animals↗
PubMed1998

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Cell Lineage↗
PubMed1998

A newly discovered class of human hematopoietic cells with SCID-repopulating activity.

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here, we identify a newly discovered human repopulating cell, distinct from previously identified repopulating cells, that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells, or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR, Thy-1 and CD34). In contrast to CD34+ subfractions, Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations, providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation.

Animals↗
PubMed1997

Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

ADP-ribosyl Cyclase↗
PubMed1997

Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

ADP-ribosyl Cyclase↗
PubMed1997

A rapid screening procedure for the identification of high-titer retrovirus packaging clones.

We have developed a viral RNA (vRNA) dot blot assay for rapid identification of high-titer retrovirus vector production by packaging cell clones. The procedure employs Trizol LS reagent to purify vRNA from packaging cell supernatants, a sensitive dot blot assay, and Phosphorlmager technology to quantify packaged viral genomes in 2 days. Experiments performed on viral supernatants of known biological titer demonstrated that the vRNA dot blot assay was extremely sensitive and that dot intensity correlated directly with viral titer. It is often necessary to analyze approximately 100 virus producing cell clones, making this method useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of candidate high-titer producer cell clones. In three independent screens the supernatant with the highest biological titer was produced by one of the previously defined candidate high-titer producer clones. Our procedure greatly facilitates virus titration by: (1) rapidly eliminating the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for further biological titration and assessment of the proviral genomic structure; and (3) reducing laborious tissue culture manipulations to a minimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lacking a drug selection marker.

Gene Transfer Techniques↗
PubMed1996

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis.

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.

Animals↗
PubMed1996

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

ADP-ribosyl Cyclase↗
PubMed1994

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.

Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

ADP-ribosyl Cyclase↗
PubMed1993

Engraftment of human lymphoid cells into newborn SCID mice leads to graft-versus-host disease.

Although there has been considerable progress in transplanting normal human hematopoietic cells into immune-deficient mice, the establishment of a functional human immune system has proved to be difficult. Current methods of introducing mature human lymphoid cells into adult SCID mice lead to oligoclonal engraftment with restricted dissemination to various organs. We have attempted to improve human lymphoid cell engraftment in mice, both qualitatively and quantitatively, by injecting human bone marrow cells and peripheral blood leukocytes intraperitoneally into newborn SCID mice. Newborn mice were used as recipients because certain immune functions such as natural killer cell activity do not develop until several weeks after birth and the numerous growth factors secreted in young mice may facilitate the engraftment and proliferation of transplanted human cells. At various times after transplantation, the presence of human cells in different organs was determined by Southern blot analysis using a human specific probe. Within 4 weeks, 70% of the mice were engrafted with human cells. Human cell engraftment of the bone marrow, spleen, lungs, kidneys, liver, and thymus exceeded 10% in at least 40% of the transplanted mice; most of these highly engrafted mice were sick. Flow cytometry and immunocytochemistry indicated these organs were heavily infiltrated with mature T and B lymphocytes. Histologic and molecular analysis showed massive human cell infiltrates within the liver, lung and spleen. The presence of human IgG and IgM antibodies against mouse red blood cells provided evidence that the engrafted human cells retained some immune function. Mice transplanted with peripheral blood leukocytes from donors that were allergic to mouse antigens engrafted to the same extent as normal cells but in addition developed the classical symptoms of acute allogeneic graft-versus-host disease (GVHD) including infiltrates of the skin, gut, and liver. The newborn SCID system provides a new in vivo model to study human xenoreactivity and GVHD.

Animals↗
PubMed1992

Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice.

Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with human mast cell growth factor, a fusion of interleukin-3 and granulocyte-macrophage colony-stimulating factor (PIXY321), or both, starting immediately or 1 month later. Immature human cells repopulated the mouse bone marrow with differentiated human cells of multiple myeloid and lymphoid lineages; inclusion of erythropoietin resulted in human red cells in the peripheral blood. The bone marrow of growth factor-treated mice contained both multipotential and committed myeloid and erythroid progenitors, whereas mice not given growth factors had few human cells and only granulocyte-macrophage progenitors. Thus, this system allows the detection of immature human cells, identification of the growth factors that regulate them, and the establishment of animal models of human hematopoietic diseases.

Animals↗
PubMed1992

PC analysis of altered speech respiration following fitting of a multi-channel cochlear implant prosthesis.

This paper describes a personal computer (PC) application for speech respiration analysis. Parameters that can be determined include lung volume changes, the relative contributions of the rib cage and abdomen to lung volume changes and the configuration of the chest wall during speech production. Data collected from two subjects fitted with Nucleus cochlear implant prostheses illustrate one application of this technique. The PC based kinematic analysis described provides previously inaccessible information about the effects of changes in the level of auditory feedback resulting from activation of a multiple channel cochlear prosthesis on the speech production mechanism.

Adult↗
PubMed1992

Differential kinetics of engraftment and induction of CD10 on human pre-B leukemia cell lines in immune deficient scid mice.

The sensitivity of the scid mouse model was assessed by comparing the growth of two pre-B acute lymphoblastic leukemia (ALL) cell lines, A1 and G2, established from patients at relapse. When cell numbers varying from 10(4) to 10(7) were injected intravenously into scid mice, advanced growth and dissemination of leukemia was observed at 10-12 weeks with the G2 cells. Bone marrow, spleen and thymus contained high levels of human leukemic cells and infiltration into lung, kidney, liver, and brain was observed. Two of three mice grafted with only 100 cells showed high levels of infiltration at 15 weeks, suggesting that 100 G2 cells was near the limiting cell number that could produce disseminated leukemia. With the A1 line, a minimum of 10(5) cells was needed to obtain dissemination to liver, lung, brain, and kidney; a low level of spleen infiltration occurred and thymus invasion was not observed. In vitro, both lines showed a density dependent growth in clonogenic assays but the cloning efficiency of the A1 line was 10-fold higher than for G2 cells. These results indicate that G2 and A1 lines have a dissimilar aggressiveness in vivo which does not correlate with clonogenic assay in vitro. Neither G2 nor A1 lines, growing in vitro, expressed CD10/CALLA on their surface, despite low levels of antigen on the freshly obtained relapse samples. Although A1 cells remained CD10-negative in the scid mice, G2 cells showed detectable levels of CD10, particularly on those cells found in the thymus. Several subclones of the G2 line were derived from isolated colonies in vitro; they were found to be CD10- in vitro, but to become CD10+ when proliferating into scid mouse thymus, suggesting the induction of CD10 by the murine microenvironment.

Animals↗
PubMed1991

Bone marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates rapidly in scid mice.

Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.

Adolescent↗
PubMed1991

Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays.

The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus-mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune-deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte-macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418-resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

Adult↗
PubMedDate not supplied

Transient-evoked and 2F1-F2 distortion product oto-acoustic emissions in dogs: preliminary findings.

Transient (click)-evoked oto-acoustic emissions (TEOAEs) and distortion product oto-acoustic emissions (DPOAEs) were recorded in a feasibility study in 7 healthy mixed-breed dogs using the ILO 92 OAE analyser (Otodynamics, Hartfield, UK). Five dogs were found to have normal hearing in both ears and 2 dogs in the left ear only following otoscopy, tympanometry and auditory brainstem response audiometry. Twelve sets of TEOAEs (click-evoked) to 80 dB peSPL click stimulus and 9 sets of DPOAEs (2F1-F2) to 8 different stimulus levels of the primary tones (L1/L2) were collected at 11 test frequencies (F2) in these normal-hearing dogs. TEOAEs were successfully recorded in 11 of the 12 ears using the default user setting and in all 12 ears using the quickscreen program. DPOAEs were successfully recorded in all 9 ears tested. While the TEOAEs parameters matched those for humans, the average signal-to-noise ratio of DPOAEs was considerably higher in the dogs. Stimulus levels at 55/55, 55/45 and 55/35 dB SPL were demonstrated to produce DPOAEs that seem to reflect the active dynamic status of the outer hair cell system. Postmortem DPOAEs at these stimulus levels and TEOAEs at 80 Db peSPL could not be elicited 5 min following euthanasia of dogs. However, DPOAEs could still be recorded albeit with reduced amplitude at stimulus levels where L1 > 55 dB SPL. The results suggest that TEOAEs and DPOAEs in dogs have the potential to provide valuable insights into their mechanisms of generation, and the specific role and behaviour of outer hair cells of the cochlea in certain pathological conditions, particularly in drug-induced ototoxicity, in humans.

Acoustic Stimulation↗