Biomedical subjects

H Glimm

Publications and source records attributed to H Glimm.

12 recordsLinked to original sources

PubMed2006

[Stem cell therapy. Biology of hematopoietic stem cells].

In recent years much progress has been made in the understanding of the biology of hematopoietic stem cells (HSC) and their involvement in normal blood cell development. Using immunophenotyping it is possible, to enrich HSC, however, so far we are not able to positively select HSC. For the identification, characterization and quantification of HSC it is necessary to use functional assay systems, such as xenotransplantation models. HSC from bone marrow, peripheral blood and in some cases also cord blood have been used for years in transplantation settings especially in patients with leukemia. A better understanding of the mechanisms underlying stem cell regulation as well as stem cell self renewal would have clinical implications e. g. for clinical transplantation strategies. A number of hematological diseases such as chronic myeloid leukemia originates from a malignant transformed HSC. A better understanding of the biology of normal as well as malignant HSC is therefore crucial not only for a better understanding of the disease, but also for the development of strategies aiming at the discrimination of normal and malignant stem cell candidates and the development of therapies targeting the leukemic stem cell.

Cell Transformation, Neoplastic↗

PubMed2005

Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients' samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-beta2microglobulin-/- mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL+/Ph+ cells. The regenerated leukemic cells included an initial, transient population derived from CD34+CD38+ cells as well as more sustained populations derived from CD34+CD38- progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34+) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.

Animals↗

PubMed2001

Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Animals↗

PubMed2001

Characterization of human hematopoietic cells with short-lived in vivo repopulating activity.

Recent studies with purified hematopoietic stem cells in vitro support a model of stem cell self-renewal control that involves distinct mechanisms regulating permissiveness to and execution of lineage restriction. Such a model predicts the existence of phenotypically separable populations of hematopoietic cells that are pluripotent and either capable or incapable of extensive self-renewal. Such populations have been previously described in the mouse. We describe here the first evidence that such cells can now be identified in humans using different types of immunodeficient mice as hosts.

Animals↗

PubMed2001

A model for the detection of clonality in marked hematopoietic stem cells.

The semirandom location of retroviral integration in the target cell genome introduces a marker in the form of a fusion sequence composed of a genomic and a proviral part that is unique for each transduced cell and its clonal progeny. High-sensitivity detection of these fusion sequences would allow the tracking of clonal contributions of individual, marked hematopoietic progenitor, and stem cells in vivo. Clone detection by Southern blot has helped to analyze models of oligoclonal repopulation but is limited in sensitivity and specificity. Inverse PCR (Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414-2419) can demonstrate the clonal identity by sequencing but does not permit simultaneous detection of multiple clones. In an efficiently transduced rhesus macaque model (Tisdale et al., Blood 92: 2681-2687; Wu et al., Mol. Ther. 1: 285-293) Kim et al. (Blood 96: 1-8) have identified more than 40 insertion sequences from marrow CFU by inverse PCR. However, no previous study has been able to directly analyze the number of clones active in vivo. Here we demonstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal contributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integration sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked hematopoiesis in small and large animals as well as in human gene transfer.

Animals↗

PubMed2001

Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice.

Increasing use of purified or cultured human hematopoietic cells as transplants has revealed an urgent need for better methods to predict the speed and durability of their engraftment potential. We now show that NOD/SCID-beta2 microglobulin-null (NOD/SCID-beta2m-/-) mice are sequentially engrafted by two distinct and previously unrecognized populations of transplantable human short-term repopulating hematopoietic cells (STRCs), neither of which efficiently engraft NOD/SCID mice. One is predominantly CD34+CD38+ and is myeloid-restricted; the other is predominantly CD34+CD38- and has broader lymphomyeloid differentiation potential. In contrast, the long-term repopulating human cells that generate lymphoid and myeloid progeny in NOD/SCID mice engraft and self-renew in NOD/SCID-beta2m-/- mice equally efficiently. In short-term expansion cultures of adult bone marrow cells, myeloid-restricted STRCs were preferentially amplified (greater than tenfold) and, interestingly, both types of STRC were found to be selectively elevated in mobilized peripheral blood harvests. These results suggest an enhanced sensitivity of STRCs to natural killer cell-mediated rejection. They also provide new in vivo assays for different types of human STRC that may help to predict the engraftment potential of clinical transplants and facilitate future investigation of early stages of human hematopoietic stem cell differentiation.

Animals↗

PubMed2000

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0).

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G(1) fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G(0)/G(1) and S/G(2)/M. Interestingly, the G(0) cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle-associated change in in vivo stem cell homing, the cultured G(0)/G(1) and S/G(2)/M CD34(+) CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor beta(1) that increased the G(0)/G(1) fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle-associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo-manipulated grafts. (Blood. 2000;96:4185-4193)

Animals↗

PubMed1999

Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture.

Recently, culture conditions that stimulate the proliferation of primitive hematopoietic cells defined by various phenotypic and functional endpoints in vitro have been identified. However, evidence that they support a high probability of self-renewal leading to a large net expansion in vitro of transplantable cells with lympho-myeloid repopulating ability has been more difficult to obtain. The present study was designed to investigate whether the low overall expansion of human repopulating hematopoietic cells seen in vitro reflects a selective unresponsiveness of these rare cells to the growth factors currently used to stimulate them or, alternatively, whether they do proliferate in vitro but lose engrafting potential. For this, we used a high-resolution procedure for tracking and reisolating cells as a function of their proliferation history based on the loss of cellular fluorescence after staining with (5- and 6-) carboxyfluorescein diacetate succinimidyl ester. The results show that the vast majority of long-term culture-initiating cells and in vivo lympho-myeloid competitive repopulating units present in 5-day suspension cultures initiated with CD34(+) human cord blood and fetal liver cells are the progeny of cells that have divided at least once in response to stimulation by interleukin-3, interleukin-6, granulocyte colony-stimulating factor, Steel factor, and Flt3-ligand. Thus, most human repopulating cells from these two sources are stimulated to undergo multiple divisions under currently used short-term suspension culture conditions and a proportion of these retain engraftment potential.

Abortion, Induced↗

PubMed1998

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.

Animals↗

PubMed1998

New developments in hematopoietic stem cell expansion.

The possibility of maintaining, manipulating, and expanding human hematopoietic stem cells in ex vivo culture could help to provide patients with autologous and allogeneic stem cell transplants improved in purity and performance and could offer access to gene therapy of the hematopoietic system. Recent advances in the ex vivo culture of immature human hematopoietic progenitor cells and human hematopoietic stem cells have led to experimental evidence for the qualitative and quantitative maintenance and possible numerical expansion of hematopoietic stem cells in ex vivo culture, making ex vivo graft engineering a realistic possibility. This review summarizes recent developments in the field, their regulatory implications and their application in hematopoietic gene therapy.

Animals↗

PubMed1997

Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector.

Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.

ADP-ribosyl Cyclase↗

PubMed1996

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus.

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4-5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 microM tetrodotoxin-sensitive Na+ current; a high-voltage-activated (HVA) Ca2+ current, which was 30 microM Cd(2+)- and partially 2 microM nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs+. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca(2+)-dependent outward current. This transient Ca(2+)-dependent K+ outward current was identified by a subtraction procedure. K+ currents recorded under conditions of blocked Ca2+ currents (after rundown of the HVA Ca2+ current or blocked by extracellularly applied Cd2+) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.

Animals↗