| 1. |
- Björgvinsdottir, Helga, et al.
(författare)
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Efficient Oncoretroviral Transduction of Extended Long-Term Culture-Initiating Cells and NOD/SCID Repopulating Cells: Enhanced Reconstitution with Gene-Marked Cells Through an Ex Vivo Expansion Approach.
- 2002
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Ingår i: Human Gene Therapy. - 1043-0342. ; 13:9, s. 1061-1073
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Tidskriftsartikel (refereegranskat)abstract
- Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.
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| 2. |
- Bryder, David, et al.
(författare)
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Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation
- 2001
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 194:7, s. 941-952
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Tidskriftsartikel (refereegranskat)abstract
- Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.
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| 3. |
- Ramsfjell, Veslemøy
(författare)
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Hematopoietic stem cells: c-mpl and TNF receptor function
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hematopoietic stem cells (HSC) are crucial to life. Daily and throughout the life span of an individual they are responsible for replacing approximately 10(12) mature blood cells. Mature cells are, for example, required for transportation of oxygen, defense against infections and to stop bleeding. Thus, our aim with this study was to characterize these rare murine (Lin-Sca1+c-kit+) and human (CD34+CD38-) bone marrow (BM) HSC with respect to their viability, proliferation, differentiation and expansion in response to cytokines. When these studies were initiated thrombopoietin (Tpo) was postulated to act primarily as a megakaryocyte lineage-specific factor. However, we demonstrated that Tpo directly and synergistically enhanced multilineage growth and differentiation from both Lin-Sca1+c-kit+ and CD34+CD38- BM cells, when combined with cytokines like c-kit ligand (KL), flt3 ligand (FL), and the interleukins 3 and 11. In addition, Tpo, more than any other early-acting cytokines, efficiently promoted viability of single CD34+CD38- cells in the absence of cell division, further implicating that Tpo directly acted on candidate stem cells. Interestingly, a direct comparison of human long-term culture-initiating cells (LTC-IC) and the more primitive extended LTC-IC (ELTC-IC), demonstrated distinct and stringent requirements for their ex vivo expansion. Specifically, ELTC-IC were potently expanded in serum-free medium in the presence of high concentrations of KL+FL+Tpo for 12 days. We also observed that Tpo-induced clonal growth of human CD34+CD38- progenitor cells was almost completely abrogated by tumor necrosis factor-a (TNF-a) and transforming growth factor-b. Additionally, we demonstrated in mice that functional signaling through the Fas and TNF receptors severely compromised HSC short- and long-term multilineage repopulating activity.
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