| 11. |
- 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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| 12. |
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| 13. |
- Bryder, David, et al.
(författare)
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Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion.
- 2005
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 7:2, s. 137-144
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Tidskriftsartikel (refereegranskat)abstract
- Background Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. Methods The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. Results We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. Conclusions These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.
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| 14. |
- Bryder, David
(författare)
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Hematopoiesis at single-cell resolution
- 2016
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 128:8, s. 1025-1026
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Tidskriftsartikel (refereegranskat)
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| 15. |
- Bryder, David, et al.
(författare)
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Hematopoietic stem cells: the paradigmatic tissue-specific stem cell
- 2006
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 1525-2191 .- 0002-9440. ; 169:2, s. 338-346
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Tidskriftsartikel (refereegranskat)abstract
- The recent prospective isolation of a wide variety of somatically derived stem cells has affirmed the notion that homeostatic maintenance of most tissues and organs is mediated by tissue-specific stem and progenitor cells and fueled enthusiasm for the use of such cells in strategies aimed at repairing or replacing damaged, diseased, or genetically deficient tissues and organs. Hematopoietic stem cells (HSCs) are arguably the most well-characterized tissue-specific stem cell, with decades of basic research and clinical application providing not only a profound understanding of the principles of stem cell biology, but also of its potential pitfalls. It is our belief that emerging stem cell fields can benefit greatly from an understanding of the lessons learned from the study of HSCs. In this review we discuss some general concepts regarding stem cell biology learned from the study of HSCs with a highlight on recent work pertaining to emerging topics of interest for stem cell biology.
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| 16. |
- Bryder, David, et al.
(författare)
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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro
- 2000
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Ingår i: Blood. - 1528-0020. ; 96:5, s. 1748-1755
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Tidskriftsartikel (refereegranskat)abstract
- Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin(-)Sca-1(+)kit(+) bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3. (Blood. 2000;96:1748-1755)
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| 17. |
- Bryder, David
(författare)
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Molecular regulation of hematopoietic stem cells
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hematopoietic stem cells (HSCs) are ultimately responsible for the all mature blood cell production. They can primarily be found in the bone marrow (BM) at a low frequency (~0.02%). Unlike other blood cells, HSCs have a high degree of self-renewal capacity, the process where upon cell division, at least one daughter cell has the same properties as the stem cell it was generated from. In addition, HSCs need to be able to form mature blood cell components, a process called differentiation. In this thesis, we investigated how in vitro regulation of HSCs would affect their capacity to self-renew and/or differentiate. Whereas we found some growth factors, including kit ligand and Thrombopoietin, to be crucial for induction of self-renewal, we failed to find any direct role of flt3 ligand in these processes. In fact, we developed an efficient strategy to isolate HSCs based on their lack of flt3. Although we, using flt3 ligand deficient mice, failed to find any role of flt3 in HSC regulation, we found flt3 to be crucial in early lymphoid development, as flt3 ligand deficient mice had severely reduced numbers of early lymphoid, but not myeloid, precursors. Since we were capable of efficiently inducing self-renewal in vitro, we were able to characterize the roles of TNF-alpha and fas on HSC self-renewal. These two molecules had previously been suggested to negatively regulate early hematopoiesis. Whereas HSCs in steady state do not seem to be regulated by fas, we found that HSCs can be efficiently targeted by fas following exposure to TNF-alpha. In all, we here demonstrate critical and distinct cytokine requirements for the HSC regulatory processes self-renewal, differentiation and apoptosis.
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| 18. |
- 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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| 19. |
- Bryder, David, et al.
(författare)
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Shaping up a lineage-lessons from B lymphopoesis
- 2010
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Ingår i: CURRENT OPINION IN IMMUNOLOGY. - : Elsevier Science B.V., Amsterdam.. - 0952-7915 .- 1879-0372. ; 22:2, s. 148-153
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Forskningsöversikt (refereegranskat)abstract
- Even though the development of B lymphoid cells from hematopoietic stem cells is one of the most carefully investigated models of cell differentiation in adult mammalians, a set of recent findings has to a large extent increased our understanding for how B lymphoid commitment is achieved. These include the identification of IKAROS, PU.1 and E2A as transcription factors responsible for lymphoid lineage priming in multipotent cells, as well as the identification of EBF1 dependent B lineage restricted progenitors among cells lacking expression of the classical B lineage markers CD19 or 8220. The insight that the B cell identity may be defined at an earlier stage then previously thought, allows for an increased understanding of B lymphoid development likely to unravel molecular mechanisms of high relevance also for other differentiation processes within as well as outside of the hematopoietic system.
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| 20. |
- Canals, Isaac, et al.
(författare)
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Rapid and efficient induction of functional astrocytes from human pluripotent stem cells
- 2018
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 15:9, s. 693-696
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Tidskriftsartikel (refereegranskat)abstract
- The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.
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