Spangrude, G. J., Heimfield, D. S. & Weissman, I. L. Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62

Stanford University, Palo Alto, California, United States
Science (Impact Factor: 33.61). 08/1988; 241(4861):58-62. DOI: 10.1126/science.2898810
Source: PubMed


Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can
proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of
these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the

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    • "Hematopoietic stem cells (HSCs) are rare cells that reside in adult bone marrow and have the potential to give rise to the entire repertoire of mature blood cells [1]. HSCs are essential for the maintenance of all blood cell compartments [2]. "
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    ABSTRACT: The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.
    PLoS ONE 08/2014; 9(8):e105525. DOI:10.1371/journal.pone.0105525 · 3.23 Impact Factor
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    • "EPC isolation was performed as reported previously with documented modifications [1,55,56]. Selected mouse tissues or tumor samples were surgically removed from euthanized nude mice. Tissues were kept in mouse endothelial media (M1168, CellBiologics, Chicago, IL) supplemented with the growth factor kit (VEGF, EGF, heparin, hydrocortisone, and L-Glumatine) and 10% fetal bovis serum (FBS) and then minced into small chunks with a #11 scalpel blade. "
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    ABSTRACT: Background Endothelial progenitor cells (EPCs) have been demonstrated to have stem-cell like as well as mature endothelial functions. However, controversy remains as to their origins, immunophenotypic markings, and contribution to the tumor vascular network and tumor survival. Methods Flow cytometric analysis and sorting was used to isolate Flk-1+/c-Kit+/CD45- cells. Matrigel and methycellulose assays, flow cytometry, and gene array analyses were performed to characterize several murine EPC cell populations. Human tumor xenografts were used to evaluate the impact of EPCs on tumor growth and vascular development. Results Flk-1+/c-Kit+/CD45- cells were present at low levels in most murine organs with the highest levels in adipose, aorta/vena cava, and lung tissues. Flk-1+/c-Kit+/CD45- cells demonstrated stem cell qualities through colony forming assays and mature endothelial function by expression of CD31, uptake of acLDL, and vascular structure formation in matrigel. High passage EPCs grown in vitro became more differentiated and lost stem-cell markers. EPCs were found to have hemangioblastic properties as demonstrated by the ability to rescue mice given whole body radiation. Systemic injection of EPCs increased the growth of human xenograft tumors and vessel density. Conclusions Flk-1+/C-Kit+/CD45- cells function as endothelial progenitor cells. EPCs are resident in most murine tissue types and localize to human tumor xenografts. Furthermore, the EPC population demonstrates stem-cell and mature endothelial functions and promoted the growth of tumors through enhanced vascular network formation. Given the involvement of EPCs in tumor development, this unique host-derived population may be an additional target to consider for anti-neoplastic therapy.
    Molecular Cancer 07/2014; 13(1):177. DOI:10.1186/1476-4598-13-177 · 4.26 Impact Factor
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    • "Then, this lineage negative population was incubated with antibodies to cell surface epitopes, the presence or absence of which, enriched for long-term repopulating cells (5). Many candidate stem cell markers were evaluated with positivity for c-kit, Sca-1, intermediate staining for Thy.1, and negativity for FLK2 (11–13) being initially defined markers and CD150 or Slam (14) and CD34 (15) also currently in vogue for definition of the stem cell. These studies showed a lack of tight correlation with the purified stem cells and CFU-S and led to the dismissal of CFU-S as a relevant stem cell; as we will develop below, this was probably a fundamental mistake. "
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    ABSTRACT: Current concepts of hematopoiesis are encompassed in a hierarchical stem cell model. This developed initially from studies of colony-forming unit spleen (CFU-S) and in-vitro progenitors for different cell lineages, but then evolved into a comprehensive model of cells with different in vivo differentiative and proliferative potential. These cells were characterized and purified based largely on expression of a variety of lineage specific and stem cell specific surface epitopes. Monoclonal antibodies bound to these epitopes and were then used to physically and fluorescently separate different classes of these cells. The gold standard for the most primitive marrow stem cells was long-term multilineage repopulation and renewal in lethally irradiated mice. Progressive work seemed to have clonally defined a lineage negative Sca-1+, c-kit+ CD150+ stem cell with great proliferative, differentiative and renewal potential. This cell was stable and in the G0 phase of cell cycle. However, continued work in our laboratory indicated that the engraftment, differentiation, homing, and gene expression phenotype of the murine marrow stem cells continuously and reversibly changes with passage through cell cycle. Most recently, using cycle defining supravital dyes and fluorescent activated cell sorting and S-phase specific tritiated thymidine suicide, we have established that the long-term repopulating hematopoietic stem cell is a rapidly proliferating, and thus a continually changing cell; as a corollary it cannot be purified or defined on a clonal single cell basis. Further in vivo studies employing injected and ingested BrdU, showed that the G0 lin-Sca-1, c-kit + Flt3- cell was rapidly passing through cell cycle. These data are explained by considering the separative process: the proliferating stem cells are eliminated through the selective separations leaving non-representative dormant G0 stem cells. In other words they throw out the real stem cells with the purification. This sys
    Frontiers in Oncology 04/2014; 4:56. DOI:10.3389/fonc.2014.00056
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