Conditions controlling the proliferation of haemopoietic stem cells in vitro.
01 Jun 1977-Journal of Cellular Physiology (Wiley Subscription Services, Inc., A Wiley Company)-Vol. 91, Iss: 3, pp 335-344
TL;DR: A liquid culture system is described whereby proliferation of haemopoietic stem cells, production of granulocyte precursor cells (CFU‐C), and extensive granulopoiesis can be maintained in vitro for several months.
Abstract: A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S), production of granulocyte precursor cells (CFU-C), and extensive granulopoiesis can be maintained in vitro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells, “epithelial” cells, and “giant fat” cells. The latter appear to be particularly important for stem cell maintenance and furthermore there is a strong tendency for maturing granulocytes to selectively cluster in and around areas of “giant fat” cell aggregations. By “feeding” the cultures at weekly intervals, between 10 to 15 “population doublings” of functionally normal CFU-S regularly occurs. Increased “population doublings” may be obtained by feeding twice weekly. The cultures show initially extensive granulopoiesis followed, in a majority of cases, by an accumulation of blast cells. Eventually both blast cells and granulocytes decline and the cultures contain predominantly phagocytic mononuclear cells.
Culturing at 33°C leads to the development of a more profuse growth of adherent cells and these cultures show better maintenance of stem cells and increased cell density.
When tested for colony stimulating activity (CSA) the cultures were uniformly negative. Addition of exogenous CSA caused a rapid decline in stem cells, reduced granulopoiesis and an accumulation of phagocytic mononuclear cells.
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TL;DR: The study of mesenchymal stem cells, whether isolated from embryos or adults, provides the basis for the emergence of a new therapeutic technology of self‐cell repair.
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TL;DR: Marrow stromal cells present an intriguing model for examining the differentiation of stem cells and have several characteristics that make them potentially useful for cell and gene therapy.
Abstract: Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells Also, they have several characteristics that make them potentially useful for cell and gene therapy
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TL;DR: The results should facilitate the development of therapeutically useful inhibitors of TNF-α release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.
Abstract: Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.
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TL;DR: It is concluded that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of H SCs by regulating niche size.
Abstract: Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.
2,949 citations
Additional excerpts
...in 1978, and is supported by the co-culture of HSCs with marrow stromal cell...
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TL;DR: Several studies which tested the use of MSCs in models of infarct (injured heart), stroke (brain), or meniscus regeneration models are reviewed within the context of M SC‐mediated trophic effects in tissue repair.
Abstract: Adult marrow-derived Mesenchymal Stem Cells (MSCs) are capable of dividing and their progeny are further capable of differentiating into one of several mesenchymal phenotypes such as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, and adipocytes. In addition, these MSCs secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities. These secreted bioactive factors suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate mitosis and differentiation of tissue-intrinsic reparative or stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of MSCs into repair tissue. Several studies which tested the use of MSCs in models of infarct (injured heart), stroke (brain), or meniscus regeneration models are reviewed within the context of MSC-mediated trophic effects in tissue repair.
2,743 citations
Cites background from "Conditions controlling the prolifer..."
...Prior to themore recent discoveries of themultiple potential of MSCs, bone marrow was known primarily as a source of osteogenic cells, and cultures of bonemarrow stromal cells were known to support hematopoiesis in culture [Friedenstein et al., 1974; Dexter et al., 1977]....
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References
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TL;DR: Counts of macroscopic splenic colonies were used to obtain an estimate of the radiation sensitivity of normal mouse bone marrow progenitor cells.
Abstract: Counts of macroscopic splenic colonies were used to obtain an estimate of the radiation sensitivity of normal mouse bone marrow progenitor cells. Reproduced from Radiation Research 1961(Feb); 14(2): 213-222 by copyright permission of the Radiation Research Society (www.radres.org).
4,451 citations
TL;DR: A simple in vitro technique for the growth of colonies from single cell suspensions of mouse bone marrow involves the plating of marrow cells in agar on feeder layers of other cells, those from 8-day-old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layer.
Abstract: A simple in vitro technique is described for the growth of colonies from single cell suspensions of mouse bone marrow. The system involves the plating of marrow cells in agar on feeder layers of other cells, those from 8-day-old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layers.
1,903 citations
TL;DR: Evidence is presented that the development of these colonies is under separate control from that of granulocytic colonies found in the same cultures.
Abstract: A culture method has been developed in which erythroid colonies are produced in vitro from hemopoietic cells from the livers of 13-day fetuses of C3Hf/Bi mice. Heme synthesis by the cultures was correlated with the presence of these colonies, and the hemoglobin produced was shown to be electrophoretically normal. The individual colonies were identified as erythroid since they were erythropoietin-dependent, positively stained by the histochemical “Lepehne” procedure for hemoglobin, and labeled by 59Fe radioautography. Evidence is presented that the development of these colonies is under separate control from that of granulocytic colonies found in the same cultures.
581 citations
TL;DR: It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells, and that nodes containing such cells can participate in an immunological response against sheep red cells.
Abstract: The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.
429 citations
TL;DR: In this paper, the theta (θ) isoantigen is determined by a single locus with two alleles: θAKR and RF mice and θC3H present in most other inbred strains of mice tested, which is found chiefly in thymus lymphocytes and brain, and to a lesser extent in peripheral lymphocytes in mice.
Abstract: THERE is an obvious need for a marker that will differentiate one type of lymphocyte from another. The need has become urgent in view of recent evidence suggesting that there are at least two populations of lymphocytes, one thymus-derived and one bone marrow-derived, which participate in different ways in the immune response1. The theta (θ) isoantigen (θ is determined by a single locus with two alleles: θAKR found in AKR and RF mice and θC3H present in most other inbred strains of mice tested), described by Reif and Allen2,3, which is found chiefly in thymus lymphocytes and brain, and to a lesser extent in peripheral lymphocytes in mice, seemed a possible antigenic marker of thymus-derived lymphocytes. To establish that θ is such a marker, it is necessary to demonstrate that there is a discrete population of peripheral lymphocytes which carry the antigen and that these cells are thymus-dependent.
375 citations