Stem Cells, Vol. 14, No. 3, 366-367,
May 1996
© 1996 AlphaMed Press
Keystone Symposium: The Hematopoietic Microenvironment
Joel Greenberger,
Armand Keating
University of Pittsburgh Pittsburgh, PA 15213 USA
The Toronto Hospital Toronto, ONT, M5G 2C4 Canada
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Introduction
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Organized by Armand Keating and Joel Greenberger Taos, New Mexico, February, 1996
The Symposium in Taos was the first major meeting in several years devoted exclusively to the hematopoietic microenvironment and was organized to bring together investigators with diverse expertise and common interests in this field. This was underscored by presentations from cell biologists, biochemists, zoologists, molecular biologists, radiobiologists, developmental biologists and clinical investigators. Another important feature of the Symposium was the inclusion of presentations by investigators working on the microenvironment of tissues other than the bone marrow such as the gut, the thymus, the central nervous system and bone. Speakers at the meeting were charged with helping to address some of the key questions concerning the microenvironment. Rather than providing a comprehensive summary of the Symposium, this brief synopsis alludes to some of the data presented that help to answer some of those questions, or, more frequently, contribute to rephrasing the questions themselves.
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What cells comprise the hematopoietic microenvironment?
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Most studies employ cells derived from the adherent layers of long-term marrow cultures. Work by Wolf (Seattle) and Charbord (Besançon) indicates the presence of subtypes of non-hematopoietic mesenchymal cells expressing muscle actins, including some that may be involved in cell adhesion (metavinculin). Recent interesting work by Simmons (Adelaide) on stromal cell progenitors uses the Stro-1 antibody as a tool. Stro-1 reacts with all CFU-F, with smooth muscle cell precursors, endothelial cells and adipocytes. A minor, bright population of Stro-1+ cells in human marrow generates a homogeneous population of CFU-F grown under serum-deprived conditions. Characterization of the Stro-1 antigen is hampered by the isotype and low affinity of the antibody. Waller (Atlanta) has identified a stromal progenitor population with the characteristics CD34(+)DR(–) CD38(–)CD50(–). These studies will be helpful in establishing lineage-stem cell relationships for microenvironmental cells, including pre-adipocytes, osteoblasts and smooth muscle cell precursors. However, considerable work on stromal cell ontogeny is required to reach a level of understanding comparable to that of hematopoiesis.
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Why are stem cells maintained in murine but not in human marrow cultures?
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The basis for differences is unclear and a human hematopoietic stem cell assay remains elusive. The exciting studies by Zanjani (Reno) showing engraftment of human hematopoietic stem cells in pre-immune sheep are a major advance but too resource-intensive and expensive to serve as a routine assay. A similar limitation applies to the SCID mouse-human stem cell assay. The work of Lebkowski (Santa Clara) looking at long-term hematopoietic reconstitution with limiting numbers of cultured hematopoietic cell subpopulations (such as thy(+)Lin(–) cells) in murine recipients is helpful but painstaking. Although conventional human long-term marrow cultures are currently sub-optimal, studies by Emerson (Philadelphia) and colleagues with bioreactor technology suggest that new insights will be gained into the role of stromal cells in supporting progenitor cell proliferation. Further studies are required to develop a simple stem cell assay that, in turn, will aid in establishing the optimal composition of an in vitro microenvironment for the culture of hematopoietic stem cells.
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How do cobblestone islands mediate stem cell quiescence?
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Gordon (London) provided some insight into this issue by demonstrating that despite adherence to stroma, progenitors in chronic myeloid leukemia are in cycle, in contrast to the observation with stromal cells from normal subjects. A potentially important notion is the possibility that hematopoietic stem cells are required to appropriately influence stromal cell biology. Perhaps cobblestone islands and their effects on hematopoiesis are dependent on stem cells as well as on stromal cells, suggesting a fruitful area of investigation.
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How does regulation of adhesion molecules and integrin expression relate to stromal cell biology in vivo?
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This question has relevance to the issue of the homing of hematopoietic cells. Interesting studies were reported by Hardy (Jackson), who showed that the neoglycoprotein, galactosyl-bovine serum albumin partially blocks hematopoietic stem cell seeding in vivo, in contrast to a fucosyl derivative, and suggests the existence of a homing receptor that may precede interactions with 
4 and ß1 integrins. Further studies and other models are required to definitively address this issue. Progress has been limited by difficulties in tracking stem cells, by the lack of data documenting differences in the microenvironments of the liver, spleen and bone marrow, and by uncertainty regarding the key functional microenvironmental cells involved and how changes in the expression of adhesion molecules can be assayed in vivo. Presentations on non-hematopoietic microenvironments, while fascinating in their own right, may provide useful models to probe this question.
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What role does the stromal cell microenvironment play in leukemogenesis?
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Recent research in this field has focused on stem cell-stromal cell interactions both at the molecular level of cellular attachment and cytokine production and the cell biology level. The stroma in chronic myelogenous leukemia (CML) may influence the biology of both normal and CML stem cells. Other studies by Verfaille (Minneapolis) suggest that the binding of CML stem cells to stromal cells may be critical to the pathogenesis of CML.
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What stromal cell factors control trafficking of stem cells and return to quiescence after movement?
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Studies comparing the biology of transplantation of cord blood stem cells, peripheral blood and bone marrow stem cells highlight the similarities in the biology of these different cell populations but also suggest differences. Bernstein (Seattle) discussed phenotypic markers on the cell surface that may not be fully explained by the stromal cell factors involved in the differentiation of the stem cell from the multilineage compartment, or by the biologic changes that accompany trafficking. The role of bone marrow stromal cells in modulating stem cell biology is a new field and requires new models and methods to study the mechanism of homing.
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Which bone marrow stromal cell phenotypes are transplantable?
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Several laboratories have provided insights into the mechanism whereby bone marrow stromal cells can proliferate in vitro and differentiate to osteoblasts—Taichman (Ann Arbor), Long (Ann Arbor), Bab (Tel Aviv); or to fibroblasts and adipocytes—Simmons (Adelaide). Work by Keating (Toronto), Hurwitz (Marlborough, MA) and Nolta (Los Angeles) suggests that stromal cells can carry relevant transgenes in vivo and provide continuous short-term production of gene products in vivo. The use of stromal cells in gene therapy will require a better understanding of the most effective way of expanding relevant cell populations in vitro, more effective ways of targeting transgenes to stromal cells, and, most importantly, facilitating molecular mechanisms for the prolonged expression of gene products in the engrafted stroma. The mechanism of homing of bone marrow stromal cells in vivo is poorly understood; however, the work of Prokop (Philadelphia) indicates that bone marrow stromal cells introduced by intravenous injection may locate at multiple sites outside the bone marrow and give rise to detectable levels of stromal cell-associated gene products at distant sites.
The meeting also stressed the importance of understanding the interaction of bone marrow stromal cells with cells of the osteo-inductive microenvironment. While controversy persists as to the precise phenotype of osteoblasts, and whether a true bi-lineage osteoblast/bone marrow stromal fibroblast exists, there was consensus that investigators working in hematopoietic stromal cell biology and researchers in osteogenesis need to communicate more frequently with one another. This might resolve some of the questions which remain about nomenclature of cell types, and the physiologic properties of stromal cells with a common role in both fields.
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Introduction
What cells comprise the...