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REVIEWS Selection and Transplantation of Hematopoietic Stem and Progenitor Cells Karen Auditore-Hargreaves,’ Shelly Heimfeld, and Ronald J. Berenson CellPro, Inc., 22322 20th Avenue SE, Bothell, Washington 98021. Received December 6, 1993 The mammalian hematopoietic system is composed of multiple cell types that provide vital infection-fighting, blood-clotting, and oxygen-carrying abilities to the body. These cells arise in the bone marrow and migrate to the periphery as they mature. The hematopoietic system represents a spectrum of differentiation encompassing, at one end, mature, morphologically identifiable cells that have little or no ability to divide. At the opposite end is a rare, but self-sustaining population of stem cells that have the ability to give rise to cells of all the hematopoietic lineages. In between these two extremes are cells termed progenitor cells which, although committed to a specific lineage, are functionally immature and retain a limited capacity for proliferation. The antigenic profile of hematopoietic cells changes during the course of differentiation, with some antigens lost and others gained (Beverley et al., 1980;Fitchen et al., 1981;Civin and Loken, 1987). This heterogeneity enables one to discriminate among and isolate cells at different points in the continuum, paving the way to a variety of research and clinical applications for the various subsets of cells. This review will focus on methods, especially immunologically based techniques, for the isolation of human hematopoietic stem and progenitor cells and will summarize clinical experience using these cells for bone marrow transplantation and gene therapy. BONE MARROW TRANSPLANTATION
Approximately 25 years ago, E. Donnall Thomas and colleagues pioneered bone marrow transplantation to reconstitute the hematopoietic system of patients exposed to myeloablative doses of chemotherapy and/or radiation for the treatment of malignancy (reviewed by Thomas et al. (1975)). Today, two types of bone marrow transplantation are practiced, autologous and allogeneic. In an autologous transplant, a portion of the patient’s own marrow is removed prior to myeloablation, stored frozen, and reinfused at the completion of the patient’s treatment. In an allogeneic transplant, marrow is obtained from a donor, usually a relative and preferably one whose HLA type matches that of the transplant recipient, and infused into a patient whose own marrow has been destroyed by radiation andlor chemotherapy. The application of allogeneic bone marrow transplantation has been limited by the inability to transplant across a major histocompatibility barrier and by the occurrence of severe, often life-threatening graft versus host disease (GVHD) in approximately one-third to one-half of transplant recipients. The severity of GVHD, which is caused at least in part by T cells present in the graft, is directly related to the degree of mismatch between donor and recipient. The availability of potent immunosuppressive drugs, such as cyclosporine, has helped to control the incidence and severity of GVHD, but it has not eliminated the problem. 1043-1a0219412905-02a7$04.5010
Autologous transplantation does not suffer from these limitations; however, it may not always be an alternative, especially in patients whose tumor involves the bone marrow. With improved methods of detection, such as immunocytochemical staining, it has become clear that tumor cell contamination of the marrow is more common than was thought when diagnosis was made by routine histology. For example, marrow metastases have been detected in 1 7 4 4 % of patients with solid tumors whose marrows appeared histologically normal at the time of harvest (Berendsen et al., 1988; Cote et al., 1988; Porro et al., 1988; Moss et al., 1991). Bone marrow is obtained by aspiration from the pelvis or sternum of anesthetized patients or donors. Between 5 and 25 mL of marrow are removed at each aspiration, with the needle moved to a new site between aspirations. A typical transplant involves collecting 1-2 L of marrow and processing that by centrifugation to yield 200-500 mL of buffy coat. Until relatively recently, this entire volume of buffy coat was then infused into the patient. Although only the stem cells in marrow are necessary for engraftment and although these constitute only a small fraction (less than 1 % )of the total cells in marrow, there was no clinically practicable method to identify and isolate these rare cells from among the much larger number of unnecessary cells. Human Stem Cell Markers. A cell surface marker unique to human hematopoietic stem cells has not yet been discovered, but several markers are known to be shared among stem cells and early progenitor cells. One such marker, the CD34 antigen, identifies a population of cells capable of mediating sustained reconstitution of the hematopoietic system. A large number of monoclonal antibodies have been identified that collectively define at least three epitopes on the human CD34 antigen (Civin et al., 1984; Tindle et al., 1984, 1985; Andrews et al., 1986; Fina et al., 1990). These epitopes are distinguished by their sensitivity to various enzymes (Sutherland et al., 1992). The CD34 antigen is a 115 kDa glycoprotein present on 1-3 % of human bone marrow cells, including virtually all committed progenitor cells, as well as more primitive progenitors, such as the long-term culture-initiating cell, detected by in vitro assays. It is not expressed at detectable levels on mature blood cells, including lymphocytes, granulocytes,erythrocytes, and platelets, nor is it expressed on most malignant cell types, with the exception of certain leukemias (reviewed in Sutherland and Keating, 1992). The CD34 antigen shows no sequence homology with any other previously described molecules at either the protein level (Sutherland et al., 1988)or the DNA level (Simmons et al., 1992), and little is known about its function in the cell membrane. Berenson and colleagues, working initially in a baboon model (Berenson et al., 1988)and subsequently in humans 0 1994 American Chemical Society
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Figure 1. CD34 antigen expression in hematopoiesis. The CD34 antigen is a 115 kDa glycoprotein which is expressed on pluripotent stem cells and committed progenitor cells in human bone marrow (depicted in the left-hand box). The level of CD34 expression gradually declines as cells differentiate along a given lineage, such that mature cells in the blood (right-hand box) are uniformly CD34 negative.
(Berenson et al., 1991; Shpall et al., 1992a,b), were the first to show that CD34+ cells could reconstitute the hematopoieticsystem following myeloablativetreatment. These i n vivo studies were critical to the validation of CD34 as a stem cell marker, as there is no definitive in vitro assay for the human stem cell. Sources of CD34+ Cells. Bone Marrow. The bone marrow is a particularly rich source of CD34+ cells. Figure 1shows a schematic representation of the distribution of the CD34 antigen on hematopoietic cells in the bone marrow. The majority of these CD34+ cells are committed progenitors, rather than true stem cells. True stem cells probably represent only about 1in lo5bone marrow cells. The fact that the CD34 antigen identifies both the stem cell and progenitor populations is an advantage for clinical applicationssuch as bone marrow transplantation. Several studies in mice have shown that engraftment is delayed when marrow depleted of progenitor cells is transplanted, thus placing the recipient at increased risk of bleeding and infectious complications (Jones et al., 1989, 1990). These same studies demonstrated that transplantation of marrow lacking stem cells leads to rapid, but transient, engraftment. Thus, stem cells as well as progenitor cells are necessary to accomplish both rapid and sustained engraftment of transplant patients. Since CD34 marks both populations, it is an ideal antigen for therapeutic protocols. Peripheral Blood. Numerous studies in animals (Cavins et al., 1964; Nothdurft et al., 1977; Fliedner et al., 1979; Storb et al., 1977;Carbonell et al., 1984)and man (Korbling et al., 1981,1986; Reiffers et al., 1986) have demonstrated that hematopoieticstem cells circulate in peripheral blood and can reconstitute hematopoiesis completely and permanently. The number of stem cells normally present in the peripheral circulation is small compared with marrow (Kessinger et al., 1986; Bell et al., 1986; Bender et al., 1991). However, their number can be increased dramati-
cally by pretreatment with an agent that mobilizes stem and progenitor cells from the bone marrow. G-CSF (Duhrsen et al., 1988),GM-CSF (Gianni et al., 1989),and cyclophosphamide(Siena et al., 1989;To et al., 1989)have all been used successfully to increase the CD34 content of peripheral blood. Consequently, it is now possible to isolate by an apheresis procedure sufficient quantities of engrafting cells from the peripheral blood to transplant myelosuppressed or ablated patients (Korbling and Martin, 1988). UmbilicaZ Cord Blood. CD34+ stem and progenitor cells have also been obtained from umbilical cord blood at parturition (Broxmeyer et al., 1989,1991, 1992). The use of cord blood CD34+ cells for transplant is attractive because the material is readily available and matching of donor and recipient may be less important than when adult cells are employed. Several groupshave reported success in treating children with malignant (Vilmer et al., 1992; Wagner et al., 1992) and nonmalignant diseases (Gluckman et al., 1989) using these cells. More recently, two children with severe combined immunodeficiency disease (SCID) have been transplanted with cord blood stem cells genetically modified to express adenosine deaminase (ADA), the enzyme missing in this disease (Kohn et al., 1993). Several groups have advocated the banking of cord blood for future use in transplantation or gene therapy (Wagner, 1993). METHODS OF CELL SELECTION
Methods for fractionating heterogeneous mixtures of cells into subpopulations include techniques based on the cells’ physical properties, such as size and density, and specific binding methods, in which cells are identified by their expression of surface markers. The technique of choice for a given application is dictated by the frequency of the cells of interest in the starting population, the degree
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of enrichment required, the desired yield, and the intended use of the selected cells. Selection of hematopoietic stem and progenitor cells is a particularly demanding application of cell selection technology because these cells are so rare in the tissues from which they are typically isolated. The less frequently a cell type occurs in a given tissue, the larger the amount of that tissue which must be processed in order to obtain a desired number of the target cell type. In order to obtain an engrafting dose of CD34+ cells, approximately 20 billion nucleated marrow cells must be processed. It is also self-evident that in any cell fractionation procedure there is a trade-off between purity and yield which must be balanced. In the stem cell transplant setting, there is probably a threshold number of CD34+ cells below which the recipient will not engraft in a clinically acceptable time frame. However, for ethical reasons, the expected dose-response relationship has been difficult to demonstrate in humans. In clinicaltransplantation studies with CD34-selected cells, engraftment has been seen with as few as 0.5 X lo6 CD34-selected cellslkg (Shpall et al., 1994),but it is likely that the actual threshold dose is even lower. In the mouse, where there are fewer ethical constraints, studies indicate that the LDm for survival of lethally irradiated animals is approximately 30 “true”stem cells (Spangrude et al., 1988). Both the number and the nature of nontarget cells contaminating a target cell preparation are important in a cell selection process where the cells are to be used clinically. The number of CD34-negative cells which can be tolerated in a CD34-selected preparation is dependent on whether or not those CD34-negative cells are clinically neutral. If they are clinically neutral, a relatively high proportion of them may be tolerated in the enriched product. In the allogeneic stem cell transplant setting, for example, the presence of granulocytes in the CD34enriched fraction is clinically inconsequential. However, contamination of this fraction with T lymphocytes is highly significant, as these cells contribute to the development of GVHD in the recipient and thus influence clinical outcome. Physical Separation Methods. Physical methods of cell separation include velocity sedimentation, density gradient centrifugation, counterflow centrifugal elutriation, and related techniques (reviewed in Kumar and Lykke, 1984). Velocity sedimentation, using automated or semiautomated cell-separating centrifuges, has been used to separate the red cells and plasma from the nucleated cells in marrow (Gilmore et al., 1983; Faradji et al., 1988)and peripheral blood (Korbling and Martin, 1988; Williams et al., 1990). This technique reduces the volume of material by about 10-fold and recovers 60-80% of nucleated cells. Density gradient separations can also be accomplished on these machines (English et al., 1989; Humblet et al., 1988). Human hematopoietic stem cells, as assayed by their ability to form colonies in methyl cellulose, are lighter (1.060-1.070 g/mL) than most other mononuclear cells in bone marrow (Lasky and Zanjani, 1985;Francis et al., 1983; Ellis et al., 1984). Thus, a density separation method can afford a modest (2- to 10-fold) enrichment of colonyforming cells relative to other cells in the marrow. Colonyforming cells in cord blood are thought to be more dense than those in marrow (Broxmeyer et ai., 1991; reviewed in Flomenberg and Keever, 1992);hence, use of the typical density media (Ficoll, Percoll) to fractionate cord blood may result in loss of 50-90% of committed progenitor
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cells (Broxmeyer et al., 1989, 1990, 1991; Nakahata and Ogawa, 1982). In counterflow centrifugal elutriation, a cell suspension is pumped into a spinning chamber in a direction opposite to the centrifugal field. Either the pump speed or the rotor speed is varied gradually, causing cells of increasing sedimentation rate to elute out of the chamber in a centripetal direction. Studies by Noga and colleagueshave shown that when this technique is applied to human bone marrow, CD34+ cells are recovered predominantly in the 100 mL/min and rotor-off fractions, where the majority of CFU-GM are also found (Gao et al., 1987; Noga et al., 1986a,b). Unfortunately, the majority of lymphocytes are found in these same fractions, making elutriation of limited utility for the purification of allogeneic marrows, because the T cells that mediate GVHD are enriched to approximately the same extent as the stem and progenitor cells. Generally speaking, the degree of purification of stem cells achieved with physical separation methods is low (
