JOURNAL OF HEMATOTHERAPY 1:115-129 Mary Ann Lieben, Inc., Publishers

The CD34

(1992)

Antigen: Structure, Biology, Clinical Applications

and Potential

D. ROBERT SUTHERLAND and ARMAND KEATING

ABSTRACT The diversity of function of mature circulating blood cells is reflected in their respective complements of cell-surface molecules and receptors. Although monoclonal antibodies have

been instrumental in the identification and characterization of many cell-surface molecules on mature hematopoietic cells, the CD34 antigen represents to date, the only molecule, similarly identified, whose expression within the blood system is restricted to a small number of primitive progenitor cells in the bone marrow. Although its precise function remains

unknown, the pattern of expression of the CD34 structure suggests that it plays an important

early hematopoiesis. The availability of CD34 antibodies has greatly aided the development of techniques for the enrichment of primitive progenitor cells for studies of hematopoiesis in vitro. Additionally, the use of CD34 antibodies for the 'positive selection' of hematopoietic stem/progenitor cells represents an alternative strategy to 'negative selection' or purging for the large-scale manipulation of bone marrow cells prior to transplantation. The availability of pure populations of the most primitive hematopoietic progenitor cells may also facilitate the development of genetic techniques for the repair of specific blood cell disorders. In this article, we review the biology of the CD34 molecule and assess some of the roles for CD34 antibodies in immunopathology and for progenitor/stem cell purification in clinical applications. role in

SEROLOGIC STUDIES

(MY 10 [Civin e/ al., 1984], B1.3C5 [Katzef at., 1985], 12.8, 115.2 [Andrews et al., ICH3 [Watt et al., 1987], and TUK.3 [Unchanska-Ziegler et al., 1989] raised against the AML-derived KG1 or KG la cell lines [Koefflerei al., 1980] and an anti-endothelial cell antibody (QBEND 10 [ Fina et al., 1990] assigned to the CD34 cluster [Civin et al., 1989] have been shown to identify an antigen that is expressed on 1-3% of normal bone marrow cells. This population has been shown by colony-forming assays to include virtually all unipotent—burst-forming units-erythroid, colony-forming units-granulocyte/

Six1986],

antibodies

macrophage, colony-forming units-megakaryocyte (BFU-E, CFU-G/-M, CFU-Meg)—and multipotent

Oncology Research, and the University of Toronto Autologous Hospital, Toronto, Ontario M5G 2C4, Canada. 115

Bone Marrow

Transplant Program,

The Toronto

SUTHERLAND AND KEATING

progenitors—colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) as well as pre-CFU (Civin etal., 1984; Katz etal., 1985; Andrews et al., 1986). Primitive T- and B-lymphocyte precursors ( (Katz et al., 1985; Ryan et al., 1986) and leukemias of primitive myeloid and lymphoid lineages also express CD34 (Katz et al., 1985; Ryan etal., 1986; Watt etal., 1987; Facklere/a/., 1990a; Schuhe al., 1990). Furthermore, single-cell cloning experiments demonstrate that highly purified CD34+ cells, which lack coexpression of any lineage-associated antigens, are capable of generating several types of colonies when grown over irradiated stromal cells in vitro (Andrews et al., 1990). Significantly, CD34+ bone marrow cells can reconstitute all lineages of the hematopoietic system in lethally irradiated baboons (Berenson etal., 1988) and rhesus monkeys (Wagermaker et al., 1990), suggesting (but not unequivocally proving) that the 'stem cells' responsible for long-term reconstitution of hematopoiesis express the CD34 antigen. A recent report of the autotransplantation of CD34+ cells after intensive therapy in patients with solid tumors suggests that this population is also capable of reconstituting hematopoiesis in humans (Berenson et al., 1991). In healthy individuals, CD34 expression is confined to panhematopoietic progenitor cells in the bone marrow, with the exception of vascular endothelial cells (see below), which are CD34+ on immunohistologic staining (Watt et al., 1987; Beschomer et al., 1985). Tumor expression of CD34 tends to mirror normal expression, and CD34 antibodies bind strongly to angioblastomas and some Kaposi's sarcomas (Fina et al., 1990; Sankey et al., 1990). Binding is especially striking around the vascular sprouts within tumors where active angiogenesis is taking place (Schlingemann etal., 1990). Essentially, all other nonhematologic tumors are unreactive (Watt et al., 1987). About 40% of acute myeloid leukemias and 65% of 'common' (pre-B) acute lymphoblastic leukemias are reactive, whereas only 1-5% of acute T-lymphoid leukemias express the CD34 antigen (Civin et al., 1989). Blasts (exhibiting the composite phenotypes of primitive T-, B-, or myeloid cells) from chronic myeloid leukemia (CML) patients in blast crisis are often reactive, whereas chronic-phase cells, other chronic leukemias, and lymphomas of more differentiated phenotypes are uniformly negative (Civin etal., 1989). EPITOPE MAPPING STUDIES From early studies, it was apparent that the CD34 antibodies MY10, B1.3C5, and ICH3 were detecting distinct, non-overlapping epitopes and that the carbohydrate moieties in general, and the terminal sialic acid residues in particular, were important for antibody binding (Watt et al., 1987). Recently, we demonstrated

that a novel proteolytic enzyme from Pasteurella haemolytica (P. h. glycoprotease), which is highly specific for glycoproteins rich in O-linked glycans (Otulakowski et al., 1991), cleaves the CD34 antigen (Sutherland et al., 1992a). We subsequently showed that the epitopes detected by five of the seven CD34 antibodies are removed by glycoprotease treatment (Sutherland etal., 1992b). All epitopes previously shown to be variably dependent upon the presence of sialic acid residues, i.e., MY10, B1.3C5, 12.8, and ICH3, are efficiently removed by the P. h. glycoprotease (Fig. 1). These epitopes were designated class I. The enzyme also removed the sialic acid-independent epitope detected by QBEND 10 (class II). The epitopes detected by TUK3 and 115.2 that were not cleaved by either enzyme were referred to as class III. Class III epitopes are therefore closer to the extracellular membrane surface than the class I and class II epitopes. As evidenced by the shape of the histograms shown in Fig. 1, it is perhaps noteworthy that antibodies that are either totally dependent (B1.3C5 and 12.8), or partially dependent (MY 10 and ICH3) on the presence of sialic acid residues for their binding, tend to generate a greater range of staining intensities on KG1 cells than the class II or III antibodies. These observations suggest that there may be some cell type variation in the glycosylation of the CD34 antigen. Indeed, using a recently derived subline of KG 1 that is incapable of efficiently adding terminal sialic acid residues to carbohydrate moieties of glycoproteins, we find striking discordance of CD34 epitope expression. The binding of all class I epitopes is either reduced (MY10 and ICH3), or is almost totally abrogated (B1.3C5 and 12.8). As expected, however, the binding of class II (QBEND10) and class III (TUK3 and 115.2) antibodies is if anything, slightly increased (M.F. Greaves and D.R. Sutherland, unpublished observations). Furthermore, at least with respect to class I and class II antibodies, there is also some discordance of CD34 epitope expression in normal hematopoietic progenitor cells (P. Grimsley, M. Siczkowski, and M.F. Greaves, personal communication). These observations, although preliminary, 116

CD34 ANTIGEN

MY10

B1.3C5

—jaSi^ f'^'TTTTTTi

'

IMnM

it—,T,m""

LFL1

Av

12.8

ICH3 ÏÎL_

.rrul "'i

^**r-

JtNJ

iii

in.

îr"

LFL1

L^**^

TÜK3

QBEND10

TV

FIG. 1. Effects of neuraminidase and Pasteurella haemolytica glycoprotease cleavage on CD34 epitopes. KG1 cells were stained with anti-CD34 antibodies as indicated in Sutherland et al. ( 1992b) and analyzed by flow cytometry. For each antibody, the lower histogram (i) represents the staining of untreated cells, the middle histogram (ii) represents the staining of neuraminidase-treated cells, and the upper histogram (iii) represents the staining of the glycoprotease-treated cells.

117

SUTHERLAND AND KEATING suggest that for most purposes such as immunophenotyping or progenitor cell purification (see below), it may be advantageous to use CD34 antibodies that detect sialic acid independent, i.e., class II or class III epitopes.

IMMUNOCHEMISTRY a monomeric structure of about 110 kD from albeit with different efficiencies la KG cells, (Fig. 2). Similar bands can be isolated from lysates fresh CD34+ acute leukemias of primitive myeloid, B-lymphoid and T-lymphoid phenotypes (Katz et al., 1985; Facklerer al., 1990a; Schuh et al., 1990). These antibodies also react with COS cells transfected with a CD34 cDNA providing conclusive evidence that they recognize the same gene product (Fina et al., 1990). Several CD34 antibodies identify denaturation-resistant epitopes in Western blots, although with widely different efficiencies (Fig. 3). MY10 and ICH3 are partially dependent and B1.3C5 and 12.8 are totally dependent on the presence of sialic acid residues for their binding, in agreement with the epitope mapping studies described above. Strikingly, the desialylated form of this structure exhibits an increased molecular weight of about 150 kD. Such aberrant mobilities in one-dimensional SDS-PAGE reflect the influence that multiple negatively charged sialic acid residues can have on glycoprotein mobility in SDS gels (Iyer & Carlson, 1971). In keeping with their inability to sialylate cell-surface glycoproteins, the CD34 antigen from sialic acid-deficient KG1 cells exhibits a molecular weight similar to that of desialylated CD34 antigen extracted from 'wild-type' KG1 cells (D.R. Sutherland and M.F. Greaves, unpublished observations). A combination of lectin-binding studies and endoglycosidase cleavage experiments demonstrated the presence of several complex-type N-linked glycans. Alkaline hydrolysis followed by gel filtration of released glycans indicated the presence of O-linked carbohydrates. Binding of the desialylated CD34 structure to peanut lectin confirmed the presence of O-linked glycans of the sialylated Galßl-3GalNAc-R type

All

seven

workshop CD34 antibodies immunoprecipitate

of KG1

or

Mr(xio-3)

/c? /

/

75% and >70% identical, respectively). The amino-terminal domains of about 145 amino acids (encoded by exons 2 and 3) are only 45% sequence identical. A (high-probability) potential chondroitin sulfate conjugation site (Ser-Gly-Ser-Gly) is additionally present in the murine sequence. However, the presence of high levels of serine and threonine residues in this domain strikingly is conserved between the species, suggesting that the O-linked carbohydrate moiety may determine the functional capabilities of this part of the CD34 structure. A comparison of the predicted human and murine CD34 structures is shown schematically in Fig. 5.

Gene

expression studies

Hematopoietic Cells: Previous studies have demonstrated that there is a close correlation between CD34 mRNA and CD34 antigen expression in a variety of cell lines and tumor tissues. Together with the observations that all workshop CD34 antibodies bind to COS cells transfected with the CD34 cDNA, these data indicate that in most situations, control of CD34 gene expression is primarily at the level of transcription (Fina et al., 1990; Simmons et al., 1992). However, as indicated above, there may be cell-type variations in the complex patterns of glycosylation that this structure exhibits, which may give rise to occasional discrepancies between gene transcription and epitope-restricted surface antigen expression. Furthermore, as also indicated above, the CD34 molecule 'turns-over' rather slowly in cell lines in vivo (Sutherland et al., 1988; Satterthwaite et al., 1990). In support for this notion, a recent study of CD34+ pre-B-cell acute leukemias demonstrated that subfractions of individual patient's lymphoblasts that did not coexpress markers of more mature B cells, contained readily detectable levels of CD34 mRNA. In contrast, the fraction of CD344 lymphoblasts that coexpressed the CD22 structure (generally considered to be a marker of more mature B-lymphoid cells) did not contain detectable CD34 mRNA transcripts (Re el al., 1991 ). Finally, it was recently demonstrated that stimulation of protein kinases C with phorbol esters caused a rapid up-regulation of surface expression of the CD34 antigen in both cell lines and normal hematopoietic progenitor cells. This up-regulation was due to the translocation of preformed CD34 antigen from intracellular locales to the surface membrane, and took place in the presence of inhibitors of gene transcription (Fackler etal., 1992). Although these observations may not accurately reflect the correlation between CD34 gene transcription and glycoprotein expression in normal hematopoiesis in vivo, we feel that it is important to consider that post-translational modifications (N-, O-glycosylation, terminal sialylation, glycosamino-glycan conjugation, phosphorylation by a variety of kinases, glycoprotein half-life, etc.) of the CD34 polypeptide may be important modulators of this structure's functional capabilities. Nonhematopoietic Cells: Despite evidence from the staining of endothelial cells in tissue sections and the unequivocal assignation of the anti-endothelial cell antibody QBEND 10 to the CD34 cluster, we were unable to immunoprecipitate the CD34 structure from lysates of cultured endothelial.cells after either surface radioiodination or metabolic labeling. Similarly, Western blots of lysates of cultured endothelial cells failed to show the CD34 structure, possibly indicating that binding to endothelial cells was due to nonspecific cross-reactions. However, with the availability of molecular probes, cultured endothelial cells were shown to express CD34 mRNA of the same size (2.5 kb) as that in hematopoietic cells (Fina et al., 1990; Simmons et al., 1992). Finally, using freshly isolated umbilical cord endothelial cells, we were able to detect the CD34 glycoprotein (Fina et al., 1990), confirming that the CD34 gene product is expressed in endothelial cells. 121

CD34 cDNA

MOUSE

HOMOLOGY

HUMAN

NH2

NH2

145aa

45% 139/152aa

4 cys cys —

i cys 4 cys 4 cys 4 cys

66aa

71% 66aa

70aa

70aa 77%

vvvvvvvvv

vvvv

J

JiAAàAhAàA.

RRSWSi

73aa

92% 73aa

SXXR SXR

COOH 9N

-

COOH

«_7N-LINKED CHO

LINKED CHO

_

354aa

+

_X GAG SITE 361 aa + SIGNAL(21aa) or 348aa + SIGNAL(34aa)

SIGNAL(19aa)

FIG. 5.

Comparative structural aspects of the predicted human and murine CD34 structures. The number and approximate location of the cysteine residues is based on Simmons et al. (1992) for human and (Brown et al. 1991) for

The minimum size ofthe human CD34 structure (354 amino acids) is based upon amino-terminal amino acid (Sutherland et al., 1988). The murine CD34 antigen is predicted to be either 348 or 361 amino acids in length (Brown et al., I99l). The location of the potential glycosaminoglycan (GAG) linkage site in the murine CD34 antigen is indicated by an X. Two of three potential sites for PKC phosphorylation of human CD34 are conserved in the murine CD34. The RRSWS(PT) sequence represents a potential substrate site for several kinases (see text). mouse CD34.

sequence data

As already shown in Fig. 3, QBEND 10 was the most efficient of the workshop antibodies at detecting the denatured CD34 structure on Western blots. Similarly, a recent report also demonstrated that this antibody, in contrast to the others, also detects a fixation-resistant epitope in paraffin-embedded bone marrow biopsies 122

CD34 ANTIGEN

(Soligo et al., 1991). Thus, ultrathin transmission electron micrographs of capillary endothelium show the CD34 antigen to be localized to the luminal surfaces and particularly to the interdigitating surfaces of adjacent cells (Fina et al., 1990); it is strikingly absent from areas of tight junctions between the endothelial cells (Fina etal., 1990). Similarly, electron microscopy (EM) has also been used to clarify what appeared to be (by light microscopy) staining of the basement membrane or stromal elements. Under EM, this staining can be clearly demonstrated

to

be

on

tissue fibroblasts (Greaves

et

al., 1992) (G. Cattoretti, D. Robertson, M.F. Greaves,

personal communication).

FUNCTIONAL CONSIDERATIONS

Although the CD34 molecule has yet to give up its functional secrets, molecular and structural studies suggest that it has several features in common with CD43 (Cyster el al., 1991) in that both are highly

O-sialo-glycosylated proteins in which the carbohydrate content may account for more than 70% of the molecular mass. In sufficient quantity, molecules like CD34, CD43, and some members of the CD45 family (Thomas, 1989; Lansdorpef a/., 1990), which are also found on primitive leukocytes, could effectively shield the cell from interactions with many macromolecules/cytokines and/or other cells. It is likely, based on comparison with other mucin-like structures (Thomas, 1989, Jentoft, 1990; Cyster et al., 1991) that the amino-terminus of the CD34 antigen protrudes beyond the glycocalyx of the cell, like an extended 'flag-pole' structure. The numerous carbohydrate moieties, could by similar analogy, represent the 'flag.' These features, together with the antigen's relatively high density, lack of tyrosine kinase activity, and very slow rate of synthesis and turnover suggest that it is not a growth factor receptor/signal transducer but is more likely to be involved in cell-cell interactions or cell-matrix adhesion. The absence of CD34 in areas of tight junctions between endothelial cells is perhaps predictable, given the highly acidic nature of the molecule. However, the localization of the CD34 antigen to 'loose' interdigitating surfaces of these cells suggests that it could be serving as a distal ligand for a lectin-like component of the extracellular matrix laid down between adjacent cells. Alternatively, because capillaries are a major site for leukocyte binding and migration to extravascular locales, the highly sialylated CD34 structure may facilitate this process by discouraging the 'tight' association of adjacent endothelial cells. In the bone marrow, proteoglycan components of the extracellular matrix such as heparan sulfate have been shown to bind growth factors such as colony-stimulating factor granulocyte-macrophage (CSF-GM) (Gordon etal., 1987). It is possible that in this environment, the carbohydrate side chains of the CD34 molecule could perform the similar role of concentrating factors for presentation in high local concentrations to adjacent cells. If, as seems likely, the carbohydrate moieties in general, and the sialic acid residues in particular, modulate the function(s) of this part of the CD34 structure, then it is entirely possible that there could be several natural ligands for it; interactions with individual ligands in different environments could be determined by differential processing of the terminal sugar residues (particularly sialic acids) on the same basic glycoprotein structure. The availability of CD34 antibodies to a range of epitopes may aid future studies in this area. Whatever the function(s) of the CD34 antigen, its expression may be modulated by protein kinases. The CD34 antigen is a substrate for, and can be phosphorylated (on serine residues) to high stoichiometry, by activated protein kinase C (Fackler et al., 1990a; Sutherland et al., 1992c). It has also been demonstrated that other protein kinases, including glycogen synthase kinäse and casein kinase II, can phosphorylate the CD34 antigen (Fackler et al., 1990b), underlining the role of modulators of protein kinases in the biology of primitive leukocytes. The cytoplasmic domain of the human CD34 contains several potential protein kinase phosphorylation sites, including one for tyrosine kinases (Simmons et al., 1992). Thus far, however, no evidence for the phosphorylation of CD34 on tyrosine residues has been found (Fackler et al., 1990a,b; Sutherland et al., 1992c). The sequence, RRSWSPT (Fig. 5), could in theory be a substrate site for several serine/threonine protein kinases (reviewed by Kennelly & Krebs 1991), including protein kinase A, casein kinase I, and calmodulin-dependent kinase as well as protein kinase C (PKC), and as such could be an important site for the modulation of CD34 expression and function. 123

SUTHERLAND AND KEATING

CLINICAL APPLICATIONS

Immunopathology progenitors is panhematopoietic, and its expression (in nothing about lineage, CD34 antibodies are an important member of the immunopathologist's antibody panel for defining the overall or 'composite phenotype' of an individual sample. In some specific instances, for example, chronic myeloid leukemia (CML), increased numbers of CD34+ cells can be a reliable indicator of the onset of accelerated phase and/or blast crisis. Similarly, CD34 antibodies can help delineate B-cell lymphoma from acute lymphoblastic leukemia. Furthermore, with the availability of antibodies like QBEND 10, which can enumerate CD34+ cells in aldehyde-fixed, decalcified, paraffin-embedded bone marrow biopsies, it should be possible to optimize morphologic and immunohistochemical observations (Soligo et al'., 1991). Observed differences in the numbers of CD34+ cells and their appearance in clusters in some myelodysplastic syndromes, suggests that this type of analysis may be of diagnostic and prognostic utility (Soligo et al., 1991).

Although

isolation) in

CD34

some

antigen expression

acute

leukemias tells

in normal

us

Transplantation Although the function of the CD34 antigen remains elusive, the ability to use this antigen for the purification of stem/progenitor cells for bone marrow transplantation may have some important implications for the intensive treatment of selected malignancies, as well as possibly providing potential target cells for genetic manipulation studies in vitro. As indicated earlier, 'positive selection' of hematopoietic stem/ progenitor cells represents an alternative strategy to purging by 'negative selection' of bone marrow cells prior to transplantation. Several studies of autologous bone marrow transplantation using autograft-s purged of malignant cells are suggestive of clinical benefit (Rowley etal., 1989; Gorin etal., 1990; Gribben etal., 1991). Such trials have employed negative selection to eliminate neoplastic cells from the autografts of patients with hématologie malignancies. Potential problems with this general approach include toxicity to primitive hematopoietic progenitor cells, inadequate reduction in tumor load, and the frequent requirement for different purging modalities for different types of tumor. Definitive studies are required in this area and, in particular, the usefulness of eliminating clonogenic malignant cells from the autografts of patients with breast cancer and neuroblastoma is of considerable interest. Such malignant clones do not express CD34, and tumor-free autografts may be best obtained by employing the positive selection approach afforded by selective CD34 expression on hematopoietic stem cells (Berenson et al., 1991). The elimination of clonogenic leukemic cells from Philadelphia chromosome-positive (Ph+) CML patients, may also be amenable to a similar strategy. There is ample evidence, at least during the early phase, that normal hematopoiesis is detectable in the marrow aspirates of many patients with CML (Coulombel etal., 1983; Barnett et al., 1989). However, because a significant proportion of the leukemogenic CD34+ progenitors coexpress lineage-associated cell-surface antigens like CD7, CD19, or CD33, it should not be difficult to remove them. Primitive leukemic progenitors remaining in the depleted fraction, contain the BCR-ABL gene and express its product, p210'"'""w, which is a potent tyrosine kinse(Konopkac/a/., 1984). It is assumed that the presence of this promiscuous kinase in primitive CML progenitors confers a growth advantage over normal hematopoietic elements. It is anticipated that CD34+ CML progenitors will reflect this by coexpressing antigens like CD38 (Terstappen et al., 1991) and HLA-DR (Keating et al., 1984) that are generally associated with undifferentiated, but activated, progenitor cells. Recent data from Verfaule et al. ( 1992) indeed suggest that small numbers of primitive normal hematopoietic stem cells with the CD34+/lin~/ HLA~DR~ phenotype can be isolated from some CML marrow aspirates. Long-term cultures initiated with these cells gave rise to Ph colonies. Although further studies are needed to confirm these interesting observations, the results suggest that isolating normal progenitors from CML marrow for autografting may be feasible.

124

CD34 ANTIGEN it is technically difficult to isolate the most primitive (i.e., normal bone marrow populations for effective reconstitution of hematopoietic function in human recipients. Current technologies fail to provide pure populations of CD34+ progenitor cells in the quantities required on a reliable and cost-effective basis. While fluorescence-activated cell-sorting produces relatively pure populations of CD34 cells for in vitro experiments, the method is currently too slow to isolate the 1-3% CD34-reactive cells on the scale required for transplantation. Panning techniques require rigorous and laborious procedures to remove naturally adherent cells (macrophages,

However,

despite

such

promising studies,

CD34+ ) progenitor cells from

even

granulocytes, etc.) prior to panning on anti-CD34-coated surfaces in large plastic vessels. CD34+ cells produced by variations of this technique may routinely be contaminated with more than 30% CD34~ cells and the overall yields of CD34f cells are low. This technique is also relatively expensive in that it requires large quantities of highly purified antibodies. Another approach, which employs the use of avidin-biotin affinity column chromatography, can generate CD34+ progenitor cells in acceptable yields (25-58%) from bone marrow with purities in the 35-92% range (Berenson etal., 1991). Other affinity-based separation systems have used magnetic immunoselection techniques. A major disadvantage has been that overnight incubation at 37°C was required to remove the magnetic microspheres from the positively sorted cells by the process of capping and antigen turnover. More recently, the proteolytic enzyme chymopapain, has been used in conjunction with magnetic affinity matrix techniques to effect the release of the purified CD34+ population rapidly from the magnetic beads (Civin et al., 1990). However, because this enzyme cleaves a number of cell-surface molecules in addition to the CD34 antigen (D.R.S., unpublished observations), its usefulness may be limited in some clinical situations. We have recently described a simple, rapid, and flexible method to isolate primitive hematopoietic progenitor cells from normal bone marrow (Marsh et al., 1992). The CD34+ cells were positively selected using the high-affinity, class II antibody QBEND 10 (see Epitope Mapping Studies section), and immunomagnetic beads. Prior depletion of naturally adherent cells was not required. After magnetic selection of CD34+ cells/magnetic beads, the cells were detached from the beads by incubation with the Pasteurella haemolytica glycoprotease, which selectively cleaves glycoproteins rich in O-linked glycans (Otulakowski et al., 1991 ). The purity of the released cells was assessed by immunofluorescence techniques using either of the class III CD34 antibodies, TUK3 or 115.2, or the non-workshop class III antibody 8G12 (Landsdorp et al., 1990) (D.R. Sutherland and P. Landsdorp, unpublished observations), whose epitopes are not removed by the glycoprotease (Sutherland et al., 1992a,b). The purity (up to 95%) and yield (up to 80%) of the enzyme-released cells were high. The purified cells, which generated normal numbers of hematopoietic colonies in semisolid media, were enriched 81-fold for multilineage progenitors and reconstituted hematopoiesis in long-term culture, indicating that the functional competence of CD34+ progenitor cells in vitro, was unaffected by glycoprotease treatment (Marsh et al., 1992). In addition to providing a rapid and efficient technique for the purification of progenitors cells from normal marrow, this procedure could serve as an initial step in the selection of normal, Ph hematopoietic stem cells from CML marrows. Glycoproteins such as CD7, CD19, CD33, CD38, and HLA-DR, which are expressed on lineage-committed progenitors or their activated precursors (see above), are not cleaved by the Pasteurella glycoprotease (D.R. Sutherland, unpublished observations). Thus, by employing secondary fluorescenceactivated cell-sorting techniques, it should be possible to separate the leukemic progenitor cells from the residual, normal stem/progenitor cells that express only CD34.

Gene

therapy

therapy has emerged as an important new area of clinical investigation as demonstrated by the increasing number of protocols for human subjects approved by the NIH Recombinant DNA Advisory Gene

Committee in the United States. An early focus for the design of human gene therapy protocols involved the hematopoietic system, because the developmental biology of hematopoietic tissue is relatively well known and bone marrow transplantation

offers

a

suitable

means

of delivering

engineered cells (Keating, 1991). 125

SUTHERLAND AND KEATING

There is now broad consensus that retrovirus-mediated gene transfer is the preferred method of transfection, despite its current limitations (Keating, 1991). An additional potential drawback, especially for protocols involving hematopoietic cells, was the observation that successful transduction is accomplished only in actively dividing cells (Miller et al., 1990). Because most stem cells are noncycling, the probability of transfecting pluripotent progenitors by retroviral infection is expected to be low. However, prior incubation of marrow cells with cytokines has considerably improved gene transfer into committed progenitors (Hughes et al., 1989;Cournoyerera/., 1991). Optimal strategy for gene therapy using hematopoietic tissue involves the transduction of cells capable of long-term hematopoietic reconstitution and the orchestration of sustained lineage-specific expression of the transgene. Further advances toward this goal are likely to be achieved with CD34+ cell fractions (that include pluripotent progenitor cells) as target cells for the following reasons. First, because increased gene transfer efficiency can be achieved by stimulation of nucleated marrow cells with cytokines such as CSF-GM, interleukein-lß (IL-Iß), IL-3, and IL-6 (Hughes etal., 1989; Cournoyere/a/., 1991), further improvements may be obtained using a well-defined population such as the CD34+ fraction. An additional increase in gene transfer efficiency and expression might be expected with stimulation of CD34+ cells with stem cell factor (also called Steel factor or c-kit ligand) in combination with other cytokines. Moreover, absence of accessory cells in such a population may obviate compensatory mechanisms that stimulate production of negative hematopoietic regulators. Another advantage in using a hematopoietic cell population enriched for CD34+ cells is that fewer cells need to be transfected to provide an adequate transduced marrow graft. More importantly, the statistical probability of insertional mutagenesis is reduced because this phenomenon is related to the total number of cells transduced (Karlsson, 1991). Finally, successful in vitro expansion/ proliferation with cytokine-marrow stromal cell combinations may yield marrow grafts with an increased capacity for rapid hematopoietic engraftment (Moore et al., 1992). Additional in vitro studies are required to investigate the progeny of transduced long-term reconstituting cells using two-stage long-term marrow culture methodology with preformed functional adherent stromal layers (Andrews et al., 1990). Recent studies would also indicate that similar populations may be amenable to investigations in the SCID mouse (Lapidot etal., 1992).

ACKNOWLEDGMENTS The authors thank Prof. M.F. Greaves and his colleagues at the Leukemia Research Fund Centre, U.K., and Dr. M.J. Fackler and her colleagues at Johns Hopkins Oncology Center, for making available to us, previously unpublished observations. We thank Dr. P.M. Lansdorp for the 8G12 antibody. This work is supported by the Medical Research Council of Canada, the National Cancer Institute of Canada (NCIC), and the Leukemia Research Fund of Canada. Dr. A. Keating is a Senior Cancer Research Scientist of the NCIC.

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I.D. (1986): Monoclonal antibody 12.8 recognizes a 115-kd molecule present unipotent and multipotent colony-forming cells and their precursors. Blood 67: 842. Andrews, R.G., Singer, J.W. & Bernstein, I.D. (1990): Human hematopoietic precursors in long-term culture: Single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells. J Exp Med 172: 355. on

both

Atkin, N.B. (1986): Chromosome 1 aberrations in cancer. Cancer Genet. Cytogenet.'21: 279. Barnett, M.J., Eaves, C.J., Philips, G., Kalousek, D.K., Klingemann, H.G., Lansdorp, P.M., Reece, D.E., Shephard, J.D., Shaw, G.J. & Eaves, A.C. (1989): Successful autografting in chronic myeloid leukemia after maintenance of marrow

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Berenson, R.J., Andrews, R.G.. Bensinger, W.I., Kalamasz, D., Knitter, G., Buckner, CD. & Bernstein, I.D. (1988):

Antigen CD34-positive marrow cells engraft lethally

irradiated baboons. J. Clin. Invest. 81: 951.

126

CD34 ANTIGEN

Berenson, R.J., Bensinger, W.I., Hill, R.S., Andrews, R.G., Garcia-Lopez, J., Kalamaz, D.F., Still, B.J., Spitzer, G., Buckner, D., Bernstein, I.D. & Thomas, E.D. (1991 ): Engraftment after infusion of CD34+ marrow cells in patients with breast

cancer or

neuroblastoma. Blood 77: 1717.

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antibody.

Brown, J., Greaves, M.F. & mouse

and

Am. J. Pathol. 119: I.

Molgaard,

H.V.

(1991): The gene encoding the

stem cell

antigen CD34

expressed in haemopoietic progenitor cell lines, brain, and embryonic fibroblasts.

is conserved in Int. Immunol. 3: 175.

Carlsson, S.R. & Fukuda, M. (1986): Isolation and characterization of leukosialin, a major sialoglycoprotein on human

leukocytes. J.

Biol. Chem. 261: 12779.

Civin, C, Strauss, L.C, Brovall, C, Fackler, M.J., Schwartz, J.F. & Shaper, J.H. (1984): Antigenic analysis of III. A hematopoietic KGla cells. J. Immunol. 133: 157.

hematopoiesis

progenitor cell surface antigen defined by

a

monoclonal

antibody

raised

against

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a

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