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Genes of the major histocompatibility complex
Cell, Vol. 28, 685-687,
April 1982,
Copyright
0 1982
by MIT
Genes of the Major Histocompatibility
Leroy Hood, Michael Steinmetz Robert Goodenow Division of Biology California Institute of Technology Pasadena, California 91 125
and
Metazoans display ceil-surface recognition systems that exhibit the ability to distinguish between self and nonself. One self-nonself recognition system, the major histocompatibility complex of vertebrates, was initially defined by the rejection of foreign skin grafts in mice. This system is now being analyzed at the molecular level. In mice, individual gene products of the major histocompatibility or H-2 complex can be detected by antisera raised in appropriate inbred congenie or recombinant strains. Genetic studies with these alloantisera, as well as with appropriate monoclonal antibodies, have demonstrated that the major histocompatibility complex of the mouse maps to chromosome 17 and encodes at least three classes of molecules (figure, A). The class I genes encode polymorphic cell-surface molecules that fall into two distinct categories. First, the classical transplantation antigens are present on all of the cells of the organism and play an important role in mediating T-cell immunosurveillance of virally infected or neoplastically transformed cells. For example, the BALB/c mouse has four serologically defined transplantation antigens-K, D, L and R. A second category includes the hematopoietic differentiation antigens, such as Qa and TL, which are closely related structurally to the transplantation antigens, but are expressed only on a subset of bone-marrow-derived cells. The class II genes encode a set of cell-surface molecules, denoted the la antigens, that are involved in interactions between macrophages and T and 6 cells and that regulate the immune response to many different antigens. The class III genes encode several components of the complement system. Structure of Class I Genes The transplantation antigens appear to represent one of the most polymorphic systems studied in eucaryotes. Indeed, more than 50 different alleles have been defined for inbred and wild mice at both the K and D loci. The constellation of H-2 alleles present in a given inbred mouse is denoted its haplotype. The BALB/c mouse has an H-2d haplotype, with the haplotype of individual gene products being designated by a superscript, for example, the Ld antigen. Transplantation antigens are transmembrane proteins composed of two chains (figure, D). A 45,000 dalton transmembrane polypeptide is encoded by the class I genes of the major histocompatibility complex on chromosome 17 in the mouse, and is noncovalently associated with a 12,000 dalton polypeptide, p2microglobulin, encoded on chromosome 2. The class I molecule is divided into three external domains (each
Complex
-90 residues); a transmembrane region (-40 residues); and a short cytoplasmic domain (-30 residues). Recently, cDNA clones encoding products of the major histocompatibility complex have been isolated, characterized and used to obtain genomic clones containing class I genes. Two mouse class I genes (clones 27.1 and 27.5) have been isolated from a hBALB/c sperm DNA library with cDNA clones as hybridization probes (Steinmetz et al., Cell 25, 683692, 1981; Moore et al., Science 215, 679-682, 1982). Analysis of their DNA sequences shows that the domain organization of the class I molecule is reflected precisely by the exon-intron structure of the gene: separate exons encode the signal peptide, each of the three external domains and the transmembrane region, and three exons encode the small cytoplasmic domain (figure, C). A human class I gene has been sequenced, and it appears to have a similar structure (Malissen et al., PNAS 79, 893-897, 1982). A comparison of the two mouse class I genes reveals that they are highly conserved; the exons are 87% homologous, and the introns are 80% homologous, except for a 1000 nucleotide insertion in the third intron of gene 27.1 (Moore et al., op. cit.). This insert is flanked by sequences that are highly repetitive in the mouse genome and belong in the so-called Alu repeat family. These elements have transposon-like structures and may therefore be responsible for the insertion or deletion of the nonhomologous sequence. Organization of Class I Genes The linkage arrangement and complexity of class I genes in the mouse have been studied by cloning 40 kb fragments of eucaryotic DNA into cosmid vectors (Steinmetz et al., Cell 28, 489-498, !982). Overlapping cosmid clones isolated from a BALB/c sperm DNA library define 13 distinct gene clusters with a total of 36 class I genes encompassing 837 kb of DNA (figure, B). One cluster contains seven class I genes spread over 191 kb of DNA. Gene 27.1 is present in this cluster. All seven genes are oriented in the same 5’ to 3’ direction. Three linked class I genes at the 3’ end of this cluster are more closely related to one another than to any of the other class I genes, and therefore constitute a closely related set or subgroup of class I genes. Southern blot analyses of congenic and recombinant congenic mouse strains have been used to localize genes or gene clusters within the major histocompatibility complex. This is feasible because individual class I genes exhibit restriction enzyme polymorphisms in different inbred strains of mice (Cami et al., Nature 297, 673-675, 1981). This approach has been used to map the gene cluster of seven genes to the Qa-2,3 region (Steinmetz et al., Cell 28, 489-498, 1982) and additional class I genes to the T/a region (Margulies et al., Nature 295, 168-l 70, 1982). Indeed, DNA fragments isolated from the flanking re-
Cdl 686
gions of class I genes in the 13 gene clusters will allow us to map the clusters and to determine whether all the class I genes map to the H-2 complex, or alternatively whether some map to other chromosomal regions. (A second approach to the mapping of class I genes with serologically identified gene products is that of gene transfer; see below.) Flanking-sequence probes from two clusters have been used to analyze the organization of these clusters in the DNAs of various inbred strains of mice. Again, subgroups of closely related class I genes were identified showing duplications and deletions in different mouse strains. Thus gene expansion and contraction, presumably by homologous but unequal crossing-over, may be an important mechanism for the generation of polymorphism in this multigene system. When probes specific for the 5’ and the 3’ halves of class I genes are used to analyze the 36 genes, it appears that the intensity of hybridization for the first two external domains (exons 2 and 3) varies 1 OO-fold, whereas the intensity of hybridization for the third external domain (exon 4) appears to be relatively constant for all class I genes. The latter observation indicates that the third external domain is highly conserved, and is consistent with the finding that there is significant homology between the third external domain and the constant-region domains of immunoglobulins (Steinmetz et al., Cell 24, 125-l 34, 1981). This homology suggests that /32-microglobulin, also conserved and homologous to immunoglobulins, interacts with the third external domain of class I molecules. Presumably, the antibody-like folds permit these domains to interact with one another much as the light- and heavy-chain domains interact in immunoglobulins. The DNA sequence homology observed in the genes encoding immunoglobulins and class I molecules, together with the similarities in exon-intron organization, suggests a common evolutionary origin for these two gene families. Reconstruction experiments, in which Southern blots of genomic DNA and pooled cosmid DNAs containing all 36 cloned class I genes are compared, indicate that the majority of the class I genes have been isolated. If most of these genes map to the major histocompatibility complex, a major fraction of the complex will have been cloned. Isolation of cosmid
clusters containing class II and class Ill genes, and chromosomal-walking experiments, should yield a complete molecular map of the major histocompatibility complex. Identification and Expression of Class I Genes Gene-transfer experiments have identified gene 27.5 as the Ld gene of the BALB/c mouse (Goodenow et al., Science 2 15, 677-679, 1982). Cotransformation of mouse L cells (H-2k haplotype) lacking the enzyme thymidine kinase with the herpes viral thymidine kinase gene and gene 27.5 results in the expression of cell-surface class I molecules that react with monoclonal antibodies to the Ld polypeptide. The Ld antigen can readily be distinguished serologically from all transplantation antigens of the H-2” haplotype. The Ld molecule isolated from transformed mouse L cells and analyzed by two-dimensional gel electrophoresis has a similar molecular weight and charge properties to normal Ld molecules isolated from spleen cells of the BALB/c mouse. Indeed, the complete sequence of gene 27.5 shows that it is identical to the available Ld protein sequence at 77 of 77 positions compared (Moore et al., op. cit.). Moreover, the Ld antigen appears to be oriented correctly in the mouse L-cell membrane, since it serves as an allogeneic target for cytotoxic T cells of C3H mice (H-2” haplotype) directed against H-2d haplotype class I antigens (Woodward et al., PNAS, in press). Preliminary experiments also suggest that the Ld molecule is recognized by the T-cell receptor as a restricting element in T-cell immunosurveillance. Mouse L cells have been transfected with most of the remaining class I genes. This approach has led to the identification of most of the serologically defined class I gene products of the major histocompatibility complex. Thus this technique has been a very powerful approach to the mapping of class I genes with serologically detectable gene products. It is not clear how many of the 36 class I genes are functional. Whereas gene 27.5 has been shown to encode an Ld antigen, it appears that gene 27.1, which maps to the Qa-2,3 region, is a pseudogene. A premature stop codon has been found for this gene at the end of the exon encoding the transmembrane domain. However, gene 27.1 might still encode a truncated class I molecule lacking a cytoplasmic domain. Indeed, Qa-2,3 antigens have been shown to be 40,000 rather than 45,000 daltons in molecular weight (Michaelson, Immunogenetics 73, 167-l 71, 19611, and class I polypeptides of 39,000 daltons also have been found in certain mutant mice (Wilson et al., Immunogenetics, in press). Thus gene 27.1 may actually encode such a shortened class I molecule. In addition, RNA transcripts from class I genes containing premature termination codons and aberrant transmembrane sequences have been observed (Reyes et al., Immunogenetics 74, 383-392, 1981; Cosman et al., Nature 295, 73-76, 1982). These class I genes
Cell 687
might encode secreted forms of the class I molecule. Transfection experiments should help to clarify these questions and to determine what fraction of the class I genes are pseudogenes. Obviously some of the class I genes may encode differentiation antigens in addition to the TL and Qa antigens that, accordingly, may have been difficult to detect by conventional serological approaches. There is a possibility that the genes for class I molecules may employ alternative RNA-splicing patterns to generate two or more polypeptides from the
Cell, Vol. 28, 687-688,
April 1982,
Copyright
0 1982
In Vitro Approaches
Edward S. Golub Department of Biological Purdue University West Lafayette, Indiana
same gene, as is seen with the heavy-chain genes for immunoglobulins, which generate the membrane and secreted molecules by a similar mechanism. Several indirect lines of evidence suggest that the multiple exons and introns encoding the cytoplasmic domain may be involved in alternative RNA-splicing patterns that would result in the synthesis of distinct transplantation antigens with the same external domains and different cytoplasmic domains (Steinmetz et al., Cell 25, 683-692, 1981).
by MIT
to Hemopoiesis
Sciences 47906
Differentiation is really a problem of regulated change; that is, the form or function of a cell or cell lineage undergoes progressive and predictable change in response to signals from the environment. With the great advances in the study of surface receptors and cell interactions, as well as gene organization and expression, questions about the mechanism of differentiation can now be approached, provided that the proper systems are available. One system that should receive a great deal of attention because of its inherent interest and its medical importance is hemopoiesis, the differentiation of the cells of the blood. This is an interesting system because animals are constantly renewing their blood cells (that is, differentiation is occurring), and the processes are controlled by some form of complex feedback mechanism. In vitro systems are now at hand so that this system, which up to now has lent itself almost exclusively to descriptive studies, may become one of the more attractive differentiating systems in which to study mechanisms. It has been known for a very long time that the bone marrow of mammals is the source of a cell or cells that could reconstitute the entire hemopoietic system of a lethally irradiated animal. The development of an in vivo assay by Till and McCulloch (Radiat. Res. 74, 213-222,1961) paved the way for the most important conceptual advance in the area of blood-cell formation. With an assay available it was possible to determine that all of the various kinds of blood cells were derived from a common cell, the hemopoietic stem
cell. This nonproliferating, multipotent and uncommitted cell gives rise to actively proliferating progenitor cells that are committed to paths of differentiation. The figure shows the flow of differentiation of blood cells. The microenvironment represents the sites in which induction occurs. Erythrocytes, granulocytes/ monocytes, platelets and lymphocytes are the final differentiated forms. Three conditions are required for an approach to the problem: first, the establishment in vitro of the environment that allows both self-renewal of the uncommitted stem cell and the process of committment to occur; second, the establishment of the in vitro microenvironment in which induction of the committed cells occurs; and third, the availability of reliable assays for the committed cells. All of these conditions either have been met or are nearly at hand. It has been proposed that hemopoietic-cell commitment is a stochastic process. Given the fact that the stem cells are not a synchronous population, the transition between the uncommitted and committed state might be governed by a single rate-limiting process. What that process or event is remains a mystery, but an in vitro system in which this can be approached has been developed that offers great promise. The Dexter bone marrow culture system allows the longterm maintenance of both stem cells and progenitor cells (Dexter and Lajtha, Br. J. Haematol. 28, 525 530, 1974). It thus fulfills the first part of the problem, providing the in vitro environment for self-renewal and commitment. The bone marrow stromal cells are maintained as an adherent monolayer, and support both uncommitted and committed cells. The monolayer consists of several cell types, and cell interactions in the cultures are probably important, although little is known about this. It is clear that these cultures in
April 1982,
Copyright
0 1982
by MIT
Genes of the Major Histocompatibility
Leroy Hood, Michael Steinmetz Robert Goodenow Division of Biology California Institute of Technology Pasadena, California 91 125
and
Metazoans display ceil-surface recognition systems that exhibit the ability to distinguish between self and nonself. One self-nonself recognition system, the major histocompatibility complex of vertebrates, was initially defined by the rejection of foreign skin grafts in mice. This system is now being analyzed at the molecular level. In mice, individual gene products of the major histocompatibility or H-2 complex can be detected by antisera raised in appropriate inbred congenie or recombinant strains. Genetic studies with these alloantisera, as well as with appropriate monoclonal antibodies, have demonstrated that the major histocompatibility complex of the mouse maps to chromosome 17 and encodes at least three classes of molecules (figure, A). The class I genes encode polymorphic cell-surface molecules that fall into two distinct categories. First, the classical transplantation antigens are present on all of the cells of the organism and play an important role in mediating T-cell immunosurveillance of virally infected or neoplastically transformed cells. For example, the BALB/c mouse has four serologically defined transplantation antigens-K, D, L and R. A second category includes the hematopoietic differentiation antigens, such as Qa and TL, which are closely related structurally to the transplantation antigens, but are expressed only on a subset of bone-marrow-derived cells. The class II genes encode a set of cell-surface molecules, denoted the la antigens, that are involved in interactions between macrophages and T and 6 cells and that regulate the immune response to many different antigens. The class III genes encode several components of the complement system. Structure of Class I Genes The transplantation antigens appear to represent one of the most polymorphic systems studied in eucaryotes. Indeed, more than 50 different alleles have been defined for inbred and wild mice at both the K and D loci. The constellation of H-2 alleles present in a given inbred mouse is denoted its haplotype. The BALB/c mouse has an H-2d haplotype, with the haplotype of individual gene products being designated by a superscript, for example, the Ld antigen. Transplantation antigens are transmembrane proteins composed of two chains (figure, D). A 45,000 dalton transmembrane polypeptide is encoded by the class I genes of the major histocompatibility complex on chromosome 17 in the mouse, and is noncovalently associated with a 12,000 dalton polypeptide, p2microglobulin, encoded on chromosome 2. The class I molecule is divided into three external domains (each
Complex
-90 residues); a transmembrane region (-40 residues); and a short cytoplasmic domain (-30 residues). Recently, cDNA clones encoding products of the major histocompatibility complex have been isolated, characterized and used to obtain genomic clones containing class I genes. Two mouse class I genes (clones 27.1 and 27.5) have been isolated from a hBALB/c sperm DNA library with cDNA clones as hybridization probes (Steinmetz et al., Cell 25, 683692, 1981; Moore et al., Science 215, 679-682, 1982). Analysis of their DNA sequences shows that the domain organization of the class I molecule is reflected precisely by the exon-intron structure of the gene: separate exons encode the signal peptide, each of the three external domains and the transmembrane region, and three exons encode the small cytoplasmic domain (figure, C). A human class I gene has been sequenced, and it appears to have a similar structure (Malissen et al., PNAS 79, 893-897, 1982). A comparison of the two mouse class I genes reveals that they are highly conserved; the exons are 87% homologous, and the introns are 80% homologous, except for a 1000 nucleotide insertion in the third intron of gene 27.1 (Moore et al., op. cit.). This insert is flanked by sequences that are highly repetitive in the mouse genome and belong in the so-called Alu repeat family. These elements have transposon-like structures and may therefore be responsible for the insertion or deletion of the nonhomologous sequence. Organization of Class I Genes The linkage arrangement and complexity of class I genes in the mouse have been studied by cloning 40 kb fragments of eucaryotic DNA into cosmid vectors (Steinmetz et al., Cell 28, 489-498, !982). Overlapping cosmid clones isolated from a BALB/c sperm DNA library define 13 distinct gene clusters with a total of 36 class I genes encompassing 837 kb of DNA (figure, B). One cluster contains seven class I genes spread over 191 kb of DNA. Gene 27.1 is present in this cluster. All seven genes are oriented in the same 5’ to 3’ direction. Three linked class I genes at the 3’ end of this cluster are more closely related to one another than to any of the other class I genes, and therefore constitute a closely related set or subgroup of class I genes. Southern blot analyses of congenic and recombinant congenic mouse strains have been used to localize genes or gene clusters within the major histocompatibility complex. This is feasible because individual class I genes exhibit restriction enzyme polymorphisms in different inbred strains of mice (Cami et al., Nature 297, 673-675, 1981). This approach has been used to map the gene cluster of seven genes to the Qa-2,3 region (Steinmetz et al., Cell 28, 489-498, 1982) and additional class I genes to the T/a region (Margulies et al., Nature 295, 168-l 70, 1982). Indeed, DNA fragments isolated from the flanking re-
Cdl 686
gions of class I genes in the 13 gene clusters will allow us to map the clusters and to determine whether all the class I genes map to the H-2 complex, or alternatively whether some map to other chromosomal regions. (A second approach to the mapping of class I genes with serologically identified gene products is that of gene transfer; see below.) Flanking-sequence probes from two clusters have been used to analyze the organization of these clusters in the DNAs of various inbred strains of mice. Again, subgroups of closely related class I genes were identified showing duplications and deletions in different mouse strains. Thus gene expansion and contraction, presumably by homologous but unequal crossing-over, may be an important mechanism for the generation of polymorphism in this multigene system. When probes specific for the 5’ and the 3’ halves of class I genes are used to analyze the 36 genes, it appears that the intensity of hybridization for the first two external domains (exons 2 and 3) varies 1 OO-fold, whereas the intensity of hybridization for the third external domain (exon 4) appears to be relatively constant for all class I genes. The latter observation indicates that the third external domain is highly conserved, and is consistent with the finding that there is significant homology between the third external domain and the constant-region domains of immunoglobulins (Steinmetz et al., Cell 24, 125-l 34, 1981). This homology suggests that /32-microglobulin, also conserved and homologous to immunoglobulins, interacts with the third external domain of class I molecules. Presumably, the antibody-like folds permit these domains to interact with one another much as the light- and heavy-chain domains interact in immunoglobulins. The DNA sequence homology observed in the genes encoding immunoglobulins and class I molecules, together with the similarities in exon-intron organization, suggests a common evolutionary origin for these two gene families. Reconstruction experiments, in which Southern blots of genomic DNA and pooled cosmid DNAs containing all 36 cloned class I genes are compared, indicate that the majority of the class I genes have been isolated. If most of these genes map to the major histocompatibility complex, a major fraction of the complex will have been cloned. Isolation of cosmid
clusters containing class II and class Ill genes, and chromosomal-walking experiments, should yield a complete molecular map of the major histocompatibility complex. Identification and Expression of Class I Genes Gene-transfer experiments have identified gene 27.5 as the Ld gene of the BALB/c mouse (Goodenow et al., Science 2 15, 677-679, 1982). Cotransformation of mouse L cells (H-2k haplotype) lacking the enzyme thymidine kinase with the herpes viral thymidine kinase gene and gene 27.5 results in the expression of cell-surface class I molecules that react with monoclonal antibodies to the Ld polypeptide. The Ld antigen can readily be distinguished serologically from all transplantation antigens of the H-2” haplotype. The Ld molecule isolated from transformed mouse L cells and analyzed by two-dimensional gel electrophoresis has a similar molecular weight and charge properties to normal Ld molecules isolated from spleen cells of the BALB/c mouse. Indeed, the complete sequence of gene 27.5 shows that it is identical to the available Ld protein sequence at 77 of 77 positions compared (Moore et al., op. cit.). Moreover, the Ld antigen appears to be oriented correctly in the mouse L-cell membrane, since it serves as an allogeneic target for cytotoxic T cells of C3H mice (H-2” haplotype) directed against H-2d haplotype class I antigens (Woodward et al., PNAS, in press). Preliminary experiments also suggest that the Ld molecule is recognized by the T-cell receptor as a restricting element in T-cell immunosurveillance. Mouse L cells have been transfected with most of the remaining class I genes. This approach has led to the identification of most of the serologically defined class I gene products of the major histocompatibility complex. Thus this technique has been a very powerful approach to the mapping of class I genes with serologically detectable gene products. It is not clear how many of the 36 class I genes are functional. Whereas gene 27.5 has been shown to encode an Ld antigen, it appears that gene 27.1, which maps to the Qa-2,3 region, is a pseudogene. A premature stop codon has been found for this gene at the end of the exon encoding the transmembrane domain. However, gene 27.1 might still encode a truncated class I molecule lacking a cytoplasmic domain. Indeed, Qa-2,3 antigens have been shown to be 40,000 rather than 45,000 daltons in molecular weight (Michaelson, Immunogenetics 73, 167-l 71, 19611, and class I polypeptides of 39,000 daltons also have been found in certain mutant mice (Wilson et al., Immunogenetics, in press). Thus gene 27.1 may actually encode such a shortened class I molecule. In addition, RNA transcripts from class I genes containing premature termination codons and aberrant transmembrane sequences have been observed (Reyes et al., Immunogenetics 74, 383-392, 1981; Cosman et al., Nature 295, 73-76, 1982). These class I genes
Cell 687
might encode secreted forms of the class I molecule. Transfection experiments should help to clarify these questions and to determine what fraction of the class I genes are pseudogenes. Obviously some of the class I genes may encode differentiation antigens in addition to the TL and Qa antigens that, accordingly, may have been difficult to detect by conventional serological approaches. There is a possibility that the genes for class I molecules may employ alternative RNA-splicing patterns to generate two or more polypeptides from the
Cell, Vol. 28, 687-688,
April 1982,
Copyright
0 1982
In Vitro Approaches
Edward S. Golub Department of Biological Purdue University West Lafayette, Indiana
same gene, as is seen with the heavy-chain genes for immunoglobulins, which generate the membrane and secreted molecules by a similar mechanism. Several indirect lines of evidence suggest that the multiple exons and introns encoding the cytoplasmic domain may be involved in alternative RNA-splicing patterns that would result in the synthesis of distinct transplantation antigens with the same external domains and different cytoplasmic domains (Steinmetz et al., Cell 25, 683-692, 1981).
by MIT
to Hemopoiesis
Sciences 47906
Differentiation is really a problem of regulated change; that is, the form or function of a cell or cell lineage undergoes progressive and predictable change in response to signals from the environment. With the great advances in the study of surface receptors and cell interactions, as well as gene organization and expression, questions about the mechanism of differentiation can now be approached, provided that the proper systems are available. One system that should receive a great deal of attention because of its inherent interest and its medical importance is hemopoiesis, the differentiation of the cells of the blood. This is an interesting system because animals are constantly renewing their blood cells (that is, differentiation is occurring), and the processes are controlled by some form of complex feedback mechanism. In vitro systems are now at hand so that this system, which up to now has lent itself almost exclusively to descriptive studies, may become one of the more attractive differentiating systems in which to study mechanisms. It has been known for a very long time that the bone marrow of mammals is the source of a cell or cells that could reconstitute the entire hemopoietic system of a lethally irradiated animal. The development of an in vivo assay by Till and McCulloch (Radiat. Res. 74, 213-222,1961) paved the way for the most important conceptual advance in the area of blood-cell formation. With an assay available it was possible to determine that all of the various kinds of blood cells were derived from a common cell, the hemopoietic stem
cell. This nonproliferating, multipotent and uncommitted cell gives rise to actively proliferating progenitor cells that are committed to paths of differentiation. The figure shows the flow of differentiation of blood cells. The microenvironment represents the sites in which induction occurs. Erythrocytes, granulocytes/ monocytes, platelets and lymphocytes are the final differentiated forms. Three conditions are required for an approach to the problem: first, the establishment in vitro of the environment that allows both self-renewal of the uncommitted stem cell and the process of committment to occur; second, the establishment of the in vitro microenvironment in which induction of the committed cells occurs; and third, the availability of reliable assays for the committed cells. All of these conditions either have been met or are nearly at hand. It has been proposed that hemopoietic-cell commitment is a stochastic process. Given the fact that the stem cells are not a synchronous population, the transition between the uncommitted and committed state might be governed by a single rate-limiting process. What that process or event is remains a mystery, but an in vitro system in which this can be approached has been developed that offers great promise. The Dexter bone marrow culture system allows the longterm maintenance of both stem cells and progenitor cells (Dexter and Lajtha, Br. J. Haematol. 28, 525 530, 1974). It thus fulfills the first part of the problem, providing the in vitro environment for self-renewal and commitment. The bone marrow stromal cells are maintained as an adherent monolayer, and support both uncommitted and committed cells. The monolayer consists of several cell types, and cell interactions in the cultures are probably important, although little is known about this. It is clear that these cultures in