Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer.

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vß gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK.

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

DNA microarrays for comparison of gene expression profiles between diagnosis and relapse in precursor-B acute lymphoblastic leukemia: choice of technique and purification influence the identification of potential diagnostic markers.

Microarrays for gene expression profiling are rapidly becoming important research tools for the identification of novel markers, for example, for novel classification of leukemias and lymphomas. Here, we review the considerations and infrastructure for microarray experiments. These considerations are illustrated via a microarray-based comparison of gene expression profiles of paired diagnosis-relapse samples from patients with precursor-B acute lymphoblastic leukemia (ALL), who relapsed during therapy or after completion of treatment. Initial experiments showed that several seemingly differentially expressed genes were actually derived from contaminating non-leukemic cells, particularly myeloid cells and T-lymphocytes. Therefore, we purified the ALL cells of the diagnosis and relapse samples if their frequency was lower than 95%. Furthermore, we observed in earlier studies that extra RNA amplification leads to skewing of particular gene transcripts. Sufficient (non-amplified) RNA of purified and paired diagnosis-relapse samples was obtained from only seven cases. The gene expression profiles were evaluated with Affymetrix U95A chips containing 12 600 human genes. These diagnosis-relapse comparisons revealed only a small number of genes (n=6) that differed significantly in expression: mostly signaling molecules and transcription factors involved in cell proliferation and cell survival were highly upregulated at relapse, but we did not observe any increase in drug-resistance markers. This finding fits with the observation that tumors with a high proliferation index have a poor prognosis. The genes that changed between diagnosis and relapse are currently not in use as diagnostic or disease progression markers, but represent potential new markers for such applications. Leukemia (2003) 17, 1324-1332. doi:10.1038/sj.leu.2402974

A novel germline mutation of PTEN associated with brain tumours of multiple lineages.

We have identified a novel germline mutation in the PTEN tumour suppressor gene. The mutation was identified in a patient with a glioma, and turned out to be a heterozygous germline mutation of PTEN (Arg234Gln), without loss of heterozygosity in tumour DNA. The biological consequences of this germline mutation were investigated by means of transfection studies of the mutant PTEN molecule compared to wild-type PTEN. In contrast to the wild-type molecule, the mutant PTEN protein is not capable of inducing apoptosis, induces increased cell proliferation and leads to high constitutive PKB/Akt activation, which cannot be increased anymore by stimulation with insulin. The reported patient, in addition to glioma, had suffered from benign meningioma in the past but did not show any clinical signs of Cowden disease or other hereditary diseases typically associated with PTEN germline mutations. The functional consequences of the mutation in transfection studies are consistent with high proliferative activity. Together, these findings suggest that the Arg234Gln missense mutation in PTEN has oncogenic properties and predisposes to brain tumours of multiple lineages.

Selective development of a strong Th2 cytokine profile in high-risk children who develop atopy: risk factors and regulatory role of IFN-gamma, IL-4 and IL-10.

BACKGROUND: The immunological processes in early life and their relation to allergic sensitization leading to a Th2 cytokine profile are still not well understood. OBJECTIVE: To analyse the environmental and genetic risk factors and immunological responses at birth in relation to the development of atopic disease at 12 months of age in a longitudinal study of high-risk children. METHODS: High-risk children were followed from birth till 12 months of age. Mononuclear cells obtained at birth and 6 and 12 months thereafter were analysed for their proliferative and cytokine responses after polyclonal and allergen-specific stimulation. RESULTS: At 12 months of age 25% children had developed an atopic disease. Two atopic parents, parental smoking and atopic dermatitis of at least one of the parents were significant risk factors. In cord blood of newborns who developed atopy, an increased percentage of CD4+CD45RO+ cells and an increased polyclonal-stimulated proliferation were observed. Furthermore, an impaired allergen-induced, but not polyclonal-stimulated IFN-gamma production was found, suggesting a regulatory defect. At 6 and 12 months of age, a strong Th2 profile (characterized by increased levels of IL-4, IL-5, and IL-13) after both polyclonal and, to a lesser extent, allergen-specific stimulation was found in the children developing atopy. Allergen-induced IL-10 production at 12 months of age was only observed in the non-atopic children. CONCLUSION: Our data indicate that the first 6 months of life represent a critical time window for the initiation of immunological changes resulting in the development of atopy. The selective development of a Th2 cytokine profile in high-risk children who develop atopy is due to increased production of Th2 cytokines, possibly caused by impaired allergen-induced IFN-gamma production in the neonatal period. Furthermore, the decreased allergen-induced IL-10 levels observed in the atopic children at 12 months of age may result in a lack of down-regulation of the inflammatory process.

Clinico-immunological heterogeneity in Comèl-Netherton syndrome.

BACKGROUND: Comèl-Netherton syndrome (CN) is characterized by atopic-eczema-like skin abnormalities combined with linear ichthyotic lesions, hair shaft abnormalities and atopy with high IgE levels. OBJECTIVE: Five children with CN are described. In 2 of the 3 CN patients still alive, analysis of cytokines regulating IgE synthesis was performed. METHODS: In peripheral blood mononuclear cells and cultures of purified T cells, mRNA expression and protein production of interleukin 4 (IL-4), IL-13, IL-5 and interferon gamma were analysed. The results were compared with the values in age-matched atopic dermatitis patients and healthy children. RESULTS: The 5 CN patients showed striking differences in disease severity and evolution. Marked differences were found in several cytokines in the 2 analysed CN patients. Low percentages of natural killer cells were observed in both CN patients. CONCLUSION: The regulation of IgE production in patients with CN is varied and complex. The CN patients were heterogeneous in terms of Th2 skewing.

Markers for early sensitization and inflammation in relation to clinical manifestations of atopic disease up to 2 years of age in 133 high-risk children.

BACKGROUND: The combination of genetic susceptibility and environmental factors induce allergic sensitization and subsequently local inflammation, resulting in atopic manifestations. OBJECTIVE: To examine whether immunological features reflecting sensitization (total and specific IgE levels, allergen-induced proliferative responses and skin tests) and markers of inflammation (plasma sE-selectin and blood eosinophils) are related to the clinical expression of atopy and whether they precede atopic disease in children up to 2 years of age. METHODS: The development of these markers during the first 2 years of life was studied prospectively in 133 newborns at high risk to develop atopic disease. RESULTS: The prevalence of atopic disease increased from 25% at 12 months to 32% at 24 months of age. The children with food allergy at 12 months, who all had atopic dermatitis (AD), turned out to have asthma-like disease in 40% and AD in 100% at the age of 24 months. Total IgE levels increased with time and from 12 months onward levels started to differ markedly between atopics and nonatopics. Food-specific IgE antibodies were significantly associated with AD (relative risk [RR] = 2.39), food (RR = 1.32) and upper-airway allergy (RR = 1.20), and house dust mite-specific IgE antibodies with upper-airway allergy (RR = 5.00). A positive skin test was significantly associated with AD (RR = 2.90) and food allergy (RR = 1.36). The inflammation markers investigated, were not related to the clinical expression or preceded atopic disease at 2 years of age in high-risk children. CONCLUSION: Positive skin tests and specific IgE to food or inhalant allergens were related to the clinical expression of different atopic diseases. The combination of AD and food allergy at 12 months reflected the strongest risk factor in this high risk cohort for the development of asthma-like disease at 24 months of age.

Differential mRNA expression and production of interleukin-4 and interferon-gamma in stimulated peripheral blood mononuclear cells of house-dust mite-allergic patients.

Optimal culture conditions were established for the analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression and protein production, as well as proliferative capacity of peripheral blood mononuclear cells (PBMC). These culture conditions permitted the analysis of differences in the responses of house-dust mite (HDM) allergic patients and healthy controls after polyclonal and allergen-specific stimulation. Proliferative responses were optimal when PBMC were cultured in RPMI, whereas for studying mRNA expression by RT-PCR and protein production by ELISA, PBMC should be stimulated in Yssels's medium. Blood holding period influenced the cytokine mRNA expression and proliferative capacity of primarily the unstimulated cells. It is thus crucial to isolate PBMC as soon as possible, and in any event no later than 7 hours after blood collection. Proliferative responses to Dermatophagoides pteronyssinus-extract were observed in HDM allergic patients (mean stimulation index (SI) = 5.3+/-0.75), but not in non-allergic subjects (mean SI = 2.3+/-0.21). After D. pteronyssinus-specific stimulation, IL-4 mRNA expression was significantly (p = 0.03) increased in HDM-allergic subjects compared to non-allergic subjects. No significant differences were found in IFN-gamma mRNA expression between HDM-allergic and non-allergic subjects. Both IFN-gamma (p = 0.04) and IL-4 (p = 0.06) protein production were increased after D. pteronyssinus-specific stimulation in HDM-allergic subjects compared to non-allergic subjects. Our data suggest activation of both Th1 and Th2-like cells, as well as CD8+ T cells in allergic patients. Furthermore, analysis of possible functional differences in PBMC between allergic and non-allergic patients, necessitates polyclonal and allergen-specific stimulation of PBMC. Moreover, proliferative responses as well as cytokine mRNA expression and protein production should be studied under optimal culture conditions to highlight the often subtle differences.

Levels of soluble intercellular adhesion molecule-1, soluble E-selectin, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptor p55 and p75 in atopic children.

During inflammation, membrane expression of adhesion molecules and tumor necrosis factor (TNF)-receptors (TNF-R) are increased, and soluble forms of these molecules are released. This study analyzed plasma levels of sICAM-1 and sE-selectin as well as TNF-alpha, sTNF-R55, and sTNF-R75 in nonallergic (NAA) and allergic asthma patients (AA), atopic dermatitis patients (AD), and healthy children (HC) by ELISA. Plasma levels of sICAM-1, sE-selectin, and sTNF-R, but not TNF-alpha, were detectable, but were not significantly different between the patient groups and healthy children. In the AA group, a significant correlation (rs = 0.78, P = 0.008) was found between sICAM-1 and sE-selectin levels. Furthermore, a significant correlation was found between sTNF-R55 and sTNF-R75 levels in the AA group (rs = 0.70, P = 0.025) and in the AD group (rs = 0.69, P = 0.027). In AD patients, a significant correlation was observed between sE-selectin and the disease severity, as measured by the SCORAD index (rs = 0.73, P = 0.038). Our data demonstrate that plasma levels of sICAM-1, sE-selectin, TNF-alpha, sTNF-R55, and sTNF-R75 were not different between atopic and nonatopic children during a stable phase of the disease. In AD patients, levels of sE-selectin seemed to be related to clinical severity of the disease.

T cell subsets and cytokines in allergic and non-allergic children. I. Analysis of IL-4, IFN-gamma and IL-13 mRNA expression and protein production.

Interleukin 4 (IL-4) and IL-13 are key cytokines inducing switching to immunoglobulin E (IgE), whereas interferon gamma (IFN-gamma) acts inhibitory on this process. We analysed whether differences existed in IL-4, IFN-gamma and IL-13 mRNA expression and protein production between T cells of children with allergic and non-allergic asthma, atopic dermatitis and health control children. IL-4 mRNA expression was increased in stimulated T cells of children with allergic asthma and atopic dermatitis, but not in those with non-allergic asthma as compared with healthy controls. Thus the increase in IL-4 expression can be considered as an underlying mechanism of the allergic disease process and not so much of the asthmatic state of the children. In unstimulated T cells of children with atopic dermatitis increased IFN-gamma mRNA expression with a reduced IFN-gamma protein production was found, indicating a post-translational defect in IFN-gamma. Differences in IL-13 expression between the groups were not significant, but IL-13 was significantly correlated with the height of the radio-allergo-sorbent test (RAST) class and with the severity scoring of atopic dermatitis (SCORAD) index. This indicates the clinical relevance of IL-13 for the degree of allergen-specific sensitization and severity of atopic dermatitis. In conclusion, the imbalance in IL-4 and IFN-gamma secretion in patients with atopic dermatitis may reflect general T cell activation in the presence of an intrinsic defect of IFN-gamma secretion.

T cells subsets and cytokines in allergic and non-allergic children. II. Analysis and IL-5 and IL-10 mRNA expression and protein production.

Interleukin 5 (IL-5) has an enhancing effect on IL-4 induced immunoglobulin E (IgE) synthesis. Furthermore, IL-5 plays an important role in the differentiation, recruitment, activation and survival of eosinophils. IL-10 has a downmodulating effect on interferon gamma (IFN-gamma) production and can exert strong anti-inflammatory activities. Therefore, we analysed whether differences were present in IL-5 and IL-10 mRNA expression and protein production between T cells of children with allergic and non-allergic asthma, atopic dermatitis and healthy control children. We demonstrated significant increases in IL-5 mRNA expression and protein production in different T cell fractions of children with allergic and non-allergic asthma and children with atopic dermatitis as compared to healthy controls. This indicates that IL-5 is not only involved in allergy, but also plays a role in the inflammatory process of non-allergic asthma. Interestingly, IL-10 mRNA expression by purified T cells of children with allergic and non-allergic asthma and children with atopic dermatitis was strongly decreased as compared with that of healthy controls. In the peripheral blood mononuclear cell (PBMC) fraction, IL-10 mRNA expression was comparable between the four groups. We hypothesize that this decreased T cell derived IL-10 expression results in a lack of immunosuppression of the inflammatory process in these diseases. However, a role of monocyte derived IL-10 cannot be ruled out.

Development of immune functions related to allergic mechanisms in young children.

The newborn immune system differs quantitatively and functionally from that of adults. Development of the immune system has important implications for childhood diseases. The immaturity of the immune system in the first years of life may contribute to failure of tolerance induction and in the development of allergic disease. T cell function is diminished, especially the capacity to produce cytokines; production of interferon (IFN)-gamma, and IL-4 is strongly reduced. IFN-gamma has been found to be even lower in cord blood of newborns with a family history of atopy. Differences in other cell types (natural killer cells, antigen-presenting cells, and B cells) could also play a role in the development of allergic disease. Current data suggest that irregularities in IgE synthesis, helper T cell subsets (Th1, Th2, CD45RA, and CD45RO), cytokines (IL-4, IFN-gamma), and possibly other cell types may play a role in the development of allergy in childhood. Moreover, the role of cell surface molecules, like co-stimulatory molecules (CD28, CD40L), activation markers (CD25), and adhesion molecules (LFA-1/ICAM-1, VLA-4/ VCAM-1) is also discussed. These variables are modulated by genetic (relevant loci are identified on chromosome 5q, 11q, and 14) and environmental forces (allergen exposure, viral infections, and smoke). The low sensitivity of current predictive factors for the development of allergic diseases, such as cord blood IgE levels, improves in combination with family history and by measurement of in vitro responses of lymphocytes and skin reactivity to allergens. New therapeutic approaches are being considered on the basis of our current understanding of the immunopathology of allergic disease, for instance cytokine therapy and vaccination with tolerizing doses of allergen or peptides.

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi-quantitative PCR.

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-gamma. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0-4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-gamma; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.

Differential adherence of murine hematopoietic stem cell subsets to fibronectin.

Growth and differentiation of hematopoietic stem cells occur in close contact with the cells and extracellular-matrix (ECM) proteins of the hematopoietic microenvironment. We observed the role of fibronectin, a matrix glycoprotein proposed to be involved in the attachment of hematopoietic cells and in the binding of stem cell subsets to bone marrow stroma, for this study. Murine bone marrow cells (BMC) were allowed to adhere to surfaces coated with human plasma fibronectin. Using the cobblestone-area-forming cells (CAFC) assay, adherent and nonadherent cell fractions were tested for their quantity of primitive and less primitive stem cell subsets. The CAFC assay is based on a time-dependent clone formation in pre-established, bone marrow-derived, irradiated stromal layers under limiting dilution conditions, and it allows in vitro enumeration of day-12 colony-forming unit-spleen (CFU-S12) (CAFC-10) and more primitive cells with long-term repopulating abilities (LTRA) (CAFC-28/35). We observed that the majority of primitive CAFC-28/35 adhered to fibronectin, while only a minority of CFU-S-like CAFC-10 did. The adherence of primitive stem cells to fibronectin could partially be blocked by high molar concentrations of oligopeptides containing the essential amino acid sequence of the central cell-binding domain of fibronectin, RGD. Adherence of the small subpopulation of CAFC-10 to fibronectin could almost entirely be prevented by oligopeptides organized in a specific fashion. These data suggest a role for RGD-binding integrins in the adherence of hematopoietic stem cells.

Selective advantage of normal erythrocyte production after bone marrow transplantation of alpha-thalassemic mice.

Anemia resulting from alpha-thalassemia in mice was corrected by transplantation of normal bone marrow cells following sublethal total body irradiation, resulting in partial hematopoietic chimerism with a preponderance of normal peripheral blood red cells. Peripheral blood red cell chimerism in recipients of graded numbers of bone marrow cells from sex-mismatched donors, determined by cytometric analysis, was directly compared with immature hematopoietic cell (CFU-S) chimerism and peripheral blood white cell chimerism. The latter two were assessed by fluorescent in situ hybridization with a murine Y-chromosome-specific probe. Peripheral blood white cell chimerism consistently corresponded with immature hematopoietic cell chimerism, emphasizing the selective advantage of normal red cell production in partially chimeric alpha-thalassemic mice.

Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units.

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light-density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild-type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240-fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.

Loss of long-term repopulating ability in long-term bone marrow culture.

We have studied the maintenance of stem cells with long-term multilineage repopulating ability from murine bone marrow, cultured on a pre-established bone marrow-derived stromal cell layer, both in a qualitative and quantitative way. Female bone marrow cells were cultured for a period of 1-4 weeks and compared with uncultured cells for their ability to establish and maintain a level of 50% chimerism in a sex-mismatched bone marrow transplantation model. Chimerism was determined in nucleated cells using fluorescence in situ hybridization with a murine Y-chromosome-specific probe. We observed a rapid decline in the ability of cultured marrow cells to repopulate the blood, bone marrow, spleen, and thymus of sublethally irradiated male recipients. After 4 weeks of culture only 5% of the long-term repopulating ability of the inoculated bone marrow cells remained. The remaining long-term repopulating cells, however, had similar qualities to establish and maintain long-term engraftment compared to fresh bone marrow, as judged from their ability to give stable chimerism over a period of 6 months. These observations are relevant for the therapeutic applications of long-term bone marrow cultures in purging protocols prior to autologous bone marrow transplantation of acute and chronic myeloid leukemic patients, and for the use of long-term marrow cultures when introducing foreign genetic material in hematopoietic stem cells.