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Transplantation of immunodeficient mice with human hematopoietic cells has greatly facilitated studies of the earliest stages of human hematopoiesis. These include demonstration of the ability of injected 'human-specific' hematopoietic growth factors to enhance the production of human cells at multiple levels of differentiation. In contrast, the effects of continuous exposure to such molecules have not been well investigated. Here, we show that nonobese diabetic severe combined immunodeficiency mice genetically engineered to produce ng/ml serum levels of human interleukin-3 (IL-3), granulocyte/macrophage-stimulating factor (GM-CSF) and Steel factor (SF) display a complex phenotype when transplanted with primitive human bone marrow (BM) or fetal liver cells. This phenotype is characterized by an enhancement of terminal human myelopoiesis and a matched suppression of terminal human erythropoiesis, with a slight reduction in human B-lymphopoiesis in the BM of the engrafted mice. Human clonogenic progenitors are more prevalent in the blood of the transplanted growth factor-producing mice and this is accompanied by a very marked reduction of more primitive human cells in the BM. Our findings suggest that long-term exposure of primitive human hematopoietic cells to elevated levels of human IL-3, GM-CSF and SF in vivo may deleteriously affect the stem cell compartment, while expanding terminal myelopoiesis.

Identification of the molecular mechanisms that can promote human hematopoietic stem cell amplification is a major goal in experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. One important class of early developmental genes determining hematopoietic development are homeobox transcription factors. Here, we report that retrovirally mediated expression of the homeobox gene HOXB4 rapidly triggers an increase in the number of human hematopoietic cord blood cells with stem cell and progenitor cell properties detected both by in vitro and in vivo assays. This growth enhancement extended across primitive myeloid-erythroid and B-lymphoid progenitors but did not lead to alterations in the balance of lymphomyeloid reconstitution in vivo, suggesting that HOXB4 does not affect control of end-cell output. These findings reveal HOXB4 as a novel, positive regulator of the primitive growth activity of human hematopoietic progenitor cells and underline the relevance of early developmental factors for stem cell fate decisions.

Objective: To study the clonality of cytogenetically normal progenitors detected in the peripheral blood (PB) of acute myeloid leukemia (AML) patients., Methods: Five female patients with cytogenetically abnormal, newly-diagnosed AML who were heterozygous for informative alleles of the androgen receptor (AR) gene were studied using the human androgen receptor allele (HUMARA) assay., Results: PB mononuclear cells and bone marrow (BM) fibroblasts from these patients were monoclonal and polyclonal, respectively. Both cytogenetically normal and abnormal colony-forming cells (CFC) were detected from 3 AML samples and the HUMARA assay determined that most of these CFC were part of the leukemic clone. The fourth sample generated colonies that were 100% normal by cytogenetics and polyclonal by HUMARA. In contrast, 5-week-old long-term culture (LTC)-derived colonies were 100% cytogenetically normal by FISH and polyclonal by HUMARA in 4 of the 5 samples. The fifth sample, which showed a small number of karyotypically abnormal LTC-derived colonies, nevertheless showed amplification of the "leukemia-associated" AR allele in 46/50 LTC-derived colonies as well as all 40 directly clonogenic cells tested., Conclusions: Thus in 4 of 5 AML samples tested, both cytogenetics and the HUMARA assay indicate that a substantial number of normal, polyclonal hematopoietic progenitors often persist in AML PB at diagnosis in spite of the predominance of malignant blasts and the severe cytopenias of normal mature blood cells that are typically seen clinically.

Efflux of Hoechst 33342 from normal hematopoietic cells identifies a "side population" (SP(+)) of negatively staining cells that, in the mouse, are largely CD34(-) and are enriched for primitive progenitors. To further characterize human SP(+) cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression. The mean percentages of SP(+) and MDR(+) cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values. The percentages of SP(+) cells that were CD34(+)CD38(-), CD34(+)CD38(+), or CD34(-) were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%). Cytogenetically abnormal cells were always detected in the SP(-)CD34(+)CD38(-) and SP(+)CD34(-) fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia-IC were detected in the former fraction. No progenitors were detected among SP(+)CD34(-) cells in any of these assays from 9 of 10 samples. In contrast, exclusively normal cells were detected in the SP(+)CD34(+)CD38(-) fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively. In contrast to murine studies, primitive progenitors are enriched among SP(+)CD34(+)CD38(-) cells from patients with AML. The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression. (Blood. 2001;97:3882-3889)

Several studies point to multiple members of the Hox transcription factor family as playing key roles in normal hematopoietic development, and they link the imbalanced expression of these transcription factors, in particular of the Abd-like A cluster HOX genes HOXA9 and HOXA10, to leukemogenesis. To test directly the hypothesis that HOXA10 is involved in human hematopoietic development, the gene was retrovirally overexpressed in human highly purified CD34(+)/GFP(+) hematopoietic progenitor cells derived from cord blood or fetal liver sources, and the impact of aberrant gene expression was analyzed on differentiation and proliferation in vitro and in vivo. HOXA10 misexpression profoundly impaired myeloid differentiation with a higher yield of blast cells in liquid culture and a greater than 100-fold increased generation of blast colonies after in vitro expansion or after replating of primary colonies first plated in methylcellulose directly after transduction (P < .01). Furthermore, aberrant HOXA10 expression almost completely blocked erythroid differentiation in methylcellulose (P < .02). HOXA10 deregulation also severely perturbed the differentiation of human progenitors in vivo, reducing B-cell development by 70% in repopulated NOD/SCID mice and enhancing myelopoiesis in the transduced compartment. The data provide evidence that the balanced expression of HOXA10 is pivotal for normal human hematopoietic development and that aberrant expression of the gene contributes to impaired differentiation and increased proliferation of human hematopoietic progenitor cells. These results also provide a framework to initiate more detailed analyses of HOX regulatory domains and HOX cofactors in the human system in vitro and in vivo.

One possible explanation for the competitive advantage that malignant cells in patients with acute myelogenous leukemia (AML) appear to have over normal hematopoietic elements is that leukemic progenitors proliferate more rapidly than their normal progenitor cell counterparts. To test this hypothesis, an overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in both colony-forming cell (CFC) and long-term culture initiating cell (LTC-IC) assays from the peripheral blood of nine patients with newly diagnosed AML. Culture of AML cells in serum-free medium with 100 ng/ml Steel factor (SF), 20 ng/ml interleukin 3 (IL-3) and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) for 16-24 h maintained the number of AML-CFC and LTC-IC at near input values (mean % input +/- s.d. for CFC and LTC-IC were 78 +/- 33 and 126 +/- 53, respectively). The addition of 20 muCi/ml high specific activity 3H-Tdr to these cultures reduced the numbers of both progenitor cell types from most of the patient samples substantially: mean % kill +/- s.d. for AML-CFC and LTC-IC were 64 +/- 27 and 82 +/- 16, respectively, indicating that a large proportion of both progenitor populations were actively cycling. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC were actively cycling (mean % kill +/- s.d.: 68 +/- 26 and 85 +/- 13, respectively). Interestingly, in six patient samples where a significant number of cytogenetically normal LTC-ICs were detected, the % kill of these cells (74 +/- 20) was similar to that of the abnormal progenitors. These data contrast with the predominantly quiescent cell cycle status of CFC and LTC-IC previously observed in steady-state peripheral blood from normal individuals but also provide evidence that a significant proportion of primitive malignant progenitors from AML patients are quiescent and therefore may be resistant to standard chemotherapeutic regimens.

Long-term cultures (LTC) and immunodeficient (NOD/SCID) mice have been used to quantitate and characterize primitive malignant progenitors from patients with acute myelogenous leukemia (AML). In 5-week-old LTC of cells from newly diagnosed patients with AML cytogenetically abnormal as well as normal progenitors could be easily detected and their numbers increased by cytokine supplements to the cultures. Sixty percent of AML samples will engraft in NOD/SCID mouse marrow. The frequency and level of engraftment of human cells detected appears to vary among the different subtypes of AML but is not generally affected by treatment of the mice with human cytokines. Both the LTC and NOD/SCID mouse assay show promise as tools to allow characterization of differences between leukemic stem cells which maintain malignant hematopoiesis in individual patients and, more importantly, between these cells and their normal stem cell counterparts.

Analysis of the mitogenic activity of interleukin-3 (IL-3), Steel factor (SF), and flt-3 ligand (FL) on acute myelogenous leukemia (AML) blasts using the short-term endpoints of proliferation in 3H-thymidine (3H-Tdr) incorporation assays or methylcellulose cultures (colony assays) showed that greater than 90% of samples contained cells that were responsive to one or more of these cytokines. With this information, culture conditions that were known to support normal long-term culture-initiating cells (LTC-IC) were tested, with or without supplements of one or more of these three growth factors, for their ability to support primitive progenitors from 10 cell samples from patients with AML. In all cases cytogenetically abnormal colony forming cells (CFC) were detected after 5 weeks when AML peripheral blood or marrow cells were cocultured on preestablished, normal human marrow feeders (HMF) and/or SI/SI mouse fibroblast feeders and the number of CFC detected in these 5-week-old LTC maintained a linear relationship to the number of input AML cells. Limiting dilution analysis, performed on 6 of the 10 samples, showed the frequency of AML cells initiating LTC (AML LTC-IC) to be 5- to 300-fold lower than the frequency of AML-CFC in the same cell sample, whereas the average number of CFC produced per LTC-IC varied from 1 to 13. Surprisingly, in each case the concentration of cytogenetically normal LTC-IC detected in AML patient blood was at least 10-fold higher than that previously observed in the blood of normal individuals. "Mixed" mouse fibroblast feeders engineered to produce human G-CSF, IL-3, and SF did not enhance detection of AML LTC-IC but did increase the output of cytogenetically normal CFC from LTC of 3 of 4 patient samples. Supplementation of AML LTC with IL-3 and exogenously provided SF and/or FL increased the output of AML-CFC from 5-week-old LTC by greater than or equal to twofold with 5 of 9 patient samples, whereas in one case exogenous addition of FL reduced the output of malignant CFC from LTC. These studies show that conditions that support normal LTC-IC also allow a functionally analogous but rare AML progenitor cell type to be detected. In addition, differences in the responses of normal and leukemic cells to various cytokines active on normal LTC-IC were revealed. Further analysis of these differences may enhance our understanding of leukemogenesis and lead to observations that could be exploited therapeutically.

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/ SCID mice) transplanted intravenously with 10(7) human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34+ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18-20 weeks. 3H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4-6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo.

To determine whether the sensitivity of the human long-term culture-initiating cell (LTC-IC) assay could be increased, we have evaluated a spectrum of different fibroblast cell lines for their abilities to influence the number of cells detectable as LTC-IC, to influence LTC-IC maintenance, and/or to influence LTC-IC differentiation into colony-forming cells (CFC) in cocultures containing various sources of LTC-IC. In a series of initial experiments with highly purified subpopulations of CD34+ cells from normal human marrow, no significant difference could be found between any of 3 different murine stromal fibroblast cells in terms of their support of either LTC-IC detection (CFC production) or maintenance (over a 6-week period), and all were equivalent to primary human marrow feeders (HMF). On the other hand, murine M2-10B4 fibroblasts engineered to produce high levels of both human granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3; 190 and 4 ng/mL, respectively), either alone or mixed 1:1 with SI/SI fibroblasts engineered to produce high levels of soluble Steel factor (SF), with or without production of the transmembrane form of SF (60 and 4 ng/ mL, respectively), stimulated the production of up to 20-fold more CFC in LTC of cells from normal human marrow, G-CSF-mobilized blood or cord blood when compared with parallel cocultures containing HMF. Limiting dilution analysis of the CFC output from all three sources of LTC-IC showed that most of this increase was due to an ability of the engineered feeders to increase the plating efficiency of the LTC-IC assay (approximately 14-fold for marrow LTC-IC and approximately 4-fold for cord blood or mobilized blood LTC-IC). Analysis of the phenotype of these additionally recruited LTC-IC from marrow showed they had the same primitive CD34+CD45RA-CD71- phenotype as conventionally defined LTC-IC. The limiting dilution studies also showed that the average number of CFC produced per LTC-IC was additionally and independently increased to yield values of 18 CFC per LTC-IC in marrow, 28 for LTC-IC in cord blood, and 25 for LTC-IC in G-CSF-mobilized blood. Replating of cells from primary LTC with different feeders into secondary LTC-IC assays containing the best combination of engineered feeders showed that LTC-IC maintenance could be significantly enhanced (up to 7-fold as compared with primary cocultures containing HMF). However, this enhancement was still not sufficient to amplify the number of LTC-IC present after 6 weeks above the input value. Thus, engineering murine fibroblasts to produce sufficient SF, G-CSF, and IL-3 can markedly enhance the detection as well as the maintenance in vitro of a very primitive population of human progenitor cells present in normal adult marrow, mobilized blood, and cord blood by providing the most sensitive assay conditions thus far described. The present findings also provide new evidence of biologic heterogeneity between different cell populations that can be operationally identified as LTC-IC, thus re-emphasizing the importance of limiting dilution analyses to distinguish between quantitative and qualitative effects on these cells.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

Mobilized peripheral blood is increasingly used as a source of cells to provide hematopoietic reconstitution in marrow ablated patients. There is controversy as to how the engraftment potential of these preparations should be evaluated. The long-term culture-initiating cell is a very primitive hematopoietic with unique phenotypic feature. The potential role and significance of these cells in peripheral blood cell autografts is discussed.

Class I homeobox (Hox) genes encode a major group of transcription factors controlling embryonic development and have been implicated in the continuing process of hematopoietic cell differentiation. They are clustered on four chromosomes and, in early development, exhibit spatially restricted expression with respect to their 3'-->5' chromosomal position. By using an improved PCR-based method for amplifying total cDNA derived from limited cell numbers, we now describe the expression of class I Hox genes in highly purified CD34+ cell subpopulations isolated from normal human bone marrow that represent functionally distinct stem and progenitor cell compartments. Our data indicate that at least 16 different Hox genes, mainly from the A and the B clusters, are expressed in one or more of these subpopulations of human hematopoietic cells. Moreover, markedly elevated expression of some of the Hox genes found at the 3' end of the A and B clusters (e.g., HoxB3) was a unique feature of the subpopulations that contained the most primitive functionally defined cells, whereas genes located in the 5' region of each cluster (e.g., HoxA10) were found to be expressed at nearly equal levels in the CD34+ subpopulations analyzed. In contrast to the findings for CD34+ cells, expression of two selected Hox genes, HoxB3 and HoxA10, was virtually extinguished in the CD34- fraction of bone marrow cells. These results demonstrate the expression of a broad range of Hox genes in primitive hematopoietic cells and point to the existence of a regulated program of Hox gene expression during their normal development.

Long-term culture initiating cells (LTC-IC) are primitive hemopoietic progenitors that give rise to clonogenic cells when provided with a supportive feeder layer of mesenchymal cells. These LTC-IC possess many of the characteristics expected of marrow-repopulating "stem cells" including high proliferative and multilineage-differentiative capacity and resistance to 4-hydroperoxy-cyclophosphamide (4-HC) killing. In addition, stem cells are known to persist and may proliferate in murine LTC, and human marrow grown in LTC has been successfully used as hemopoietic support for myeloablative therapy. LTC-IC, as well as clonogenic precursors, circulate in normal peripheral blood, and the concentration of both progenitor types can be increased by cytotoxic chemotherapy and/or growth factors. When mobilized peripheral blood cells are used as hemopoietic support for high-dose chemo/radiotherapy, engraftment has often been more rapid than that achieved with autologous marrow. Thus, primitive hemopoietic cells circulate in human blood, which can enable hemopoietic reconstitution following aggressive therapy for malignant disease.

The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin (IL)-6 and IL-3, both alone and in combination, stimulate hematopoietic cells in short-term in vitro assays and in vivo. To study their ability to influence hematopoiesis in a system that mimics many features of the marrow microenvironment, long-term cultures (LTC) were produced by co-cultivating normal human marrow cells on feeder layers of murine marrow-derived stromal cells (M2-10B4 cells) genetically engineered to produce human IL-6 and/or IL-3. Feeders stably producing 20 ng/ml IL-6 slightly increased the output of clonogenic progenitors in these LTC but did not change the production of mature (total nonadherent) cells as compared to control cultures. Feeders producing 50 ng/ml IL-3 increased both clonogenic progenitor output (approximately threefold) and the output of mature cells (six-fold) as compared to controls. Feeders producing both factors also increased the output of both progenitors and mature cells. At the time of the weekly half-medium change when primitive clonogenic progenitors in the adherent layer are quiescent, such progenitors were actively cycling in all cultures with factor-producing feeders, as shown by [3H]thymidine suicide assays. Similarly, three sequential daily additions of 20 ng/ml of IL-6 also stimulated the quiescent progenitors to enter S-phase 2 days later, although single doses of recombinant IL-6 as high as 100 ng/ml failed to do so. The combined presence of IL-6- and IL-3-producing feeders, but neither alone, was also able to enhance more than twofold the maintenance and early differentiation of cells capable of generating clonogenic cells for at least a further 5 weeks in secondary LTC. Thus, the provision of a continuous source of IL-6 or IL-3 to primitive hematopoietic cells even in the LTC system can enhance late events in the hierarchy of hematopoietic cell differentiation, but a combination of the two factors is required to stimulate early multipotent progenitors.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.

Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems, but their role as microenvironmental regulators is poorly understood. To address this problem, we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early, intermediate, and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]), clonogenic cells, and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders, cells at all stages of hematopoiesis decreased to very low levels. In contrast, maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However, this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand, in the presence of GM-CSF-producing feeders, the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications.

Cells at two distinct early stages in the development of mature human blood cells from primitive totipotent hematopoietic stem cells can now be defined and quantitated by separate in vitro assays. Current evidence suggests that most, if not all, colony-forming cells--that is, cells that give rise to colonies of mature progeny within one to three weeks in semisolid culture systems, represent an intermediate stage of hematopoietic progenitor. These cells are not self-sustaining; if they are used to initiate hematopoiesis on competent marrow stromal layers, they rapidly disappear as they differentiate or die. However, clonogenic cells can be generated in such cultures from another cell type over a period of four to eight weeks. We have, therefore, assigned the term long-term culture initiating cell (LTC-IC) to this latter type of clonogenic precursor cell. The production and differentiation of cells in both of these compartments in LTC are dependent on, and regulated by, nonhematopoietic "stromal" cells that form a heterogeneous adherent layer in which close-range interactions with hematopoietic cells take place. The use of separate endpoints to monitor the maintenance, differentiation, and reversible activation or arrest of cycling of these cells has recently revealed different molecular mechanisms regulating their respective functions. However, an important common feature appears to be the relative local concentration of positive and negative regulators to which the target hematopoietic cell is exposed. Both gene expression and growth factor release measurements as well as results obtained using genetically engineered stroma and repeated soluble growth factor addition implicate G-CSF as an endogenous positive regulator of primitive hematopoietic cells. Similarly, gene expression, factor production, factor addition, and neutralizing antibody experiments implicate TGF-beta as an endogenous inhibitor of primitive hematopoietic cells.

These results suggest a potentially important role for the LTC system, not only in the development of a quantitative assay for hematopoietic cells, but also clinically as a procedure for manipulating autologous marrow in culture in a variety of ways for therapeutic advantage. These include strategies for immune modulation, gene marking and gene therapy, and perhaps eventually for stem cell amplification.

To study the feasibility of using retroviruses for gene transfer into human hemopoietic cells, various cell types were exposed to virus carrying the gene for neomycin resistance (neor). In preliminary studies using K562 cells as targets, we found that high viral titer and co-cultivation with viral producer cells rather than incubation in medium exposed to viral producer cells were important variables for achieving high frequencies of G418 resistant (G418r) colonies. The maximum frequency of G418r K562 colonies after co-cultivation with cells producing a neor virus titer of 4 X 10(6) cfu/mL was 60%. When primary human progenitors from normal marrow, fetal liver, or chronic myelogenous leukemia blood were exposed to high titer viral stocks, both with and without helper virus, under conditions optimized for K562 cells, maximum frequencies of G418r colonies were 3% to 16% for granulocyte macrophage progenitors and 2% to 6% for primitive erythroid progenitors. The presence of the neor gene in both G418r K562 and primary hemopoietic colonies was verified by Southern blot. Expression of the neor gene was shown by RNA spot blot. These data demonstrate efficient transfer and expression of the neor gene in both K562 cells and primary human hemopoietic cells from normal and leukemic individuals.

When marrow cells from Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) patients are placed into long-term culture, Ph-positive progenitor numbers typically decline rapidly. Karyotypically normal cells derived from persisting, but previously undetectable, precursors can then frequently be demonstrated. To investigate the origin of these cytogenetically normal progenitors, long-term cultures were established with marrow from a girl with CML who was also heterozygous for the glucose-6-phosphate dehydrogenase (G6PD) enzyme variants A and B as shown by studies of her skin fibroblasts. Erythroid and granulocytic colonies generated in methylcellulose assays of fresh marrow from this patient were all Ph-positive and expressed the enzyme G6PD-A as found in the mature blood cells. In assays of 4- and 6-week-old long-term cultures, numbers of progenitors present were low and most were Ph-positive, but some (2/11) were Ph-negative. G6PD analysis showed that a similar proportion was not part of the neoplastic clone (4/30 were G6PD-B and 26/30 were G6PD-A). Thus, long-term marrow culture in conjunction with G6PD analysis was able to reveal the presence of a nonclonal hemopoietic progenitor population in a CML patient where such cells would not otherwise have been detected. This approach may offer a novel strategy for the demonstration and selection of populations of normal hemopoietic progenitors in other patients where the malignant cells cannot be distinguished by chromosome analysis.

The present study was undertaken to investigate the hemopoietic cell from which malignant change evolves in juvenile dyshemopoiesis with monosomy 7. Two male patients, aged 18 and 5 months, were studied using progenitor assays combined with cytogenetics. Both had hepatosplenomegaly, cytopenias and a cellular marrow. The karyotype in direct marrow was 45,XY-7/47,XY,+8/46,XY in patient 1 and 45,XY,-7/46,XY in patient 2. Patient 1 received chemotherapy but developed acute nonlymphocytic leukemia after 17 months and died 20 months after diagnosis. During this time marrow metaphases with 45,XY,-7 increased to 100% (25/25). Patient 2 received an allogeneic marrow transplant 4 months after diagnosis which did not engraft. In both patients progenitors of both small (CFU-E) and large (BFU-E) erythroid colonies were present at normal frequencies. However, the colonies produced were small and poorly hemoglobinized with some erythropoietin-independent maturation. Progenitors of large granulocyte/macrophage colonies (CFU-GM) were present at an elevated frequency in the marrow of patient 1 and in the blood all progenitor classes were markedly increased. Cytogenetic analysis of colonies from this patient showed BFU-E to be 45,XY,-7 or 47,XY,+8 and CFU-GM to be 45,XY,-7 or 47,XY,+8 or 46,XY. In patient 2, most BFU-E were 45,XY,-7, although a few were 46,XY. These data indicate that malignant change in this disease involves hemopoietic stem cells capable of erythroid and in at least some cases, myeloid differentiation.

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony-stimulating factor and interleukin-1 beta or medium conditioned by a human marrow "stromal" cell line to medium conditioned by agar-stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.