Factors controlling haemopoiesis in ovine long-term bone marrow cultures

There is increasing interest in both standardization and simplification of methods for enumeration of CD34+ hematopoietic progenitor cells (HPC) to facilitate cellular therapies and to improve interinstitutional comparison of clinical and laboratory results. We evaluated a novel method for CD34+ cell enumeration based on microvolume fluorimetry (MVF) compared with our laboratory's routine flow cytometric method on samples of peripheral blood and leukapheresis products. The MVF method is semiautomated and uses a 633-nm light from a helium-neon laser to scan fluorochrome-labeled cells held in stasis in a capillary known volume. The performance of the MVF assay for enumeration of CD34+ cells was found to be comparable to our routine flow cytometric assay in linearity and accuracy in the range of 5-1500 cells per microliter. Precision of MVF for replicate assays on the same instrument was demonstrated by coefficient of variation (CV) values of 8.4% at a CD34+ cell concentration of 284/microliters for a sample volume of 0.8 microliters, and 15.7% at 12/microliters for a sample volume of 3.2 microliter. Precision among three different instruments was demonstrated, using sample volumes of 1.6 microliters, by CV values of 44% at 6 cells/microliters and 4.6% at 733 cells/microliters. In a field sample evaluation, precision of the entire assay system for paired measurements on 0.8-microliter sample volumes was demonstrated by CV values of 50%, 31%, and 15% for peripheral blood samples with concentrations of 0-10, 10-20, and 20-100 CD34+ cells/microliters, respectively, and 6.3%, 8.1% and 6.5% for leukapheresis samples with concentrations of 0-100, 100-1,000, and 1,000-2,500 CD34+ cells/microliters, respectively. The MVF assay was easy to perform, required minimal technical training time, and had a turnaround time of 40 min, of which less than 10 min was actual technical time. These observations suggest that the MVF method for CD34+ cell enumeration may prove useful to clinical laboratories providing support for HPC collection, processing, and transplantation services that require relatively simple, rapid assays for product quality control or to guide real-time clinical decisions.     

Globin synthesis in normal human bone marrow

Globin synthesis has been studied by in vitro labelling with radioactive amino acids in 60 normal human bone-marrow samples. Under the conditions routinely used to fractionate alpha and beta chains by chromatography alpha/beta production ratios ranging from 0.5 to 1.0 were obtained, depending on the method of sample treatment. This variation was due entirely to the presence of non-haem proteins derived from white cells which chromagraphy with globin on CM-cellulose. Purification of globin on Sephadex G100 and fractionation of alpha and beta globin chains by a modified chromatographic system resulted in alpha/beta ratios of unity. The relevance of these findings to the study of marrows in which there is unbalanced globin chain production is discussed.     

Increased osteoclast-like cells formation in long-term bone marrow cultures from patients with a spinal cord injury

A simple and sensitive method for analysis of three tetracyclic antidepressants, maprotiline, mianserin, and setiptiline, in human whole blood was developed using headspace-solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). A vial containing a blood sample, sodium hydroxide, and imipramine as an internal standard was heated at 120 degrees C. The extraction fiber of the SPME was exposed for 45 min in the headspace of the vial. The compounds absorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.005 to 5.0 microg/g for mianserin and setiptiline and from 0.025 to 25 microg/g for maprotiline. No interferences were found, and the time for analysis was 60 min for one sample. In addition, this proposed method was applied to a medicolegal case in which the cause of death was suspected to be acute setiptiline poisoning. Setiptiline was detected in the left and right heart blood samples of the victim at concentrations of 1.77 and 0.78 microg/g, respectively.     

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.     

Effects of droperidol and fentanyl on human bone marrow cultures

The effects of droperidol and fentanyl on three samples of human bone marrow with regard to their colony forming in culture was studied in more than 256 multiple experiments. There was no evidence of any toxic effect of droperidol and fentanyl on the bone marrow cells.     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

Improved method of enrichment for immature myeloid cells from normal human bone marrow

A method of enrichment for immature myeloid cells from normal human bone marrow has been described. The method is based on 4 consecutive steps: 1. Density cut centrifugation. After centrifugation all cells above the pellet were collected. This suspension contained 93% of the originally present myeloblasts and promyelocytes. The majority of normoblasts and granulocytes was found in the pellet. 2. Nylon wool filtration. This procedure was performed to remove the majority (87%) of the monocytes. Recovery of myeloblasts and promyelocytes after filtration was 74%. 3. Centrifugation on a continuous density gradient. This procedure resulted in an additional purification of the myeloblasts and promyelocytes within a specific fraction. By centrifugation the concentration of myeloblasts and promyelocytes was increased from 19% to 38%. 4. Velocity sedimentation at 1 g. This technique produced a 82% pure immature myeloid cell suspension, comprising 57% myeloblasts and promyelocytes. All figures are the mean of 10 experiments.     

Toxicity of cytostatic drugs to normal bone marrow cells in vitro

Metastatic melanomas are often resistant to chemotherapy. To study whether the p53 mutational status affects chemosensitivity, we compared the responses to chemotherapy of four melanoma cell lines containing the wild-type p53 and four cell lines carrying the mutant p53. Cisplatin, at 10 microM, virtually killed all the cells in the wild-type p53 cell lines, while 57-95% of the cells in the mutant p53 cell lines survived (P = 0.005). After treatment with 100 nM of vincristine, on average 18% of the wild-type p53 melanoma cells survived compared with 55% of the mutant p53 cells (P = 0.04). After treatment with 40 nM, 200 nM or 1 microM of camptothecin the survival rates were, on average, 16%, 8% and 4% for the wild-type p53 melanoma cells, compared with 89%, 67% and 38% for the mutant p53 cells, respectively (P = 0.00004, P = 0.003 and P = 0.04, respectively). The anticancer agents were not toxic to normal melanocytes at doses inducing cytotoxicity in wild-type p53 melanoma cells. The main mechanism of cytotoxicity appears to be drug-induced apoptosis. Cisplatin, camptothecin and vincristine all induced apoptosis in wild-type p53 melanoma cells, but not in mutant p53 cells. Our results suggest that chemotherapy-induced apoptosis in melanoma cells is p53 dependent, and mutation of the p53 gene is an indicator of drug resistance in melanoma.     

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture

Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions. We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma in long-term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34+ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF. Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6. Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines. However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors.     

Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay

A case of Morris' syndrome in which the diagnosis has been realized only in old age is reported. A 69 year-old patient, with female external genitalia and secondary sexual characteristics, was referred to us with a diagnosis of a mass in the right inguinal region. Her personal history was based on a primary amenorrhoea, which was unsuccessfully investigated since she was adolescent. At the age of 63, during surgery for a left inguinal hernia realized in another hospital, a testis-like mass with the spermatic cord was casually found. During our hospitalization, a surgical removal of the right inguinal mass was performed, and the histologic examination showed the presence of a dominant sclerohyalin testicular tissue without evidence of seminal epithelium and sparse focuses of Leydig cells hyperplasia. Besides, the determination of gonadotropins and sex hormones yielded an increased production of LH, FSH, estradiol, testosterone and androstenedione. A cytogenetic analysis showed a 46, XY karyotype. The diagnosis realized only in old age has compelled the patient to live all her life, from sexual maturity, with indecision and doubt, and without a clinical explanation of fundamental utility even from the psychological point of view. Finally, in our patient the absence of cytologic aspect of malignant transformation in the removed testes in a six years period, seem fortuitous. It is always necessary to consider Morris' syndrome among the possible diseases causing primary amenorrhoea in the clinical evaluation of young phenotypic female patients.     

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.     

Deficient expression of adhesion molecules by human CD5- B lymphocytes both after bone marrow transplantation and during normal ontogeny

Despite the relatively early reconstitution of blood B-lymphocyte counts observed in patients treated with bone marrow transplantation (BMT), these patients undergo a prolonged phase of humoral immunodeficiency. Adhesion molecules perform relevant functions in many cell types. The present study examines the expression of several adhesion molecules on human B lymphocytes newly formed after BMT. Blood B cells from 38 patients were studied by flow cytometry and three-color analysis. Blood CD5- B lymphocytes obtained at an early stage after BMT (2 to 4 months) showed a markedly low expression of the adhesion molecules CD54, CD44, CD11a, and CD62L. However, these cells exhibited a normal expression of other molecules including CD29, CD19, CD20, and DR. This deficiency was progressively corrected, reaching normal levels in the late post-BMT period (12 to 15 months). In contrast, CD54, CD44, CD11a, and CD62L expression on the patients' CD5+ B lymphocytes was found to be consistently normal. Deficient adhesion molecule expression on CD5- B cells in the early post-BMT period was similarly observed in patients treated with either an allo-BMT (n = 24) or an auto-BMT (n = 14). Because the post-BMT period mimics normal ontogeny, adhesion molecule expression was also investigated in cord-blood B lymphocytes. Cord-blood CD5- B lymphocytes, in contrast to CD5+, also expressed CD54, CD44, CD11a, and CD62L at levels much lower than those found in normal adults. Present data suggest that progressive expression of CD54, CD44, CD11a, and CD62L seems to be a part of the maturational program of CD5- B lymphocytes during both post-BMT and normal development periods. This observation may help to explain the humoral immunodeficiency observed in both conditions.     

Receptor tyrosine kinase expression in human bone marrow stromal cells

A case of crossed aphasia is presented in a strongly right-handed 77-year-old white female without history of familial sinistrality or prior neurological illness. She developed a right middle cerebral artery infarction documented by CT and accompanied by obvious clinical signs of a conduction aphasia with some resolution but continuing obvious language defect after 9 weeks in rehabilitation. Comprehensive neuropsychological and aphasia testing suggested anomalous lateralization of phonologic-output aspects of language, emotional prosody, motor planning and body schema modules with usual lateralization of lexical-semantic aspects of language and visuo-spatial functions. Experimental validation of the uncrossed lexical-semantic aspects of language using tachistoscope methods found support for the Alexander-Annett theory that different aspects of language can be dissociated in their lateralization. The subject had difficulty identifying a semantic associate of a picture presented to the left visual field (7 errors out of 10) relative to right visual field presentation (2 errors out of 10). Bilateral free naming errors (6 and 5 errors in the left and right visual fields, respectively) occurred consistent with the aphasic presentation, suggesting phonologic-output dysfunction from the right cerebral vascular accident. Implications of the results for aphasia classification are discussed.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Associations between TGF-beta1 receptors in human bone marrow stromal cells

Bone marrow stromal cells are required for sustained haemopoiesis. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine present in the bone marrow microenvironment which regulates the expression of several cytokines, cytokine receptors and cell adhesion elements. The TGF-beta receptors type I and II, and endoglin, mediate TGF-beta1 binding to the membrane of human bone marrow stromal cells. [125I]TGF-beta1-affinity labelling experiments showed that three different anti-endoglin monoclonal antibodies co-immunoprecipitated a 68 kD TGF-beta1-labelled polypeptide together with TGF-beta1/endoglin complexes. Here, we have shown that the 68 kD receptor corresponds to the type I receptor, indicating that endoglin and the type I receptor associate on the membrane of these cells upon ligand binding. The expression of endoglin by stromal cells was found to be up-regulated by TGF-beta1, but not by IL-1beta. The association of endoglin with signalling components of the TGF-beta receptor system on the membrane of bone marrow stromal cells might modulate TGF-beta1 access to the signalling pathways, and therefore it could regulate TGF-beta1-mediated stromal cellular responses.     

Stage-specific expression of polycomb group genes in human bone marrow cells

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68-86. The TCR beta chains of these disease-causing T cells show three distinct features: they are almost exclusively Vbeta8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68-86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery--ie., that such beta chains are conformationally the most appropriate for triggering by an epitope of 68-86 complexed to class II RT1.BI MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vbeta8.2+, CDR3 AspSer+ and use few N region additions. We conclude that T cells with these TCR beta chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.     

Expansion of the nonadherent myeloid cell population by monoclonal antibodies against tenascin-C in murine long-term bone marrow cultures

Tenascin-C, a predominantly mesenchymal extracellular matrix protein, has a restricted distribution in adult tissues. It has previously been shown that this protein is expressed in the bone marrow. In this paper we show that murine myeloid and lymphoid long-term bone marrow cultures differ in their expression of tenascin-C splice variants. In the adherent stromal layer of myeloid cultures, the 260-kDa polypeptide encoded by the 8-kb mRNA was the major splice variant, whereas in the stromal layer of lymphoid cultures both the shorter 210-kDa polypeptide encoded by the 6-kb mRNA and the 260-kDa polypeptide were abundantly expressed. However, in both culture systems the larger 260-kDa tenascin-C polypeptide was the major isoform secreted in the culture supernatant. This finding is in agreement with previous reports indicating that the smaller 210-kDa isoform is preferentially deposited in the stroma, whereas the alternatively spliced segment in the 260-kDa tenascin-C may contain anti-adhesive domains. Glucocorticoids in myeloid long-term bone marrow cultures and in the MC3T3-G2/PA6 cell line downregulated the expression of tenascin-C. In the present study we observed that this was due primarily to downregulation of the 8-kb major splice variant of the tenascin-C mRNA. We also studied the possible role of tenascin-C in the bone marrow by using antibodies against tenascin-C in long-term bone marrow cultures. We found that three monoclonal antibodies against the carboxyterminal type III fibronectin repeats of tenascin-C (TNCfn 7-8) increased the number of the non-adherent myeloid cells in myeloid long-term bone marrow cultures. It has recently been suggested that the TNCfn 6-8 domain of tenascin-C binds to the alpha8beta1 integrin. Using Northern blotting, we found that the integrin alpha8 subunit was expressed in adherent cells in bone marrow cultures, raising the possibility that tenascin-C acts in bone marrow cultures by binding to the alpha8beta1 integrin.     

Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice

Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34- CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum values at 5 weeks posttransplant, after which they declined. More mature cell types peaked after another 5 weeks and then declined. A 2-week course of thrice weekly injections of human Steel factor, interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (administered just before the mice were killed for analysis) did not alter the pace of regeneration of either primitive or mature human hematopoietic cells, or their predominantly granulopoietic and B-lymphoid pattern of differentiation, although a significant enhancing effect on the level of human cell engraftment sustained after 3 months was noted. Cycling studies showed the human CFC present at 4 to 5 weeks posttransplant to be rapidly proliferating even in mice not given human growth factors. However, by 10 weeks and thereafter, only quiescent human CFC were detected; interestingly, even in mice that were given the 2-week course of growth factor injections. These studies indicate the use of this model for future analysis of the properties and in vivo regulation of primitive human hematopoietic cells that possess in vivo repopulating ability.