Normal Human Marrow Cells Research Articles

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p < 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

Marrow cell genetic phenotype change induced by human lung cancer cells

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.

Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 μM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.

Expression of CD34 on human B cell precursors

CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re-analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow.

Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo

AbstractNotch transmembrane receptors are known to play a critical role in cell-fate decisions, with Notch1 shown to enhance self-renewal of hematopoietic stem cells and cause T-cell leukemia. Four Notch receptors exist, and the extent of redundancy and overlap in their function is unknown. Notch4 is structurally distinct from Notch1 through Notch3 and has not been extensively studied in hematopoiesis. By polymerase chain reaction (PCR) we find Notch4 transcript expression in human marrow cells and in both CD34+ and CD34– populations. When constitutively active Notch1 or Notch4 was overexpressed in normal human marrow or cord cells, we found reduced colony-forming and short-term proliferative ability while the primitive progenitor content of myeloid long-term cultures was significantly increased. Notch4–intracellular domain (Notch4-IC)–transduced cord cells transplanted into β2-microglobulin–/– nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in significantly higher levels of engraftment of both green fluorescent protein–positive (GFP+) and GFP– populations as compared with controls. GFP+ cells in bone marrow and spleen of animals that had received transplants gave rise to an immature CD4+CD8+ T-cell population, whereas B-cell development was blocked. These results indicate that activation of Notch4 results in enhanced stem cell activity, reduced differentiation, and altered lymphoid development, suggesting it may influence both stem cells and the fate of the common lymphoid progenitor.

Constitutive Expression of the Fas Receptor and Its Ligand in Adult Human Bone Marrow: A Regulatory Feedback Loop for the Homeostatic Control of Hematopoiesis

ABSTRACT Fas ligand (FasL) mediated apoptosis in the bone marrow may contribute to suppression of hematopoiesis in myelodysplastic syndromes (MDS) and in aplastic anemia, and also to the regulation of normal erythropoiesis. To identify potential effector and target cells in this regulatory pathway, we examined the constitutive expression of Fas receptor (Fas) and FasL (total and cell-surface) in myeloid and lymphoid cells and subsets of CD34 + cells in normal healthy adult human bone marrow using multiparameter flow cytometry. A high proportion of CD34 + cells constitutively expressed cell-surface FasL. However, none of the CD34 + cells expressed Fas alone. A reciprocal gradient of expression of FasL and Fas was observed in subsets of CD34 + cells: as compared to primitive CD34 +/HLA-DR − (DR −) cells, a higher proportion of committed CD34 +/HLA-DR ++ (DR ++) cells expressed FasL but fewer expressed Fas; the expression of both molecules was intermediate in CD34 +/HLA-DR dim cells. Also, the intensity of FasL expression was higher in DR ++ than in DR − cells. These results suggest that the homeostatic regulation of myelopoiesis in normal bone marrow is mediated via an autoregulatory feedback loop by myeloid cells and progenitors themselves, at least partly via the Fas-FasL pathway. This notion is also consistent with our recent observation that overexpression of FasL by myeloid cells in MDS correlates directly with anemia, transfusion requirements, and shorter survival, an example of dysregulation of this pathway.

In Response:

In our study, we classify CD34+Lin−Thy-1+ cells as human stem cells because the Thy-1+ subpopulation of CD34+ cells have self-renewal characteristics of human stem cells and are capable to generate all hematopoietic lineages. Effectively, primitive hematopoietic stem cells in normal human peripheral blood and bone marrow have been characterized [1Udomsakdi C. Lansdorp P.M. Hogge D.E. Reid D.S. Eaves A.C. Eaves C.J. Characterization of primitive hematopoietic cells in normal human peripheral blood.Blood. 1992; 80: 2513PubMed Google Scholar, 2Srour E.F. Brandt J.E. Briddell R.A. Leemhuis T. van Besien K. Hoffman R. Human CD34+ HLA-DR-bone marrow cells contain progenitors capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.Blood Cells. 1991; 17: 287PubMed Google Scholar, 3Baum C.M. Weissman I.L. Tsukamoto A.S. Buckle A.M. Peault B. Isolation of a candidate human hematopoietic stem cell population.Proc Natl Acad Sci U S A. 1992; 89: 2804Crossref PubMed Scopus (888) Google Scholar]. These cells are CD34+Lin−Thy-1+, constitute less than 0.05% of whole bone marrow, are capable of multilineage differentiation, including myeloid, B-, and T-cell lineages, and have extensive self-renewal capacity [3Baum C.M. Weissman I.L. Tsukamoto A.S. Buckle A.M. Peault B. Isolation of a candidate human hematopoietic stem cell population.Proc Natl Acad Sci U S A. 1992; 89: 2804Crossref PubMed Scopus (888) Google Scholar, 4Kyoizumi S. Baum C.M. Kaneshima H. McCune J.M. Yee E.J. Namikawa R. Implantation and maintenance of functional human bone marrow in SCID-hu mice.Blood. 1992; 79: 1704PubMed Google Scholar]. CD34+Lin−Thy-1+ cells resemble hematopoietic stem cells based on in vitro assays for long-term culture-initiating cells and in vivo assays for hematopoietic stem cells utilizing the SCID-hu bone assay system [5Craig W. Kay R. Cutler R.L. Lansdorp P.M. Expression of Thy-1 on human hematopoietic progenitor cells.J Exp Med. 1993; 177: 1331Crossref PubMed Scopus (421) Google Scholar] and SCID-hu thymus assay system [6Peault B. Weisman I.L. Baum C. McCune J.M. Tsukamoto A. Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ Precursor cells.J Exp Med. 1991; 174: 1283Crossref PubMed Scopus (130) Google Scholar]. CD34+Lin−Thy-1+ cells have been shown to be able to fully reconstitute hematopoiesis in laboratory animals including non-human primates [7Donahue R.E. Kessler S.W. Kirby M.R. et al.Autologous bone marrow transplantation in non-human primates using a subpopulation of CD34+ cells expressing Thy-1.Blood. 1993; 82: 1175PubMed Google Scholar]. In humans, CD34+Lin−Thy-1+ cells can be mobilized in the peripheral blood using a high dose of cyclophosphamide and growth factor regimens and isolation in sufficient numbers to potentially permit hematologic reconstitution following myeloablative regimen. It was observed that the percentage of Thy-1+ in the CD 34+ population of successfully mobilized PBSC was regularly increased well above that seen in resting bone marrow, and it is far from clear what that means. The mobilization phenomenon may cause selective release of ontologically more primitive cells. Our study aimed at finding the minimal effective dose, which at that time was thought to be very low, and the median dose finally used in our study reflected the cell-dose-de-escalation design of the study with an actual median dose of 0.69. Therefore it was decided to base the analysis on a 0.8 × 106 CD+Lin−Thy-1+/kg threshold dose reported by Systemix in a more extensive analysis of a multidisease population. CD34+Lin−Thy-1+ cells are capable of producing fast and durable hematopoietic engraftment at cell doses greater than 0.8 × 106 CD34+ Thy−1+/kg, and the two patients (8 and 30) who experienced delayed engraftment developed sepsis, followed (for patient 30) by an hemolytic and uremic syndrome. Considering the number of CD34+ cells required for rapid engraftment, our results are comparable to those obtained after the infusion of 2.5 × 106 to 15 × 106 CD34+cells. Moreover, it has been demonstrated that the rapidity of hematopietic reconstitution was related to the number of CD34+ cells infused [8Ketterer N. Salles G. Raba M. et al.High CD 34+ cell count decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation.Blood. 1998; 91: 3148PubMed Google Scholar].

Enhanced cytotoxicity of mitomycin C in human tumour cells with inducers of DT-diaphorase.

DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. We have shown that DT-diaphorase can be induced in human tumour cells by a number of compounds, including 1,2-dithiole-3-thione. In this study, we investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumour cell lines representing four tumour types. DT-diaphorase was induced by many dietary inducers, including propyl gallate, dimethyl maleate, dimethyl fumarate and sulforaphane. The cytotoxicity of MMC was significantly increased in four tumour lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumour cells. This study demonstrates that induction of DT-diaphorase can increase the cytotoxic activity of MMC in human tumour cell lines, and suggests that it may be possible to use non-toxic inducers of DT-diaphorase to enhance the efficacy of bioreductive anti-tumour agents. © 1999 Cancer Research Campaign

Radon decay in bone marrow fat cells and the possible induction of leukaemia

The alpha particle radiation dose to haemopoietic stem cells and marrow was determined with Monte Carlo modeling using input data generated from autopsy specimens of normal marrow and compared with the dose estimates obtained using the Bristol image analysis and dose calculation system on those same marrow specimens. There was very good agreement between the two dose calculation methods. The project confirmed that the spatial distribution of haemopoietic stem cells in normal human marrow was random, and a trimodal distribution of haemopoietic stem cell diameters was described (possibly due to different stages of the cell cycle, as previously described in the mouse). Modeling of the dose to marrow with different fat fractions showed a positive linear relationship between the fat fraction and both the proportion of haemopoietic stem cells “hit” and the proportion of “hit” survivors. Modeling of the dose to haemopoietic stem cells in the marrow of the underground miners showed a positive linear relationship between the radon concentration in external air and both the proportion of haemopoietic stem cells “hit” and the proportion of “hit” survivors. In all the modeling the mean number of alpha particle passages per haemopoietic stem cell was approximately 1.02, with doses of 0.86, 0.20, and 0.12 Gy for haemopoietic stem cells of diameter 5.71, 11.63, and 14.79 μm, respectively.

Kinetic Evidence of the Regeneration of Multilineage Hematopoiesis From Primitive Cells in Normal Human Bone Marrow Transplanted Into Immunodeficient Mice

AbstractBased 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.

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

The present study was undertaken to define parameters that may limit the cytokine-mediated expansion of primitive hematopoietic cells in stirred suspension cultures of normal human marrow cells. In a first series of experiments, parallel measurements of the rate and extent of progenitor expansion and cytokine depletion from the medium were made for such cultures in which the cells were exposed to different cytokine concentrations. Supplementation of the medium with 2 ng/mL of interleukin-3 (IL-3), IL-6 and IL-11 plus 10 ng/mL of Flt-3 ligand (FL) and Steel factor (SF) allowed a 45-fold expansion of directly clonogenic cell (CFC) numbers within 2 weeks along with a 2.5-fold expansion of their precursors, detectable as longterm culture-initiating cells (LTC-IC). The addition of 5-fold higher levels of these cytokines enhanced the 2 week output of both CFC and LTC-IC numbers (to 66-fold and 9-fold above input respectively). However, this was also associated with an increase in the individual average rates of depletion of immunoreactive IL-3, SF and FL. As a result, even biweekly addition of fresh medium supplemented with the highest concentrations of cytokines tested failed to prevent a continuing decline in their levels relative to the input medium levels. A similar dependence of the IL-3 depletion rate on its extracellular concentration was demonstrable in suspension cultures of Mo7e cells, an IL-3-dependent human leukemic cell line.Additional experiments with various highly purified marrow cell fractions showed that the rate of cytokine depletion varied according to the type of responding cell as well as the specific cytokine. CD34(+)CD38(-) cells exhibited the greatest average cell-specific cytokine depletion rates (35-fold higher than unseparated bone marrow cells). These findings establish new principles that will be important for the optimization of hematopoietic cell bioreactors. In addition, they suggest that cytokine depletion may provide a novel feedback control mechanism in vivo which would contribute to the control of primitive hematopoietic cell proliferation and differentiation.

Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of > 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However, this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.

Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haemopoietic cell development.

Basic fibroblast growth factor (FGF-2) and acidic fibroblast growth factor (FGF-1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF-1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF-R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF-1 or 2 on survival of early progenitor cells in serum-free culture. To explain these findings, we examined progenitor cells for expression of the FGF-R at the mRNA and protein level using RT-PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF-R (FGF-R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF-R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF-R1 expression was detected on subpopulations of MNC and on cells derived from day-6 CFU-GM and BFU-E colonies. Accordingly, FGF-R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF-Rs are expressed on more mature cells.

Quantitation of the Quiescent Fraction of Long-Term Culture-Initiating Cells in Normal Human Blood and Marrow and the Kinetics of Their Growth Factor-Stimulated Entry Into S-Phase In Vitro

A method for quantitating the proportion of cycling long-term culture-initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions, as long as 3H-Tdr is not present. In addition, we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally, any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However, within 72 hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF, most had entered S-phase, although there was no net change in their numbers. This suggests that, under certain conditions in vitro, self-renewal divisions of LTC-IC can occur and, at least initially, balance any losses of these cells due to their differentiation or death. In contrast, many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating, although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications.

Influence of human granulocytemacrophage colony stimulating factor/interleukin-3 fusion protein (PIXY321) on the hematopoietic toxicity associated with anti-viral drugs (zidovudine and didanosine) in vitro using normal human marrow cells

The antiviral drugs didanosine (ddl) and zidovudine (AZT), synthetic nucleoside analogs, have been used in the treatment of acquired immunodeficiency syndrome (AIDS). Although clinical use of zidovudine (AZT) is still widely used, it is associated with the development of virus disease resistance and toxicity to the hematopoietic system. Alternative nucleoside reverse transcriptase derivatives such as didanosine (ddl) have been developed in order to reduce the incidence of virus disease resistance and hematological toxicity. We report here studies designed to evaluate the toxicity profile comparing didanosine (ddl) with zidovudine (AZT) when used alone or in combination with normal non-adherent, T-cell depleted human marrow cells plated in the presence or absence of the. human cytokine fusion protein of granulocyte-macrophage colony stimulating factor and interleukin-3 (PIXY321). As expected, didanosine (ddl) was less toxic for human hematopoietic progenitor Cells, I.e, CFU-GEMM, CFU-GM, CFU-Meg, and BFU-E than zidovudine. Toxicity was additive when didanosine (ddl) and zidovudine (AZT) were combined. In the absence of drugs PIXY321 colony formation was increased for all progenitor cells cultured. In the presence of didanosine (ddl) or zidovudine (AZT), either as single-agents or combined, PDCY321 reduced toxicity significantly. These results demonstrate PIXY321 is an effective cytokine capable of reversing the toxicity associated with anti-viral drugs when used in vitro where didanosine (ddl) is less toxic than zidovudine (AZT); however their suppression of hematopoietic progenitors is additive when combined.

Immunophenotype of c‐kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117+ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients. Our results show that most of normal BM CD117+ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117+ cells are CD34+, these cells displaying a different FSC/SSC distribution when compared to the CD117+/CD34- cells. No CD117+/CD15+ and CD117+/CD10+ cells were detected and very few CD117+ cells (< 1 x 10(-3) expressing the HLADR-/CD34-, CD33+/HLADR- and CD34+/HLADR- phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117+/CD15+ blast cells and eight had the CD117+ phenotypes detected at low frequencies (< 1 x 10(-3)) in normal BM. In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117+/CD15+ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.

Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor

Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB- 02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose- dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c- kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.

Stem Cell Factor Influences the Proliferation and Erythroid Differentiation of the MB-02 Human Erythroleukemia Cell Line by Binding to a High-Affinity c-kit Receptor

AbstractStem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB- 02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose- dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c- kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.© 1993 by The American Society of Hematology.

Alternative Mechanisms With and Without Steel Factor Support Primitive Human Hematopoiesis

AbstractAs a first approach to defining the molecular requirements for supporting human hematopoietic stem cell maintenance and differentiation in vitro, we have analyzed and compared the ability of various factors to support the maintenance and initial differentiation of human long-term culture-initiating cells (LTC-ICs), a distinct, rare primitive hematopoietic cell type whose progeny after 5 weeks include cells detectable as colony-forming cells. Normal human marrow cells highly enriched in LTC-ICs (approximately 1% pure) were placed in cultures containing either preestablished, irradiated human marrow adherent feeder layers, or feeders consisting of Steel factor-deficient SI/SI, or normal +/+ murine fibroblasts, or no feeders. In some groups, either Steel factor alone, granulocyte colony-stimulating factor (G- CSF) and interleukin-3 (IL-3), or all three factors combined were also added repeatedly. SI/SI murine fibroblasts were equivalent to +/+ controls and to normal human marrow feeders in supporting both LTC-IC maintenance and clonogenic cell output over a 5-week period. Soluble Steel factor alone could, however, effectively substitute for human marrow feeders to support LTC-IC maintenance, although clonogenic cell output was markedly reduced under these conditions. Conversely, soluble Steel factor with G-CSF and IL-3 or with feeders (or all together) did not further enhance (or depress) LTC-IC maintenance, although under these conditions clonogenic cell output was markedly increased. These findings confirm previous evidence that LTC-IC maintenance and clonogenic cell production are differentially regulated and show for the first time that LTC-IC maintenance can be supported by different nonsynergizing factors that may, but need not, include Steel factor.

Alternative mechanisms with and without steel factor support primitive human hematopoiesis

As a first approach to defining the molecular requirements for supporting human hematopoietic stem cell maintenance and differentiation in vitro, we have analyzed and compared the ability of various factors to support the maintenance and initial differentiation of human long-term culture-initiating cells (LTC-ICs), a distinct, rare primitive hematopoietic cell type whose progeny after 5 weeks include cells detectable as colony-forming cells. Normal human marrow cells highly enriched in LTC-ICs (approximately 1% pure) were placed in cultures containing either preestablished, irradiated human marrow adherent feeder layers, or feeders consisting of Steel factor-deficient SI/SI, or normal +/+ murine fibroblasts, or no feeders. In some groups, either Steel factor alone, granulocyte colony-stimulating factor (G- CSF) and interleukin-3 (IL-3), or all three factors combined were also added repeatedly. SI/SI murine fibroblasts were equivalent to +/+ controls and to normal human marrow feeders in supporting both LTC-IC maintenance and clonogenic cell output over a 5-week period. Soluble Steel factor alone could, however, effectively substitute for human marrow feeders to support LTC-IC maintenance, although clonogenic cell output was markedly reduced under these conditions. Conversely, soluble Steel factor with G-CSF and IL-3 or with feeders (or all together) did not further enhance (or depress) LTC-IC maintenance, although under these conditions clonogenic cell output was markedly increased. These findings confirm previous evidence that LTC-IC maintenance and clonogenic cell production are differentially regulated and show for the first time that LTC-IC maintenance can be supported by different nonsynergizing factors that may, but need not, include Steel factor.