TY - JOUR
AU - Lipschitz, David A.
AB - Abstract The multifunctional cytokine interleukin 6 (IL-6) is involved in the regulation of inflammatory and immune responses, and influences many bone and bone marrow functions. In this report we show high concentrations of IL-6 in the supernatant of murine long-term bone marrow cultures (LTBMC). The concentration increases following medium change peaking 12 h later. IL-6 plays a critical role in the generation and maintenance of myelopoiesis in LTBMC. The addition of monoclonal anti-IL-6 antibody to culture significantly suppresses myeloid cell production. IL-6 is also necessary for stromal layer development and the initiation of myelopoiesis in LTBMC. Horse sera (HS) containing low concentrations of IL-6 did not support LTBMC stromal layer development or myeloid cell production, whereas those with high concentrations did. LTBMC initially set up with horse serum containing high IL-6 concentration produced higher concentrations of colony-stimulating activity and IL-6 at the fifth week after culture initiation than those with low concentrations. The ability of a deficient serum to support myelopoiesis could be improved by the addition of recombinant IL-6 to culture. Similarly, the addition of an anti-IL-6 antibody to culture impaired the ability of a HS to initiate and support myelopoiesis in LTBMC. These results suggest that IL-6 is one of the factors that play an essential role in the formation and function of hematopoietically active LTBMC. Bone marrow culture, Interleukin 6, Horse serum, Stroma, Myelopoiesis Introduction The pleiotropic cytokine interleukin 6 (IL-6) is produced by bone marrow stromal cells [1-6] and a variety of other normal cells, cell lines and tumor cells [7, 8]. IL-6 stimulates growth and differentiation of B and T lymphocytes, is involved in the regulation of inflammatory responses, and influences many bone and bone marrow functions [1, 8-11]. IL-6 enhances the proliferation of murine [12-15] and human [16, 17] multipotential hematopoietic progenitor cells, acts as a survival factor for human colony forming units-granulocyte-macrophage (CFU-GM) [18] and is necessary for the generation of CFU-GM and myeloid cell production in murine long-term bone marrow cultures (LTBMC) [4]. IL-6 receptors have been identified on human CD34+ hematopoietic stem cells [19]. In studying the kinetics of IL-6 production in hematopoietically active primary LTBMC, we observed very high IL-6 bioactivity in the culture supernatant. In this report, we have evaluated the role of IL-6 in myelopoiesis in vitro LTBMC. Materials and Methods Animals Four- to six-month-old female C57BL/6 mice, obtained from Charles River Laboratories (Wilmington, MA), were housed in groups of four per cage and fed a standard rodent chow and water ad libitum. They were killed by cervical dislocation under CO2 anesthesia and the marrow was removed from the femora and tibiae by flushing with 1 ml alpha-modification of Eagle's medium with Earle's salt (α-MEM) (Flow Laboratories; McLean, VA). Cell numbers were determined using a model ZF Coulter counter (Coulter Electronics; Hialeah, FL). LTBMC and Horse Sera Over the last years, we have pretested many different lots of horse serum (HS) for supplementing LTBMC. Each was aliquoted and stored at –20°C. The following 10 lots have now been tested for IL-6 bioactivity and colony-stimulating activity (CSA): Lot 12449043 and 12449031 from Hazelton Biologics (Lenexa, KS); Lot 9M0302 and IM1000 from Whittacker Bioproducts (Walkersville, MD); Lot 70H-0493, Lot 122H-0636 and Lot 122H-0647 from Sigma (St. Louis, MO); Lot 3311-2043 from HyClone (Logan, UT); Lot 12449050 from JRH Biosciences (Lenexa, KS); and Lot H11-512 from Intergen (Purchase, NY). The method employed for LTBMC was similar to that initially described by Dexter et al. [20], and subsequently modified [21]. To each T25 flasks, 20 × 106 bone marrow cells were added in a total of 10 ml culture medium consisting of 25% pretested HS, 10% hydrocortisone hemisuccinate (10–6 M final concentration; Sigma Chemicals Company), and 65% α-MEM. Cultures were incubated in a water-jacketed incubator at 33°C in an atmosphere containing 5% CO2 in humidified air. At weekly intervals, the supernatant was removed, the cells were recovered by centrifugation and the total supernatant cell count was determined. The LTBMC was refed with 60% fresh medium and 40% cell-free supernatant removed at the time of medium change. The total supernatant culture medium was changed prior to measure CSA or IL-6 and replaced by fresh culture medium. At medium change, CSA and IL-6 were measured in the spent supernatant during the preceding week. At indicated times, cell-free supernatant was aliquoted and stored at -20°C to subsequently determine CSA and IL-6. Fixation and Staining of Stromal Layer At indicated times after culture initiation, the supernatants were removed, the adherent layers were washed in 0.1 M phosphate-buffered solution, pH 7.2 for 1 min, then fixed in absolute methanol for 10 min at room temperature, rehydrated in a special phosphate buffer (SPB: 1.66 g KH2PO4, 0.75 g Na2HPO4 + 7H2O in 1,000 ml H2O, pH 6.8) for 2 min, and stained with May-Grünwald stain (Sigma Diagnostics; St. Louis, MO) diluted 1:1 with SPB for 3 min. After washing in SPB for 30 sec, the staining was completed with Giemsa (Sigma Diagnostics), diluted 1:28 with buffered water (water: SPB = 5:1) for 12 min, followed by a rinse with buffered water and then air-dried [22]. CSA CSA in the supernatant of LTBMC was measured by assessing colony number when 5 × 104 fresh murine bone marrow cells were cultured for CFU-GM in the presence of 10% of various HS or cell-free supernatant from the LTBMC as described [23]. We expressed its units (U) as colonies/ml HS or as colonies/ml culture supernatant. IL-6 Bioassay IL-6 levels in the HS or culture supernatants were determined using the IL-6-dependent mouse-mouse 7TD1 hybridoma cell line (Batch #F-10658; American Type Culture Collection; Rockville, MD) [24] and the MTT colorimetric assay [25] for cell growth and survival. Briefly, 4 × 103 7TD1 cells/well of a 96-well flat-bottom microtiter plate were cultured with a series of dilutions of LTBMC supernatants in a final volume of 100 μl color-free RPMI 1640 (Sigma), supplemented with 5 × 10–5 molar of 2-mercaptoethanol (Sigma), and 10% fetal bovine serum (Sigma, Cell Culture). After 46 to 48 h incubation at 37°C in a humidified atmosphere containing 5% CO2, 25 μl of MTT solution (5 mg/ml dissolved in phosphate-buffered solution and filtered prior to use) were added for 3 h. Then, 130 μl isopropylalcohol were added to each well and vigorously mixed to dissolve the dark blue crystals, followed by 7 min centrifugation at 2,000 rpm. 180 μl were carefully transferred from each well to a new microtiter plate and the optical density was read with a microtiter reader at 570 nm wavelength. IL-6 was determined from a parallel set-up standard curve with known concentrations of recombinant mouse IL-6 (rIL-6; Upstate Biotechnology Inc.; Lake Placid, NY). The IL-6 concentrations were calculated from the best fitting curve of logistic dose response equations by using nonlinear curve fitting software (TableCurve; Jandel Scientific) and Math Transform (SigmaPlot). Statistical Analysis The results reported are expressed as mean ± standard error of means (±SE). Data sets were compared using the Student's independent t-test and Pearson's correlation coefficient to identify significant differences; p values <0.05 were considered to be significant. Results Kinetics of IL-6 and CSA Concentrations in Murine LTBMC In hematopoietically active LTBMC grown in plastic flasks, IL-6 bioactivity and CSA significantly increased following medium change (Fig. 1). The concentration of CSA increased from 0 to a maximum concentration of 350 ± 25 U/ml 24 h later (p < 0.01). IL-6 concentration increased from 1,859 ± 391 mU/ml at time 0 ( = medium change) to 26,026 ± 7,690 mU/ml 12 h later. In other experiments, at days 2, 3 and 4 following medium change, we measured a continuous slow decrease of CSA and IL-6 to the baseline levels (data not shown). Figure 1. Open in new tabDownload slide Kinetic of absolute values of CSA and IL-6 bioactivity in the supernatant of LTBMC with HS No. 2043 grown in plastic flasks immediately prior to and following medium change. Mean values (± SE) of three flasks for CSA and six flasks for IL-6 are given. Figure 1. Open in new tabDownload slide Kinetic of absolute values of CSA and IL-6 bioactivity in the supernatant of LTBMC with HS No. 2043 grown in plastic flasks immediately prior to and following medium change. Mean values (± SE) of three flasks for CSA and six flasks for IL-6 are given. IL-6 Bioactivity and CSA in Different Horse Sera In the development of the bioassay for IL-6 in the supernatant of LTBMC, we noted measurable levels of this cytokine in the HS we employed to initiate the cultures. The level varied markedly between the various HS lots (Fig. 2A). IL-6 bioactivity ranged from a low of 165 ± 25 mU/ml HS to a maximum concentration of 796 ± 151 mU/ml. We also noted varying concentrations of CSA in these lots of HS ranging from a low of 3 ± 3 U/ml to a high of 140 ± 23 U/ml (Fig. 2B). The three selected HS lots (HS-high = lot No. 043; HS-medium = lot No. 302; and HS-low = lot No. 512) contained significantly different concentrations of both CSA and IL-6. There was no correlation between the measured concentrations of IL-6 and CSA. Figure 2. Open in new tabDownload slide IL-6 bioactivity (A) and CSA (B) in 10 different pretested HS lots. CSA concentrations were measured employing the CFU-GM assay and IL-6 levels by employing the factor-dependent mouse-mouse 7TD1 hybridoma cell line and the MTT colorimetric assay, as described in Materials and Methods. *Statistical significance is described in the Results section. Figure 2. Open in new tabDownload slide IL-6 bioactivity (A) and CSA (B) in 10 different pretested HS lots. CSA concentrations were measured employing the CFU-GM assay and IL-6 levels by employing the factor-dependent mouse-mouse 7TD1 hybridoma cell line and the MTT colorimetric assay, as described in Materials and Methods. *Statistical significance is described in the Results section. We felt it essential that we determined the specificity of our IL-6 bioassay for LTBMC supernatant and HS. This was done by the addition of anti-IL-6 monoclonal antibody rat-antimouse (Upstate Biotechnology Inc.) to the sample being measured. Antibody addition completely neutralized IL-6 bioactivity in cell-free supernatant from LTBMC. Specificity was further confirmed by demonstrating neutralization of recombinant murine IL-6 added to supernatants. By contrast, IL-6 bioactivity in HS was not neutralized by this specific antibody (Fig. 3). Figure 3. Open in new tabDownload slide Effect of antimouse IL-6 monoclonal antibody on the IL-6 bioassay. The antimouse IL-6 monoclonal antibody was able to inhibit totally and dose-dependently the activity of both rIL-6 (1 U/well; •) and IL-6 bioactivity in the supernatant of murine LTBMC (5%;▪). In contrast, the measured IL-6 bioactivity in HS (here HS-high 5%/well;△) was not inhibited by the anti-IL-6 antibody. Figure 3. Open in new tabDownload slide Effect of antimouse IL-6 monoclonal antibody on the IL-6 bioassay. The antimouse IL-6 monoclonal antibody was able to inhibit totally and dose-dependently the activity of both rIL-6 (1 U/well; •) and IL-6 bioactivity in the supernatant of murine LTBMC (5%;▪). In contrast, the measured IL-6 bioactivity in HS (here HS-high 5%/well;△) was not inhibited by the anti-IL-6 antibody. Stromal Formation and Hematopoietic Activity of LTBMC We then determined if the IL-6 or CSA concentration of the HS affected its ability to support myelopoiesis in LTBMC. Sera containing less than 270 mU/ml IL-6 were unable to support the development of hematopoietically active LTBMC. By contrast, the concentration of CSA in the HS did not affect the ability of the HS to support myelopoiesis in LTBMC. Figure 4 compares myeloid cell production in the supernatant of LTBMC initiated with HS containing a high concentration of IL-6 (HS-high: 796 ± 151 mU/ml; lot No. 043), a medium concentration (HS-medium: 391 ± 83 mU/ml; lot No. 320) and a low concentration (HS-low: 170 ± 43 mU/ml; lot No. 512). Myeloid cell production in the supernatant was significantly higher in LTBMC initiated with HS-high compared to HS-medium, and was minimal in cultures supplemented with HS-low (Fig. 4). Already one week after culture initiation, LTBMC supplemented with HS-high and HS-medium contained significantly more myeloid cells in the supernatant than HS-low; the absolute counts were 2.18 ± 0.24 × 106 cells (p < 0.01), 1.89 ± 0.09 × 106 cells (p < 0.01), 0.84 ± 0.15 × 106 cells, respectively. One week after culture initiation, myeloid progenitors in the supernatant measured by employing the CFU-GM assay were not significantly different between the cultures containing different HS; the mean values ranged between 2,500 and 3,800 CFU-GM per flask. Cultures initiated with HS-high or HS-medium formed the typical morphologic network-like stromal pattern containing hematopoietically active areas. However, stromal formation was impaired in LTBMC initiated with HS-low (Fig. 5). Figure 4. Open in new tabDownload slide Hematopoietic activity of LTBMC containing different HS. The results are produced from simultaneous set up of LTBMC. Weekly measurement of myeloid cell production in the supernatant of cultures containing HS-high (▪)(lot No. 043), HS-medium (▴) (lot No. 302), and HS-low (▾) (lot No. 512). The mean values (± SE) of three individual flasks for each result are given. *p < 0.05. Figure 4. Open in new tabDownload slide Hematopoietic activity of LTBMC containing different HS. The results are produced from simultaneous set up of LTBMC. Weekly measurement of myeloid cell production in the supernatant of cultures containing HS-high (▪)(lot No. 043), HS-medium (▴) (lot No. 302), and HS-low (▾) (lot No. 512). The mean values (± SE) of three individual flasks for each result are given. *p < 0.05. Figure 5. Open in new tabDownload slide Macroscopic comparison of representative fixed and stained stromal layers of cultures containing HS-high (flask 1 at the left side), HS-medium (flask 2 in the middle), and HS-low (flask 3 at the right side). Flask 1 shows the typical stromal layer of a hematopoietically active LTBMC with the fine stromal network formed by cell cords and cell-free cavities between. Flask 2 consists of a stromal network with thicker cords and some dense areas. In flask 3 there is no network formation, the darker areas predominantly consist of fat-containing cells, whereas fibroblastoid cells and macrophages are in a loose arrangement to form pale areas. Figure 5. Open in new tabDownload slide Macroscopic comparison of representative fixed and stained stromal layers of cultures containing HS-high (flask 1 at the left side), HS-medium (flask 2 in the middle), and HS-low (flask 3 at the right side). Flask 1 shows the typical stromal layer of a hematopoietically active LTBMC with the fine stromal network formed by cell cords and cell-free cavities between. Flask 2 consists of a stromal network with thicker cords and some dense areas. In flask 3 there is no network formation, the darker areas predominantly consist of fat-containing cells, whereas fibroblastoid cells and macrophages are in a loose arrangement to form pale areas. Stromal Function of LTBMC After five weeks of culture, we measured supernatant CSA and IL-6 concentration in LTBMC initiated with HS-high, HS-medium and HS-low, respectively (Figs. 6A and 6B). Measurements were obtained at the time of medium change (time 0, corresponding to the “spent medium” during the preceding week) and 18 h later. The 18-h time point was chosen because we found that IL-6 peaked at 12 h and CSA at 24 h after medium change (Fig. 1). At medium change, LTBMC supplemented with HS-high contained significantly higher CSA concentrations than HS-low, averaging 82 ± 22 U/ml and 16 ± 1 U/ml (p = 0.04), respectively (Fig. 6A). CSA production following medium change was significantly higher in HS-high and HS-medium cultures compared to HS-low. Similarly, baseline IL-6 concentration was significantly higher in HS-high compared to HS-low, averaging 659 ± 87 mU/ml and 278 ± 100 mU/ml, respectively (Fig. 6B). Post-medium change LTBMC containing HS-low showed no significant IL-6 increase, whereas in cultures supplemented with HS-medium and HS-high, a significant increase in IL-6 bioactivity occurred. Figure 6. Open in new tabDownload slide Stromal function of LTBMC containing different HS. CSA (A) and IL-6 bioactivity (B) in the supernatant of five-week-old LTBMC. The supernatant was collected at medium change (= 0 h) and 18 h after medium change to determine CSA by using the CFU-GM assay and IL-6 bioactivity by using an IL-6-dependent cell line. The statistical significance for graph A and B is described in the Results section. Figure 6. Open in new tabDownload slide Stromal function of LTBMC containing different HS. CSA (A) and IL-6 bioactivity (B) in the supernatant of five-week-old LTBMC. The supernatant was collected at medium change (= 0 h) and 18 h after medium change to determine CSA by using the CFU-GM assay and IL-6 bioactivity by using an IL-6-dependent cell line. The statistical significance for graph A and B is described in the Results section. Effects of Anti-IL-6 and rIL-6 Addition on LTBMC To determine if IL-6 was important in stromal cell development and myelopoiesis, we examined the effects of adding 10 μg/ml of anti-IL-6 rat-antimouse monoclonal antibody to LTBMC containing HS-high. Antibody was added at the time of culture initiation and at weekly medium change for the first three weeks of culture. The addition of antibody significantly suppressed myeloid cell production (Fig. 7). Although affecting myeloid cell production, the addition of antibody had no effect on the morphologic appearance of the marrow stroma. The importance of IL-6 in the initiation of myelopoiesis was confirmed by showing that the addition of 100 U/ml rIL-6 to cultures initiated with HS-low caused a significant increase in cumulative myeloid cell production (Fig. 8). Despite improving myeloid cell production, the addition of rIL-6 did not ameliorate the stromal morphology of cultures initiated with HS-low. The addition of 10 U rIL-6 to cultures initiated with HS-low had minimal effects on myeloid cell production in LTBMC. Figure 7. Open in new tabDownload slide Weekly myeloid cell counts in the supernatant of LTBMC containing HS-high (•) and additionally 10 μg/ml anti-IL-6 monoclonal antibody (♦). The anti-IL-6 antibody was added during the first three weeks after culture initiation. The mean values (± SE) of three individual flasks for each result are given. *p < 0.05. Figure 7. Open in new tabDownload slide Weekly myeloid cell counts in the supernatant of LTBMC containing HS-high (•) and additionally 10 μg/ml anti-IL-6 monoclonal antibody (♦). The anti-IL-6 antibody was added during the first three weeks after culture initiation. The mean values (± SE) of three individual flasks for each result are given. *p < 0.05. Figure 8. Open in new tabDownload slide Weekly cumulative myeloid cell production in the supernatant of LTBMC containing only HS-low (•) and cultures supplemented with 10 U/ml (△) or 100 U/ml (♦) rIL-6. The mouse rIL-6 was added each time at medium change during the first three weeks after culture initiation. The mean values (± SE) of five individual flasks for each result are given. *p < 0.05. Figure 8. Open in new tabDownload slide Weekly cumulative myeloid cell production in the supernatant of LTBMC containing only HS-low (•) and cultures supplemented with 10 U/ml (△) or 100 U/ml (♦) rIL-6. The mouse rIL-6 was added each time at medium change during the first three weeks after culture initiation. The mean values (± SE) of five individual flasks for each result are given. *p < 0.05. Discussion Since its introduction in the 1970s by Dexter et al. [20], numerous studies have attempted to define factors involved in the regulation of hematopoiesis in LTBMC. For unexplained reasons, HS is required for stromal cell development and the initiation and maintenance of myelopoiesis. It is well known that the ability to support this culture system varies greatly in various lots of HS [26]. For this reason, extensive pretesting is required to identify an ideal HS. In this report, we provide evidence that IL-6 concentration in the HS may be important. In recent years, a great deal of attention has focused on the critical role played by IL-6 in marrow stromal and hematopoietic function [2, 3, 5, 9, 27], in the pathophysiology of multiple myeloma [28-31], and in the development of osteoporosis [11, 32, 33]. There is compelling evidence that this cytokine plays an important role in the regulation of stromal cell function and hematopoiesis. Possible actions include a role in differentiation and survival of hematopoietic stem cells [12-17, 27], interactions between stromal fibroblasts and fat cells [2] and the suppression or stimulation of other cytokines. Studies on different stromal cell lines [2, 4, 34, 35] have shown that antiadipogenic cytokines, such as IL-1, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta and other substances, induce IL-6 on the transcription level and increase IL-6 protein release. In mice, IL-1 and TNF induced IL-6 [36, 37], and IL-6 was a requisite mediator of both IL-1 and TNF-α-induced hematopoietic recovery after lethal irradiation [38]. Neta et al. concluded that interdependence and synergistic interactions of these three cytokines are mandatory for many of their biological effects [38]. In contrast, IL-6 suppressed IL-1 and TNF production in human blood mononuclear cells [39], and TNF production in cultured monocytes, U937 cells and mice [40]. In vitro, IL-6 reversibly inhibited the proliferation of macrophage progenitor cells as well as more differentiated macrophages [41]. The exact mechanisms of action remain ill-understood. IL-6 is important in LTBMC as studies have shown that myelopoiesis is suppressed when anti-IL-6 is added to culture. In this report, we identify extremely high concentrations of IL-6, both in the basal state and following medium change in five-week-old LTBMC grown in plastic flasks and employing bone marrow cells from four- to six-month-old female C57BL/6 mice. Our values were more than 10 times higher than reported by Hudak et al. [4] measured at the third and eighth week after culture initiation with an enzyme-linked immunosorbent assay in the supernatant of LTBMC from CBA/J mice using 10% HS and 10% fetal bovine serum. In contrast to the LTBMC system employed by Hudak et al., our LTBMC were primary hematopoietically active cultures without reseeding of bone marrow stem cells at the third week. This difference cannot be explained by lack of assay specificity. We determined the specificity of our IL-6 bioassay by demonstrating a dose-dependent inhibition of supernatant IL-6 or rIL-6 bioactivity by the simultaneous addition to the assay of an anti-IL-6 monoclonal antibody rat-antimouse. The activity of this antibody was very specific against mouse IL-6 and had no crossreactivity with horse IL-6, as demonstrated in Figure 3, and human IL-6 according to information from the manufacturer. The role of IL-6 on the formation and function of LTBMC was studied by adding a monoclonal antibody against murine IL-6 during the first three weeks. The formation of the adherent stromal layer was not affected histologically. The most obvious explanation is that horse IL-6 was not neutralized by the antimouse monoclonal antibody, as has been shown by the IL-6 bioassay (Fig. 3), and therefore was able to support the formation of the typical murine stromal layer. Unfortunately, we could not find a neutralizing antibody against horse IL-6. Therefore, we could not finally exclude that IL-6 does not play an essential role forming the stromal layer. However, the monoclonal antibody against murine IL-6 significantly suppressed the myeloid cell count in the supernatant, but not completely. The significant decrease of granulocytes in the supernatant continued during the following four weeks after removal of the antibody and then recovered. A similar phenomena was seen by treating LTBMC with anti-colony-stimulating factor antibodies. The antibody could not block myelopoiesis [42]. It seems that in the microenvironment of the adherent stromal layer, the cytokine-cell interaction is not neutralized, which may be due to protection via extracellular matrix substances. Therefore, the stroma adherent more primitive myeloid progenitors survive and myelopoiesis is recovered after omitting the antibody. In human LTBMC, Verfaillie demonstrated that primitive progenitors are conserved to a greater extent under direct contact with stroma compared to physically separated conditions [43]. In contrast to other groups [4], we initiated primary LTBMC without recharging the cultures with fresh bone marrow cells three weeks after culture initiation. It has been shown that IL-6 is a survival factor for hematopoietic stem cells [27] and IL-6 receptors are present on these cells [19]. An additional important factor to produce primary hematopoietic active LTBMC may be a low concentration of CSA in the HS, preventing hematopoietic stem cells to differentiate immediately after culture initiation. Our results indicate that IL-6 plays an essential role in the formation and function of murine LTBMC. Furthermore, the culture conditions are very crucial to the kind of study system being used. In addition, these data show that it is very important to define exactly the system used prior to functional studies in any long-term cultures, and to evaluate carefully the time points at which one is measuring a functional parameter such as cytokine expression. It is also clear that IL-6 is only one of the essential factors in “pretested HS” required to produce primary hematopoietic active LTBMC. Acknowledgements The authors would like to thank Virginia Fitzhugh for her excellent technical assistance. This work was supported by Grants AG 07473 and AG 09458 from the National Institutes of Health, by the Arkansas EPSCoR program funded by the National Science Foundation, Arkansas Science and Technology Authority and the University of Arkansas for Medical Sciences and by funds from the Department of Veterans Affairs. Simon P. Hauser was partly supported by a grant from the Swiss Cancer League, Bern, Switzerland. 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TI - The Pivotal Role of Interleukin 6 in Formation and Function of Hematopoietically Active Murine Long-Term Bone Marrow Cultures
JF - Stem Cells
DO - 10.1002/stem.150125
DA - 1997-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-pivotal-role-of-interleukin-6-in-formation-and-function-of-EFeOctJOSm
SP - 125
EP - 132
VL - 15
IS - 2
DP - DeepDyve
ER -