supplementation with hematopoietic growth factors In ...

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

1991 78: 3155-3161

In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors RM Schwartz, SG Emerson, MF Clarke and BO Palsson

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In Vitro Myelopoiesis Stimulated by Rapid Medium Exchange and Supplementation With Hematopoietic Growth Factors By Richard M. Schwartz, Stephen G. Emerson, Michael F. Clarke, and Bernhard 0. Palsson We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3). granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, OM-CSF, and Epo were added t o the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led t o a greater thar12.5-fold expansion in terms of the number of nonadherent cells produced over a 6- t o E-week

period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach t o hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro. 8 1991 by The American Society of Hematology.

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(SCF; c-kit ligand) have been reported to synergize with the multipotential activities of GM-CSF and IL-3.’”’’ Other HGFs, such as erythropoietin (Epo) and macrophage-CSF (M-CSF), have shown biologic activity primarily on a single progenitor cell population.” The hematopoietic microenvironment and locally produced HGFs are believed to direct hematopoiesis to produce specific hematopoietic cells in vivo. Therefore, the longevity and productivity of LTBMCs may be limited by insufficient production of HGFs by hematopoietic and stromal cells ex vivo in addition to suboptimal medium exchange rates. Therefore, we asked whether the combination of rapid culture medium exchange with the addition of the human HGFs IL-3, GM-CSF, and Epo would substantially increase the proliferation and differentiation of human LTBMCs. The results presented here show that increased medium exchange with HGF addition significantly improves hematopoietic culture proliferation and hematopoietic cell differentiation over increased medium exchange or HGF addition alone.

UCCESSFUL RECONSTITUTION of prolific human bone marrow ex vivo has long been desired as it would enable a broad range of basic biologic and clinical studies. Such cultures would have great potential value for the analysis of normal and leukemic hematopoiesis, experimental manipulation of bone marrow, eg, for gene therapy, autologous and heterologous transplantation, and, if successfully scaled up, for the large-scale production of blood cells. However, long-term human bone marrow cultures (LTBMCs) have been largely disappointing. Unlike similar cultures from species such as the tree shrew,’ mouse?’ and rat,6 human liquid marrow cultures fail to produce a significant number of hematopoietic progenitor (clonogenic) cells. Further, the cumulative number of mature cells produced is less than the number of cells seeded initiall~.~ The production of progenitor and mature cells decreases exponentially with time and cell production typically ceases by 8 to 10 In vivo, active hematopoiesis shows no evidence of senescence over a person’s lifetime. Therefore, the failure to reconstitute prolific bone marrow ex vivo is most likely technical and due to deficiencies of the culture systems used. Using the in vivo hematopoietic conditions as a guide, we have already shown that the medium exchange rate used in human bone marrow cultures is suboptimal, and that increasing the medium exchange rate to 50% medium and serum replacement per day results in substantially increased culture effi~iency.~.“ Hematopoiesis in vivo is directed by a complex interaction of hematopoietic growth factors (HGFs), which are known to act on stromal, progenitor, and stem cells.”.’* These HGFs are critical for the maintenance of hematopoietic differentiation and have been classified by their ability to support the growth and differentiation of hematopoietic progenitor cells in vitro. Several of the HGFs, such as interleukin-3 (IL-3) and granulocyte-macrophage colonystimulating factor (GM-CSF), are known to be multipotent, having demonstrated stimulatory activity on several different progenitor cell p o p ~ l a t i o n ~ . ~Other ~ ’ ~ ~HGFs ’’ such as IL-6, granulocyte-CSF (G-CSF), and stem cell factor Blood, Vol78, No 12 (December 15). 1991: pp 3155-3161

MATERIALS AND METHODS

Cells. Human bone marrow cells were obtained, after informed consent, from heparinized aspirates of the iliac crest bone marrow under a protocol approved by the University of Michigan Human

From the Departments of Chemical Engineering, Internal Medicine, and Pediatrics, and the Program in Cell and Molecular Biology, The University of Michigan, Ann Arbor. Submitted June 7,1991; acceptedAugust 20,1991. Supported by Aastrom Biosciences Inc, Ann Arbor, MI. Address reprint requests to Stephen G. Emerson, MD, PhD, Department of Intemal Medicine and Pediatrics, The University of Michigan, MSRB 4 Room 5510B, 1150 W Medical Center Dr, Ann Arbor, MI 48109. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1991 by The American Society of Hematology. 0006-4971191 17812-0001$3.0010 3155

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. 3156

Investigation Committee. The bone marrow &s separated by a Ficoll-Paque (Pharmacia, Uppsala, Sweden) deffsity-gradientcentrifugation and the low-density cells ( < 1.077 g / m 3 )were collected and washed three times with Iscove’s Modified Dulbecco’s Medium (IMDM, Cat. No. 430-2200; GIBCO Laboratories, Grand Island, NY).The cells were counted between the second and third washes. The cells were then seeded onto 6-well tissue culture plates (Costar, Cambridge, MA; No. 3406) or collagen-coated 6-well plates (rat tail !ype 1 collagen, Biocoat, Collaborative Research, Inc, Bedford, MA; Cat. No. 40400) in duplicate 5 x lo6cells/mL at 1.5 mL/well. Culture medium. The medium used was IMDM containing 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 10% horse serum (Hyclone Laboratories), 1% penicillin/streptomycin (Sigma, St Louis, MO; 10,000U/mL penicillin G and 10 mg/mL streptomycin, Cat. No. P3539), and mol/L hydrocortisone (17-Hydroxycorticosterone, Sigma; Cat. No. H0888). HGFs. Due to the frequent culture supplementation via rapid medium exchange, HGFs were added to the medium at approximately 1/20 of the concentrations found to promote maximal 21 The concentrations used colony formation in clonal were 1 ng/mL of IL-3 (a gift from Genetics Institute, Cambridge, MA), 1 ng/mL of GM-CSF (Amgen Biologicals, Thousand Oaks, CA, Cat. No. 13050), 0.1 U/mL of Epo (Terry Fox Labs, Vancouver, Canada), 10 U/mL IL-6 (Collaborative Research Inc), and 0.1 ng/mL G-CSF (Amgen Biologicals). Hematopoieticprogenitor cell assay and morphologic assays. Nonadherent hematopoietic cells removed from culture were counted and plated at 1 x lo5cells/mL or fewer cells in methylcellulose.” GM-CSF and Epo were added to the methylcellulose at 20 ng/mL and 2 U/mL, respectively. The cells were plated in 24-well plates at 0.25 mL/well and incubated at 37°C for 14 days. The colonies were then counted under an inverted microscope and colonies greater than 50 cells were scored as GM colony-forming units (CFU-GM), erythroid burst-forming unit (BFU-E), or granulocyte erythroid megakaryocyte macrophage colony-forming unit (CFU-GEMM). Aliquots of removed cells were cytocentrifuged after counting, air-dried, stained with Wright-Giemsa, and differential cell counts performed. LTBMC conditions. The cultures were incubated at 37°C in a humidified 5% C02/95%air atmosphere and mediumchanged at a rate of 50% daily medium exchange. During the first week in culture, all cells removed during the daily medium exchange were centrifuged and returned to the original wells. After the first week in culture, 50% of the total nonadherent cells were removed from the cultures on a biweekly basis during the medium exchange, mononucleated cells counted, and fresh medium returned to the wells. The remaining 5 days per week when the cells were not counted, 50% of the medium was removed from each of the culture wells and replaced with fresh medium, the removed medium was centrifuged, the medium decanted from the cell pellet, and the cells returned to their original wells. Statistical analysis. The probability of significant differences between groups of cultures was determined by comparing the normalized cumulative cell production values from the rapid medium exchanged cultures supplemented with HGFs with the matched untreated control cultures using a paired t-test. Statistical significance was taken at the 5% level. There were no statistical differences between matched rapid medium exchanged LTBMCs cultured on tissue culture plastic and type I rat tail collagen at the 5% level. Therefore, the data for the plastic and collagen matrix were combined for presentation in this and all other figures and statistical analysis was performed on the combined data.

SCHWARTZ ET AL

RESULTS

Kinetics of cell production in rapid medium exchanged growth factor-supplemented LTBMCs. As a first test of the hypothesis that the longevity and productivity of LTBMCs is limited by insufficient production of HGFs, we maintained rapidly exchanged ex vivo bone marrow cultures that were supplemented with IL-3 or Epo. In these cultures, 50% of the medium was removed daily and replaced with an equal volume of fresh medium supplemented with IL-3 or Epo. The cells removed were then centrifuged, the medium decanted and discarded, the cells resuspended, and the cells returned to the original cultures. IL-3 and Epo individually enhanced the cell productivity of rapidly exchanged LTBMCs (Fig 1A). The cultures containing Epo alone initially had a high cell production rate due to substantial terminal erythroid differentiation. However, by week 4 erythropoiesis had ceased and the cell production rate had decreased to the level of the control cultures. IL-3 and Epo induced an average increase in nonadherent cell production over controls throughout the 18 weeks of culture of 175% and 173%, respectively (Fig 1B). Combinations of growth factors proved to be more effective in increasing the nonadherent cell production rate (Fig 2A). The highest rate of cell production was observed

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Weeks Fig 1. (A) Biweeklycell production of LTBMCs supplemented with IL-3 or Epo and (8)cell production as a percent of unsupplemented controls for the same data. Shown is the average number of cells removed per culture (with only the +SEM for clarity) for untreated control ( W 1, IL-3 (O),or Epo (0) supplemented cultures.

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. RAPID PERFUSION STIMULATES MYELOPOIESIS

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Weeks Fig 2. (A) Biweekly cell production of LTBMCs supplemented with IL-3 Epo, IL-3 GM-CSF, or IL-3 GM-CSF Epo and (B) cell production as a percent of unsupplemented controls for the same data. Shown is the average number of cells removed per culture (with only the +SEM for clarity) for untreated control ( W ), IL-3 + Epo (O), IL-3 GM-CSF (O), or IL-3 GM-CSF Epo (0) supplemented cultures.

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for the combination of IL-3 + GM-CSF + Epo. These cultures produced approximately 25% of the number of cells inoculated biweekly during the first 6 weeks in culture and had an average 4.8-fold increase in nonadherent cell production over controls during weeks 2 through 8 (Fig 2B). The combination of IL-3 + GM-CSF produced an average 3.5-fold increase in nonadherent cells as compared with controls through week 8. In separate experiments, adding either IL-6 or G-CSF to the combination of IL-3 + GM-CSF + Epo did not improve the nonadherent cell production rate, but instead resulted in cell production rates indistinguishable from the cultures containing the combination of IL-3 + GM-CSF (data not shown). In all cases, the stimulatory effect on cell production induced by the addition of HGFs was maximal between weeks 0 and 8 and after 14 weeks in culture. After 14 weeks in culture, HGF supplementation caused cell production to stabilize or slowly decrease whereas cell production in the unsupplemented controls continued to decrease. The combinations of HGFs lead to high absolute numbers of nonadherent cells produced in rapidly exchanged LTBMCs. The productivity of the cultures can be shown by

comparing the cumulative number of cells produced over time (Zr=lC,, with C, being the number of nonadherent cells collected at time i), relative to the number of cells inoculated (C,) by plotting the ratio (Z:=, C,/C,) as a function of time. When this ratio exceeds unity, a culture has produced more cells than were inoculated and the culture has led to an expansion in cell number. The combination of IL-3 + GM-CSF + Epo induced cumulative cell production that was more than threefold greater than the number of cells inoculated (Fig 3). The cell production rate was the highest during the first 6 weeks in culture, during which time the cultures produced approximately as many cells as were inoculated every 2 weeks. This maximum cell production rate was 15% of the estimated in vivo bone marrow cell production rate where 50% of the myeloid cell mass is generated daily. The combination of IL-3 + GM-CSF resulted in a more than twofold expansion in cell number and at rates comparable with the combination of IL-3 + GM-CSF + Epo during weeks 3 through 7 in culture. Untreated rapidly exchanged (50% daily medium exchange) and slowly exchanged (50% medium exchange biweekly) control cultures not supplemented with HGFs produced approximately 1 and 0.37 times the number of cells inoculated after 18 weeks, respectively (Fig 3). More importantly, more than half of all cells removed from these unsupplemented cultures came from the first two samplings, indicating that many of these cells were from the original inoculum and that supplementation of the cultures with HGFs is required to induce significant cycling of progenitor and stem cells. Morphologic analysis of nonadherent cells. The addition of multiple HGFs also increased the variety of myeloid cells

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Fig 3. Normalized cumulative cell production in rapidly exchanged IL-3 (A), LTBMCs supplemented with HGFs; untreated controls (-), Epo (O), IL-3 Epo (0). IL-3 + GM-CSF (0). and IL-3 GM-CSF Epo (0) for 18 weeks in culture and LTBMCs exchanged at 50% biweekly 1-1 but otherwise treated similarly t o the rapidly exchanged untreated control cultures. Data shown are the cumulative number of cells removed during the biweekly sampling divided by the number of cells initially plated in each culture with the cumulative kSEM shown at week 18. The increase in cell production in cultures supplemented with IL-3 + OM-CSF Epo, IL-3 GM-CSF, and IL-3 Epo was statistically significant at the 1%level. IL-3 alone induced significant increases in the cell production at the 5% level, while Epo alone did not induce a significant increase in the cell production.

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SCHWARTZ ET AL

produced in the cultures. The control cultures produced nonadherent cells that were predominately macrophages after week 3 in the culture. Production of erythroid cells decreased rapidly with few erythroid cells detected after week 5 (Fig 4). The cultures containing Epo (Epo alone, IL-3 Epo, and IL-3 GM-CSF Epo) produced a transient increase in erythroid cell production, with a high percentage (55% to 75%) of nonadherent cells being erythroid through week 3. When IL-3 Epo f GM-CSF was present, the cultures continued to produce erythroid cells throughout the 16 weeks in culture with about 5% to 15% of the nonadherent cells being typed as erythroid (Fig 4). Thus, in the presence of IL-3 Epo, a low level of erythropoiesis was maintained throughout the culture. IL-3 f Epo led to a nonadherent cell population that was predominately (60% to 70%) late granulocytes (LGs) at week 5 (Fig 4). The percentage of LGs steadily decreased until it reached about 20% at week 18. The percentage of macrophages increased correspondingly. When GM-CSF was added to IL-3 ~fr Epo, the high percentage of LGs persisted through 18 weeks (Fig 4). Thus, the combination of IL-3 GM-CSF led to active granulopoiesis for 18 weeks in culture, and the addition of Epo maintained erythropoiesis as well. Photomicrographs of the control and IL-3 GM-CSF + Epo supplemented cultures at 5.5 weeks in

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culture (Fig 5 ) show the dramatic enhancement in culture density and variety of cells produced. Kinetics ofnonadherentprogenitor cellproduction. Progenitor cell production increased with the addition of multiple HGFs. The production of CFU-GMs in the untreated controls was prolonged and steady for over 18 weeks (Fig 6A), which is consistent with our earlier results in rapidly exchanged LTBMC.7 CFU-GM produced in the IL-3 GM-CSF and IL-3 Epo GM-CSF cultures was approximately 10-fold higher than controls during weeks 3 through 5, while total CFU-GM production was greater than twofold higher than controls through 17 weeks (Fig 6B). BFU-E production in human LTBMC has been reported to be low and cease quickly! The rapidly exchanged, untreated controls exhibited a rapid decrease in BFU-E production, although low levels of BFU-E were produced through 17 weeks in culture (Fig 7A). The addition of Epo alone did not significantly influence the number of BFU-Es produced. IL-3 alone induced a mild short-lived stimulation of BFU-E production in weeks 3 through 5. On the other hand, IL-3 plus either Epo or GM-CSF induced a 10- to 30-fold elevation of nonadherent BFU-E levels compared with that of controls during weeks 3 through 5 of culture (Fig 7B). Total BFU-E production was 1.7-fold higher in the IL-3 + Epo and IL-3 + GM-CSF than the unsupplemented controls (Fig 7B).

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DISCUSSION

Hematopoiesis is a highly dynamic and complex process of cell differentiation that is influenced by a number of biologic and physical variables. Heretofore, efforts to reconstitute a functional hematopoietic process ex vivo have met with limited success. The LTBMC systems used in most laboratories provide for conditions that are very different from those believed to prevail in vivo. The data presented here support the hypothesis that mimicking the physiologic bone marrow medium exchange rate in vitro combined with supplementation with picomolar quantities of hematopoietic growth factors can lead to active LTBMCs and bone marrow expansion ex vivo. Slowly exchanged human LTBMCs produce fewer number of myeloid cells than the number inoculated into culture, indicating rapid failure of the culture microenvironment. Increased medium exchange has previously been shown to provide an LTBMC microenvironment that supports increased myelopoiesis compared with slowly exchanged LTBMCS.~The unsupplemented rapidly exchanged control cultures in this previous study produced approximately as many cells as were inoculated through 18 weeks in culture. However, supplementation of these rapidly exchanged LTBMCs with picomolar quantities of HGFs leads to significantly increased myeloid cell production over unsupplemented controls. Our optimum cultures, including IL-3 + GM-CSF Epo, induced stable nonadherent myeloid cell production for 6 weeks in culture where the cultures produced approximately 25% of the number of cell inoculated biweekly. This note of cell production is a significant improvement over our unsupplemented control cultures and other reported cultures that exhibit a decrease in cell number from culture initiation.'

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Fig 4. Cell typing data for averaged replicate samples eSEM: control, Epo, IL-3, IL-3 Epo, IL-3 GM-CSF, IL-3 GM-CSF Epo. Data in each panel are shown as the percentage of erythroid cells (A), granulocytes (mature granulocytes and bands ( O ) ,immature granuloand macrophages cytes (metamyelocytes and less mature cells (0).

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Fig 5. Photomicrographs of the LTBMCs at 5.5 weeks in culture (originalmagnification x25). (A) Untreated control culture shows a myeloid colony on the stroma, while (B) the IL-3 GMCSF Epo supplemented culture shows the dense cellularity of the culture with many dark erythroid cells.

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The variety of cells produced in rapidly exchanged LTBMCs was significantly affected by HGF addition. Supplementation of the LTBMCs with IL-3 induced increased granulocyte production through 16 weeks in culture, suggesting a direct effect of IL-3 on granulopoiesis. Supplementation with Epo induced a rapid burst in erythropoiesis during the first 4 weeks in culture, indicating its ability to induce the differentiation of erythroid progenitors that were present in the inoculum, but not to recruit myeloid progenitors into the erythroid lineage. IL-3 + Epo and IL-3 + GM-CSF + Epo induced a burst in cell production during the initial 2 to 4 weeks in culture due to differentiation of the erythroid progenitors that were present in the inoculum (similar to Epo alone), but also induced low level production of

erythroid cells throughout the 18 weeks in culture. This finding suggests that the combination of IL-3 + Epo is sufficient for recruitment of primitive myeloid cells into the erythroid lineage. IL-3 + GM-CSF and IL-3 + GM-CSF + Epo induced significant increases in granulocyte production resulting in 40% to 70% of the cells produced through week 18 in culture being granulocytic. This result differs from previous LTBMCs where granulopoiesis decreases more rapidly than does macrophage production as seen in the control, Epo, IL-3, and IL-3 + Epo cultures. Therefore, culture supplementation with both IL-3 and GM-CSF resulted in the optimum long-term stimulation of granulopoiesis in these studies. Total GM-progenitor cell production was also increased


SCHWARTZ ET AL

CSF. However, increased serum/medium exchange rates can induce production of GM-CSF by stromal cells in Therefore, increased medium exchange and addition of HGFs may also induce other HGF production such as c-kit ligand by stimulating hematopoietic or accessory cells in the c ~ l t u r e s . Therefore, ~ ~ ~ * ~ increasing the medium exchange rate may provide LTBMCs with benefits other than just increasing metabolite and decreasing waste product levels. Long-term progenitor cell production in our system indicates that primitive hematopoietic cell division and maintenance are occurring for over 10 weeks in culture. This result may have implications for use of this LTBMC system for hematopoietic gene therapy as well as for stem cell selection in vitro, both of which require long-term stem cell division and/or maintenance. The stem cell division suggested by the high level of progenitor cell production during the first several weeks in culture should theoretically allow retroviral vectors to achieve high therapeutic gene transfer levels into cycling stem cells, improving the possibility for successful gene therapy. In summary, the results indicate that modeling LTBMCs on the in vivo physiologic bone marrow environment results in significant advantages over previous LTBMCs. Our attempts to mimic the bone marrow medium exchange rate in vitro, combined with daily supplementation of LTBMCs with low levels of IL-3 + GM-CSF + Epo, induced ex vivo

Weeks Fig 6. (A) Number of CFU-GM and (B) BFU-E removed per culture in untreated controls and CSF-supplemented LTBMCs. Data are shown as untreated control ( ), Epo (O), IL-3 (A), IL-3 Epo (0). IL-3 GM-CSF (01,and IL-3 GM-CSF Epo (0). Cells were assayed for CFU-GM and BFU-E every fourth sampling from the cultures.

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with HGF supplementation of the rapidly exchanged LTBMCs. Increased CFU-GM and total cell production in the rapidly exchanged HGF-supplemented cultures suggests that there is increased cycling of stem cells or increased survival of primitive hematopoietic cell capable of producing increased numbers of myeloid progenitor cells. Nonadherent BFU-E production was stimulated by combinations of HGFs but not single HGFs. Cultures supplemented with IL-3 Epo or IL-3 GM-CSF but not Epo or IL-3 alone showed increased BFU-E production, suggesting that there is a synergistic effect of Epo and GM-CSF with IL-3 on BFU-E production. The role of Epo in this synergism could be indirect, perhaps mediated by endothelial cells that have been reported to be stimulated by Epo.** These data show significant improvements over other recent reports8which have shown that although cell production increases over untreated controls with addition of HGFs to standard slowly exchanged human LTBMCs, the increases are smaller and shorter in duration than are reported here. This discrepancy suggests that physiologic exchange stimulates hematopoiesis by a mechanism independent of, and synergistic with, the effects of IL-3, GM-CSF, or Epo. For example, results in this lab suggest that unstimulated confluent stromal layers such as would be present in LTBMCs do not produce HGFs such as GM-

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RAPID PERFUSION STIMULATES MYELOPOIESIS

expansion of human bone marrow with significant increases in CFU-GM and BFU-E production Over This system should have widespread application to the lay and manipulation of hematopoiesis in vitro, and the groundwork for further advances in the complete ex vivo reconstitution of human bone marrow.

ACKNOWLEDGMENT

We thank Jerry Caldwell for his invaluable assistance with the culture maintenance, Kelly Hardesty and Diane Giannola for their technical assistance with the progenitor cell assays, Christin Martin for her assistance with the cytospins and cell typing, and Rane Curl for his advice on statistical analysis of the data.

REFERENCES

1. Moore MAS, Sheridan APC, Allen TD, Dexter TM: Prolonged hematopoiesis in a primate bone marrow culture system: Characteristics of stem cell production and hematopoietic microenvironment. Blood 54:775, 1979 2. Dexter TM, Allen TD, Lajtha LG: Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 91:335,1977 3. Greenberger JS: Sensitivity of corticosteroid-dependent insulin-resistant lipogenesis in marrow preadipocytes of obese-diabetic (dbidb) mice. Nature 275:752,1978 4. Sakaheeny MA, Greenberger JS: Longevity of granulopoiesis in corticosteroid-supplemented continuous bone marrow culture varies significantly between 28 inbred mouse strains and outbred stocks. J Natl Cancer Inst 68:305,1982 5. Testa NG, Dexter TM: Long-term production of erythroid precursor cells (BFU) in bone marrow culture. Differentiation 9:103,1977 6. Naughton BA, Preti RA, Naughton G K Hematopoiesis on nylon mesh templates. 1. Long-term culture of rat bone marrow cells. J Med 18:219, 1987 7. Schwartz RM, Palsson BO, Emerson SG: Medium perfusion rate significantly alters the cell productivity and longevity of human bone marrow cultures. Proc Natl Acad Sci USA 88:6760,1991 8. Coutinho LH, Will A, Radford J, Schiro R, Testa NG, Dexter TM: Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture. Blood 75:2118,1990 9. Greenberger JS: Long-term hematopoietic cultures, in Methods of Hematology (vol 11). New York, NY, Churchill Livingstone, 1984, p 203 10. Caldwell J, Locey B, Clarke MF, Emerson SG, Palsson BO: Influence of medium exchange schedules on metabolic, growth, and GM-CSF secretion rates of genetically engineered NIH-3T3 cells. Biotech Prog 7:1,1991 11. Metcalf D: The Molecular Control of Blood Cells. Boston, MA, Harvard, 1988 12. Dexter TM, Garland JM, Testa NG: Colony-stimulating factors: Molecular and cellular biology, in Dexter TM, Garland JM, Testa NG (eds): Immunology (vol 49). New York, NY, Dekker, 1990 13. Bot FJ, van Eijk L, Schipper P, Lowenberg B: Effects of human interleukin-3 on granulocytic colony-forming cells in human bone marrow. Blood 73:1157,1989 14. Sieff CA, Ekern SC, Nathan DG, Anderson J W Combinations of recombinant colony-stimulating factors are required for

optimal hematopoietic differentiation in serum-deprived culture. Blood 73:688,1989 15. Lu L, Briddell RA, Graham CD, Brandt JE, Bruno E, Hoffman R: Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells. Br J Haemato1 70:149,1988 16. Ferrero D, Tarella C, Badoni D, Caracciolo D, Bellone G, Pileri A, Gallo E: Granulocyte-macrophage colony-stimulating factor requires interaction with accessory cells or granulocytecolony stimulating factor for full stimulation of human myeloid progenitors. Blood 73:402,1989 17. Migliaccio G, Migliaccio AR, Visser JWM: Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation. Blood 72:944,1988 18. McNiece IK, Langley KE, Zsebo KM: Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol19:226,1991 19. Emerson SG, Yang YC, Clarke SC, Long MW: Human recombinant GM-CSF and IL-3 have overlapping but distinct hematopoietic activities. J Clin Invest 82:1282, 1988 20. Bodine D, Kar!sson S, Nienhuis A Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci USA 86:8897,1989 21. Katayama K, Koizumi S, Ueno Y, Ohno I, Ichihara T, Horita S, Miyawaki T, Taniguchi N: Antagonistic effects of interleukin 6 and G-CSF in the later stage of human granulopoiesis in vitro. Exp Hematol 18:390,1990 22. Anagnostou A, Lee ES, Kessimian N, Levinson R, Steiner M: Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proc Natl Acad Sci USA 875978, 1990 23. Caldwell J, Palsson BO, Emerson SG: Culture perfusion schedules influence the metabolic activity and growth factor production rates of human bone marrow stromal cells. J Cell Physiol147:344, 1991 24. Zsebo KM, Wypych J, McNiece IK, Lu HS, Smith KA, Karkare SB, Sachdev RK, Yuschenkoff VN, Birkett NC, Williams LR: Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell 63:195, 1990 25. Williams DE, Eisenman J, Baird A, Rauch C, Van Ness K, March CJ, Park Is,Martin U, Mochizuki DY, Boswell H S Identitication of a ligand for the c-kit proto-oncogene. Cell 63:167, 1990