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Studies on canine bone marrow long-term culture: effect of stem cell factor
Veterinary Immunology and Immunopathology 61 Ž1998. 1–16
Studies on canine bone marrow long-term culture: effect of stem cell factor Evelin Neuner a , Michael Schumm a , Eva-Maria Schneider b, Wolfgang Guenther b, Elisabeth Kremmer a , Christiane Vogl a , c Matthias Buttner , Stefan Thierfelder a , Hans-Jochem Kolb a,b,) ¨ a
Institut fuer Immunologie, GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit, Muenchen, Germany b Institut fuer Klinische Haematologie, GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit, Muenchen, Germany c Bundesforschungsanstalt fur ¨ Viruskrankheiten der Tiere, Tuebingen, Germany Accepted 15 September 1997
Abstract Long-term culture of canine marrow cells allows in vitro studies of the hematopoietic system of the dog and characterization of early progenitor cells. Colonies of fresh marrow cells grew equally good in both agar or methylcellulose supplemented with fetal calf serum, while colonies of long-term cultures required agar-based medium containing human serum. Optimum colony growth was obtained when stem cell factor ŽSCF. and granulocyte-macrophage-colony-stimulating factor ŽGM-CSF. were used as growth stimuli of colony forming units ŽCFU.. Similar results were achieved with several cell culture media. Addition of hydrocortison to long-term cultures improved clonogenic growth of cultured cells. Addition of 2-mercaptoethanol had no effect. Strong differences were observed in long-term culture with different horse serum lots and the addition of fetal calf serum to long-term culture suppressed CFU growth of cultured cells.
Abbreviations: Avg, average; BFU-E, burst forming unit-erythroid; BSA, bovine serum albumine; CD, cluster of differentiation; CFU, colony forming units; CFU-G, colony forming unit-granulocyte; CFU-GEMM, colony forming units-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM, colony forming unitgranulocyte-macrophage; FCS, fetal calf serum; rhu-GM-CSF, recombinant human granulocyte, macrophagecolony-stimulating factor; HS, horse serum; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; LTC-IC, long-term culture-initiating cells; MC, methylcellulose; MEM, minimal essential medium; 2-ME, 2-Mercaptoethanol; MNC, mononuclear cells; PHA-LCM, phythemagglutinin-stimulated dog lymphocyte-conditioned medium; rcan-SCF, recombinant canine stem cell factor; SD, standard deviation ) Corresponding author. GSF-Institut fuer Immunologie, Marchioninistr. 25, 81377 Muenchen, Germany. Tel.: q49 89 7099 327; fax: q49 89 7099 300. 0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 2 6 - 8
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Recharging of cultures with fresh marrow cells on day 7 of culture improved CFU growth only in the following week but had little effect on the outcome. Adding SCF to long-term cultures led to differentiation of more primitive cells and destruction of the stromal layer. Investigation of purified and cultured cell populations was possible when preestablished long-term cultures as stromal layers were used. Loss of long-term culture-initiating ability could be demonstrated in this system with lineage negative marrow cells expanded ex vivo with SCF and GM-CSF. q 1998 Elsevier Science B.V. Keywords: Canine; Long-term culture; Marrow; Hematopoiesis; Stem cell factor
1. Introduction The dog provides an important animal model for studies on transplantation and gene therapy ŽLadiges et al., 1990; Schuening et al., 1991.. The long-term culture-initiating cell has been proven to be the earliest hematopoietic progenitor cell which can be detected in ‘in vitro’ assays and seems to be the equivalent of the pluripotent stem cell with repopulating ability ŽBerardi et al., 1995; Moore, 1991.. Bone marrow long-term culture mimics the hematopoietic system in vitro comprising stromal cells and hematopoietic cells of all differentiation stages. Early progenitor cells reside in close contact to stromal cells and produce differentiating progenitor cells, which are released into the supernatant. Long-term cultures can be maintained for several months as long as very early cells with the capacity of self-renewal continue to produce differentiating descendants. These committed cells can then be determined by their ability to form lineage specific colonies in semisolid medium: BFU-E Žburst forming unit-erythroid., CFU-G Žcolony forming unit-granulocyte., CFU-GM Žcolony forming unit-granulocyte-macrophage., CFU-GEMM Žcolony forming units-granulocyte, erythroid, macrophage, megakaryocyte. ŽAllen and Dexter, 1984; Dexter et al., 1977; Sutherland et al., 1994.. The amount of these colony forming cells after 5–8 weeks of long-term culture has been closely related to the number of long-term culture-initiating cells ŽLTC-IC. in the beginning ŽSutherland et al., 1990.. LTC-IC are the earliest cells to be detected in culture. They represent the in vitro equivalent of the omnipotent stem cell and until now long-term culture is the only ex vivo proof of these cells ŽMoore, 1991.. The long-term culture of marrow cells allows the study of hematopoiesis during culture. Multiple substances, for example, drugs for purging of leukemic cells, cytokines and growth factors may be tested for their action on normal stromal and hematopoietic cells ŽAihara et al., 1990; Fraser et al., 1990; Galvani and Cawley, 1990.. For the investigation of purified hematopoietic cells, a ‘two-step’ long-term culture is favorable. Here, a primary long-term culture is initiated to establish a stromal layer within 2–4 weeks and irradiated to inactivate hematopoiesis. Allogeneic hematopoietic cells separated with monoclonal antibodies and immunomagnetic systems or a cell sorter can be placed on the stromal layer and investigated for their content of early progenitor cells. In this system, cell fractions need not contain stromal cells and even small numbers of purified progenitor cells can be investigated ŽSutherland et al., 1989; Verfaillie et al., 1990..
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Long-term cultures in one- or two-step technique follow the same conditions. Therefore, we had to optimize our system for the one step culture of marrow cells by testing multiple parameters which may influence the microenvironment in culture.
2. Material and methods 2.1. Dogs In our studies, 1- to 5-year-old beagles of both sexes were used as marrow donors. These animals were raised at the GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit. All dogs were healthy, dewormed and vaccinated against distemper, leptospirosis, hepatitis, and parvovirus. 2.2. Comparison of agar- and methylcellulose-based media for determination of colony forming cells Detection of differentiating colonies requires optimum growth conditions. Several components of the culture medium were therefore investigated on fresh marrow cells first. Marrow cells were separated by density gradient centrifugation over Ficoll Ž d s 1.077. and 2 = 10 5 mononuclear cells ŽMNC. were cultured in 2 ml of medium. - Agar-based medium consisted of 0.3% agar ŽDifco Laboratories, Detroit, USA. in Mc Coy’s 5A medium supplemented with 20% heat-inactivated fetal calf serum ŽPansystems, Aidenbach, Germany., 0.75% sodium bicarbonate, 1.25% sodium pyruvate, 0.5% Eagles MEM vitamins, 1% Eagles MEM amino acids, 0.5% nonessential amino acids, 0.5% L-glutamine Žall Gibco, Eggenstein, Germany., 0.5% L-serine Ž21 mgrml. 0.2% L-asparagine Ž10 mgrml, both Merck, Darmstadt, Germany., 100 Urml penicillin and 100 m grml streptomycin ŽGibco., and 20 Urml rhu-erythropoietin ŽBoehringer, Mannheim, Germany., 200 ngrml recombinant human granulocyte, macrophage-colony stimulating factor Žrhu-GM-CSF, Essex, Munich, Germany., 1% hemin ŽSigma, Deisenhofen, Germany. and 20 ngrml recombinant canine stem cell factor Žrcan-SCF, Amgen, Thousand Oaks, USA.. - Methylcellulose-based medium consisted of 1.25% of methylcellulose ŽFluka, Neu-Ulm, Germany. in Iscove’s modified Dulbecco’s medium ŽIMDM, Gibco. supplemented with 20% heat-inactivated fetal calf serum ŽFCS, Pansystems., 1% L-glutamine ŽGibco., 1% penicillin-streptomycin ŽGibco. and 20 Urml rhu-erythropoietin ŽBoehringer., 200 ngrml rhu-GM-CSF ŽEssex., 1% hemin ŽSigma. and 20 ngrml recombinant canine stem cell factor Žrcan-SCF, Amgen.. All cultures were incubated for 14 days at 388C in a humidified atmosphere of 5% CO 2 in air in 35-mm plastic Petri dishes and colonies Ž) 50 cells. were counted thereafter.
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2.3. Comparison of culture supplements and growth stimuli In some experiments, granulocyte-macrophage-colony-stimulating factor ŽGM-CSF. was replaced with either rhu-Interleukin 6 Ž100 Urml. ŽBoehringer., 1% bovine serum albumin ŽMerck., 380 m grml transferrin ŽBehring, Marburg, Germany. or 10% phythemagglutinin-stimulated dog lymphocyte-conditioned medium ŽPHA-LCM.. PHALCM was prepared by culture of peripheral blood leukocytes from 5 young, healthy dogs in the presence of 10 ngrml PHA-M ŽSigma. and 10% normal dog serum for 4 days. 2.4. Determination of colony forming cells from long-term culture No CFU growth was observed with cells from long-term cultures in methylcellulose and FCS. We therefore performed several experiments to ascertain this observation. Cells after long-term culture were cultured in either agar or methylcellulose as described above in the presence of either 20% human AB-serum or fetal calf serum, using rcan-SCF, rhu-GM-CSF and rhu-Epo as growth stimuli. 2.5. Comparison of cell culture media for canine long-term bone marrow culture Marrow cells were centrifuged over Ficoll-Hypaque Ždensity 1.077. at 800 g for 20 min and the low-density cells were collected, washed once in phosphate buffered saline and then twice in phosphate buffered saline containing 10% horse serum ŽSigma. at 600 = g for 10 min, respectively. Mononuclear marrow cells were then cultured in 25-cm2 canted-neck flasks ŽCostar, Bodenheim, Germany. at 2 = 10 6 MNCrml in 10 ml of the following media. Ži. Basal Iscove medium ŽSeromed-Biochrom, Berlin, Germany. supplemented with 20% prescreened heat-inactivated horse serum ŽSigma., 1% L-glutamine Ž200 mM, 100 = , Gibco., 1% penicillin Ž5000 Urml. ŽGibco. and 1% streptomycin Ž5000 m grml. ŽGibco.. Žii. RPMI-1640 medium ŽGibco. containing 20% horse serum, 1% nonessential amino acids, 1% sodium pyruvate, 2% L-glutamine, 1% penicillin and 1% streptomycin. Žiii. McCoy’s 5A medium containing 20% horse serum, 0.75% sodium bicarbonate, 1.25% sodium pyruvate, 0.5% Eagles MEM vitamins, 1% Eagles MEM amino acids, 0.5% nonessential-amino acids, 0.5% L-glutamine, 0.5% L-serine Ž21 mgrml., 0.2% L-asparagine Ž10 mgrml. and 1.5% penicillin-streptomycin. Živ. IMDM Ž340 mosMrkg. according to Gibson et al. Ž1995., containing 12.5% FCS, 12.5% horse serum or 20% horse serum only. 2.6. Addition of media supplements and cytokines to long-term culture Addition of hydrocortisone has been described favorable for the long-term culture of murine ŽGreenberger, 1978., human ŽGartner and Kaplan, 1980. and canine ŽSchuening et al., 1989. marrow cells. We used Hydrocortison-21-phosphate at a concentration of 10y7 molrl in all media unless indicated otherwise.
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2-Mercaptoethanol and a-Thioglycerol is widely used for retarding oxidation in culture media. We investigated the effect of both of these substances on canine long-term culture in parallel at a concentration of 10y4 Mrl. Stem cell factor is described as a very early acting factor in hematopoiesis and therefore possibly advantageous for the long-term culture of progenitor cells ŽBernstein et al., 1991.. We added 10 ngrml rcan-SCF to long-term cultures in weekly intervals at the time of medium change. Cultures without SCF were performed in parallel. 2.7. Comparison of different sera for canine long-term culture 2.7.1. Human serum For human marrow, Sutherland et al. Ž1994. proposed equal parts of fetal calf and horse serum for optimum growth of progenitor cells in long-term culture. We investigated the effect of fetal calf serum in parallel cultures by replacing horse serum to get 0, 5 and 10% of fetal calf serum, respectively. 2.7.2. Canine serum Additional experiments were performed using 20% normal dog serum instead of horse serum. 2.7.3. Horse serum The following horse sera were tested for suitability in canine long-term culture: a1 horse serum lot no. 31P4127 ŽGibco, Gaithersburg, USA., a2 horse serum lot no. 001C ŽSeromed-Biochrom., a3 horse serum lot no. 002C ŽSeromed-Biochrom., a4 horse serum H6762, lot no. 73H0765 ŽSigma., a5 horse serum H9767, lot no. 88F-0428 ŽSigma., a6 fetal equine serum, lot no. F7763 ŽSigma., a7 horse serum H1270, lot no. 43H0691 ŽSigma. and a8 horse serum Hybrimax w , H1263, lot no. 73H0721 ŽSigma.. Long-term cultures were maintained at 378C in a humidified atmosphere of 5% CO 2 in air. At weekly intervals, non-adherent cells and medium were removed from flasks and centrifuged at 600 g for 10 min and fresh medium was added. Half of the used medium was returned into flasks. The non-adherent cell population was assayed for clonogenic growth as described. After 5 weeks, non-adherent cells were harvested and adherent cells suspended by trypsinization. An aliquot of combined non-adherent and adherent cells was then assayed for colony forming units. 2.8. Recharging of canine long-term cultures We studied a marrow ‘boost’ by recharging flasks after 1 week of culture which has been reported as favorable in respect to cell and CFU recovery after long-term culture ŽSchuening et al., 1989.. Marrow was aspirated from the dog used for establishing the stromal layer, separated over Ficoll Ž d s 1.077. and 5 = 10 6 MNC were added to each culture flask.
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2.9. Determination of LTCIC after ex ÕiÕo expansion in stromaless liquid culture The ex vivo expansion of marrow cells by hematopoietic growth factors has been proposed to facilitate bone marrow transplantation. A combination of SCF and GM-CSF has been effective in expanding canine CFU in a stromaless liquid culture system ŽSchumm et al., 1995.. We investigated the effect of ex vivo expansion on LTCIC by long-term culture of marrow cells before and after expansion for 7 days. Mononuclear marrow cells were depleted from differentiated cells with monoclonal antibodies against lineage markers ŽCD5: Dog 15, granulocytes: Dog 17, B-cells: Dog 22 ŽCobbold and Metcalfe, 1994.. and sheep-anti-rat immunomagnetic beads ŽDynal, Oslo, Norway. and cultured for 7 days in Iscove medium supplemented with 20% fetal calf serum, 1% L-glutamine and 1% penicillinrstreptomycin in the presence of 10 ngrml rcan-SCF and 200 ngrml rhu-GM-CSF. The content of LTC-IC before and after liquid culture was determined by seeding cells from liquid culture onto a pre-established, irradiated stromal layer Ž15 Gy., and CFU growth was determined weekly. Control cultures with fresh marrow cells were performed on stromal layer from the same marrow aspiration.
Fig. 1. Clonogenic growth of fresh canine marrow: Comparison of stimuli and media. Clonogenic growth of fresh canine marrow cells relative to CFU growth of marrow cells stimulated with SCF and PHA-LCM in agar culture Žcontrol.. Media consisted of McCoy’s 5A medium supplemented 20% of fetal calf serum, 20 Urml erythropoietin and ingredients as indicated. ŽMC: methylcellulose, BSA: bovine serum albumine, PHA-LCM: PHA-lymphocyte-conditioned medium.. Given is the average"standard deviation ŽSD. of CFU growth relative to control cultures from 3 independent experiments Žcontrol: 105, 137, 438 coloniesr2=10 5 MNC, respectively..
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3. Results 3.1. Clonogenic assays for determination of differentiating cells The highest numbers of CFU were observed in both agar and methylcellulose when recombinant canine stem cell factor Žrcan-SCF. and rhu-GM-CSF were added as growth factors. PHA-conditioned medium was less effective in stimulating CFU growth as well as the addition of bovine serum albumin ŽBSA. with or without human transferrin which enhanced the number of BFU-E but not the total number of colonies ŽFig. 1.. The addition of SCF also resulted in larger colonies. CFU growth differed substantially between fresh and cultured cells. Unlike fresh marrow cells, cells from long-term culture grew only in agar supplemented with human serum. Here, colonies were mainly compact CFU-GM ŽFig. 2., but other types were also observed. Results of the 3 experiments are given in Table 1. 3.2. Cell culture media for bone marrow long-term culture Highest numbers of CFU were achieved when long-term cultures contained RPMI 1640 or basal Iscove medium and both stromal layers were of identical morphology. McCoy’s 5A medium produced an incomplete stromal layer with a large number of free floating cells after one week. The stromal layer did not reach confluency during the culture period of 5 weeks resulting in a lower number of cumulative CFU. Fewer
Fig. 2. Colonies from cells after 5 weeks of long-term culture grown in agar supplemented with 20% of human serum, SCF, GM-CSF and erythropoietin. Both non-adherent and adherent cell fractions were used.
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Table 1 CFU growth of non-adherent cells from long-term culture Total number of clonogenic cellsrflask Methylcellulose
Experiment 1 Experiment 2 Experiment 3
Agar
With human serum
With FCS
With human serum
With FCS
3 0 25 clusters
0 0 0
6480 3593 2485
0 1 0
CFU growth in methylcellulose and agar of non-adherent cells from week 3 of long-term culture initiated with 2=10 7 MNC.
macrophages and more fibroblasts were observed with IMDM Ž340 mosMrkg. accompanied with less CFU growth. In conclusion, all media were able to sustain long-term culture of canine marrow cells, but the highest number of CFU were recovered with basal Iscove medium and RPMI 1640 ŽTable 2.. 3.3. Supplements and sera We investigated the effect of hydrocortisone in 5 independent experiments. In 4 experiments, the addition of hydrocortisone resulted in 4–26 fold higher cumulative CFU growth in long-term culture for 5 weeks. Detailed results are given in Table 3.
Table 2 CFU growth of cells from canine long-term culture using different media Week
Experiment
RPMI-1640
Basal Iscove
McCoy’s 5A
IMDM
1
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
112 3534 6076 128 13 500 990 168 22 434 53 135 263 273 10 500 5670 734 27 691 13 433
75 14 876 6365 36 4995 828 63 56 784 84 104 195 30 16 500 5968 288 36 531 14 140
297 9234 5967 26 328 728 6 76 702 0 11 360 17 125 5368 346 9774 13 125
120 4725 2220 84 120 553 6 17 38 0 14 42 276 5988 933 486 10 864 3786
2
3
4
5
Ý
CFU growth of non-adherent cells Žweeks 1–4., adherent and non-adherent cells Žweek 5. and cumulative number of CFU from long-term culture using different culture media.
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Table 3 Effect of hydrocortisone on canine long-term culture Experiment
Without hydrocortisone coloniesrflask
With hydrocortisone coloniesrflask
CFU index
1 2 3 4 5 Avg"SD
3531 5737 274 110 23 580
4957 26 025 5272 2932 17 834
1,4 4,5 19,2 26,7 0,8 10,5"11.7
Cumulative growth after 5 weeks of long-term culture with or without the addition of hydrocortisone-21-phosphate. CFU index: CFU growth relative to control.
We found no benefit of 2-Mercaptoethanol in 5 experiments. The cumulative number of CFU was 0.75 fold Ž0.57–1.16 fold. lower in cultures with 2-mercaptoethanol Ž2-ME.. Same results were obtained when a-Thioglycerol was added. The morphology of the stromal layer did not differ from control cultures in respect to cellularity and composition. 3.3.1. Fetal calf serum To examine the effect of fetal calf serum on canine long-term cultures we replaced horse serum by fetal calf serum stepwise. Results are shown in Fig. 3. In all experi-
Fig. 3. Effect of fetal calf serum on canine long-term culture. Cumulative CFU growth in long-term cultures for 5 weeks. RPMI 1640 with fetal calf serum ŽFCS. and horse serum ŽHS. as indicated.
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ments, the cumulative number of CFU after 5 weeks of culture decreased with increasing concentration of fetal calf serum. Morphology of the stromal layer changed when fetal calf serum was added: with horse serum macrophages dominated the stromal layer which were stepwise replaced by fibroblasts with increasing concentrations of FCS. 3.3.2. Normal dog serum Heat-inactivated serum from healthy dogs was not able to build a confluent stromal layer, after 2 weeks of culture, all cells were found in the supernatant. Serum lots differ widely in their suitability for long-term cultures ŽDexter et al., 1977; Schuening et al., 1989.. We observed considerable differences between serum lots in developing a stromal layer and producing colonies in culture. All serum lots were tested with the addition of hydrocortisone. Results of 5 experiments are given in Fig. 4. Fetal equine serum was unable to build a stromal layer. Only few cells adhered to the bottom of the flask and within 3 weeks all cells died. Confluency of the stromal layer correlated with the output of CFU: only confluent layers were able to yield high numbers of colonies.
Fig. 4. Effect of serum lot on canine long-term culture. Cumulative CFU growth in long-term culture using different lots of horse serum. Average and standard deviation of 5 independent experiments are given in percent of a reference serum Ža1. tested in advance for suitability. Ža6. Fetal equine serum. ) Significant difference in Wilcoxon test for matched pairs.
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Fig. 5. Recharging of long-term culture with fresh marrow cells at day 7. Weekly CFU yield with or without recharging of canine long-term culture with 5=10 6 autologous marrow cellsrflask at day 7 of culture.
3.4. Influence of culture temperature Several authors proposed lower incubation temperatures for long-term culture of marrow cells ŽCoulombel et al., 1983; Dexter et al., 1977.. Five independent long-term
Fig. 6. Effect of rcan stem cell factor on canine long-term culture. Weekly CFU yield from long-term culture with or without addition of 10 ngrml rcan SCF. AvgqSD of 4 independent experiments.
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Table 4 Long-term culture of cytokine expanded marrow cells Weeks of long-term culture
0 1 2 3 4
Total number of CFUrflask With fresh marrow cells
With cells after stromaless liquid culture
506 1500 12 733 1116
444 25 77 0 0
Clonogenic growth in long-term culture of cells after expansion with SCF and GM-CSF in stromaless culture for 7 days. Mean of 3 experiments.
cultures were performed in parallel at 33 and 378C, respectively, to investigate the influence of incubation temperature. At both temperatures, marrow cultures could be maintained for more than 5 weeks. However, confluency of the layer was achieved within 1 week at 378C and within 3 weeks at 338C. The cumulative number of CFU after 5 weeks was about 1.77 fold higher Ž0.92–2.85 fold. at 378C. 3.5. Marrow recharging after one week Recharging of long-term cultures with marrow cells one week after initiation increased the number of CFUs from culture at week 2 Ž6.6–42 fold higher numbers of CFU. but had no influence thereafter Ž0.8–1.5 fold.. One representative experiment out of 5 is shown in Fig. 5. 3.6. Addition of canine stem cell factor to long-term culture Addition of rcan-SCF to long-term culture enhanced CFU growth of cells from culture within the first 3 weeks Ž1.8–68.6 fold.. However, the stromal layer lost confluency thereafter and the yield of CFUs decreased Ž0–0.6 fold.. The results of 4 experiments are shown in Fig. 6. 3.7. Two step long-term culture for inÕestigation of cells after stromaless liquid culture The expansion of marrow cells in the presence of SCF and GM-CSF was not able to increase the number of LTCIC. CFU growth of expanded marrow cells declined rapidly in long-term cultures in contrast to control cultures with fresh marrow cells. Results of 3 independent experiments are shown in Table 4.
4. Discussion Several protocols exist for short-term clonogenic assays using mostly agar or methylcellulose as matrix ŽDexter et al., 1977; Konwalinka et al., 1983; Sutherland et al., 1994. supplemented with fetal calf serum and growth stimuli like PHA-conditioned
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medium, post-irradiation serum or recombinant cytokines ŽHahn et al., 1991; Konwalinka et al., 1984, 1983; Kreja et al., 1988.. Using fresh marrow cells, we found equally good growth using agar or methylcellulose stimulated with SCF, GM-CSF and erythropoietin. The addition of SCF did not only improve the number of colonies but did also enhance the number of cells per colony. If cultured for more than 14 days, it even stimulated colonies with more than 50 000 cells. These HPP-CFC Žhigh proliferative potential colony forming cells. which are derived from more primitive progenitor cells than CFU-GM ŽMcNiece et al., 1987. were not seen with PHA-LCM. However, cells from long-term culture only grew in the presence of human AB-serum, indicating the need of additional growth factors. All cell culture media which were investigated were suitable for long-term culture. IMDM with elevated osmolarity which has been described for human cultures ŽGibson et al., 1995. was less effective than other media with lower osmolarity. Other media were well suited for canine cells and in our experiments Iscove medium seemed to be more stable than RPMI 1640 or McCoy 5A medium. Dexter et al. Ž1984. found that free corticosteroids are responsible for the qualification of a serum for long-term cultures. Schuening et al. Ž1989., however, proposed that well-suited sera did not need addition of steroids and CFU growth can even be detrimental. In our experiments, CFU-growth could be enhanced by addition of hydrocortisone, but the influence of the serum lot was much higher than the influence of hydrocortisone. Addition of fetal calf serum is widely used in human long-term cultures ŽCoulombel et al., 1983; Sutherland et al., 1994. and Carter et al. Ž1990. reported the use of fetal calf serum in canine long-term cultures. We found dose dependent decrease of CFU production when FCS was added to canine long-term cultures confirming observations of Schuening et al. Ž1989.. When FCS was added to cultures, the morphology of the stromal layer changed: macrophages were replaced by fibroblasts and the stromal layer lost confluency. As a consequence, the number of CFU declined rapidly. The structure of the canine long-term culture differs from other species. Here, a dense fibroblastic layer covers early progenitors which can be detected as dark cobblestones under the layer. Differentiating cells are found on top of the fibroblasts and in the supernatant ŽGartner and Kaplan, 1980; Linenberger and Abkowitz, 1992; Ploemacher et al., 1989.. Canine cells are not covered by fibroblasts and can therefore be lost easily during medium change and typical cobblestone areas can hardly be found ŽFig. 7.. Early progenitor cells survive in the presence of a stromal layer. Therefore, it seems to be useful to first establish a stromal layer and then recharge the culture with a second autologous marrow. Dexter et al. Ž1977. and Schuening et al. Ž1989. found improved CFU production with this procedure. We also observed improved CFU growth especially in week 2, but the CFU output returned to control values at the end of the culture. Obviously, the stromal layer is able to support the survival of LTC-IC from the beginning and additional LTC-IC have little effect. The development of recombinant cytokines offers the possibility to substitute the cell derived growth factors and to improve culture conditions. Stem cell factor is one of the earliest acting cytokines on hematopoietic progenitors and allows the survival of canine cells in stroma-free cultures ŽShull et al., 1992.. Moreover, cell-bound stem cell factor can be detected in a canine stromal cell line which enables long-term culture of canine
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Fig. 7. Stromal layer at day 16 of canine long-term culture in Iscove Medium supplemented with 20% of horse serum.
marrow cells ŽHuss et al., 1995.. In our experiments, stem cell factor obviously induced a transformation of the adherent stromal layer cells into non-adherent cells. As a consequence, the stromal layer disappeared completely towards the end of the culture and the number of CFU declined. This transformation has been reported by Huss et al, who postulated a differentiation of CD34 negative stromal cells into CD34 positive hemopoietic cells ŽHuss et al., 1995.. Ex vivo expansion of marrow cells mostly has been evaluated for the increase of cells or CFU. We could show the detrimental effect of SCFrGM-CSF on LTCIC in liquid culture, an observation reported by other groups for different factor combinations ŽShull et al., 1992.. Long-term culture experiments can give a more precise answer on the marrow repopulating ability of ex vivo expanded marrow than short-term CFU assays. In conclusion, canine marrow cells show special growth requirements after long-term culture which differ from fresh marrow cells. Addition of stem cell factor leads to a decrease of CFU production possibly due to an outgrow of early progenitor cells. The long-term culture system is suitable for the examination of the ex vivo expansion of very early progenitor cells. Acknowledgements We thank Karin Oettrich, Sabine Sagebiel-Kohler, Michael Hagemann, Christine Voss and Andrea von Arco-Zinneberg for excellent technical assistance. The study was supported by the Wilhelm-Sander-Foundation, Neustadt a.d. Donau, Germany.
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References Aihara, M., Sikic, B.I., Blume, K.G., Chao, N.J., 1990. Assessment of purging with multidrug resistance ŽMDR. modulators and VP-16: results of long-term marrow culture. Exp. Hematol. 18, 940–944. Allen, T.D., Dexter, T.M., 1984. The essential cells of the hemopoietic microenvironment. Exp. Hematol. 12, 517–521. Berardi, A.C., Wang, A., Levine, J.D., Lopez, P., Scadden, D.T., 1995. Functional isolation and characterization of human hematopoietic stem cells. Science 267, 104–108. Bernstein, I.D., Andrews, R.G., Zsebo, K.M., 1991. Recombinant human stem cell factor enhances the formation of colonies by CD34q and CD34qlin- cells, and the generation of colony-forming cell progeny from CD34qlin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocytemacrophage colony-stimulating factor. Blood 77, 2316–2321. Carter, R.F., Kruth, S.A., Valli, V.E., Dube, I.D., 1990. Long-term culture of canine marrow: cytogenetic evaluation of purging of lymphoma and leukemia. Exp. Hematol. 18, 995–1001. Cobbold, S., Metcalfe, S., 1994. Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop ŽCLAW.. Tissue Antigens 43, 137–154. Coulombel, L., Eaves, A.C., Eaves, C.J., 1983. Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62, 291–297. Dexter, T.M., Allen, T.D., Lajtha, L.G., 1977. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell Physiol. 91, 335–344. Dexter, T.M., Spooncer, E., Simmons, P., Allen, T.D., 1984. Long-term marrow culture: an overview of techniques and experience. Kroc. Found. Ser. 18, 57–96. Fraser, C.C., Eaves, C.J., Szilvassy, S.J., Humphries, R.K., 1990. Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells. Blood 76, 1071–1076. Galvani, D.W., Cawley, J.C., 1990. The effects of alpha interferon on human long-term bone marrow culture. Leuk. Res. 14, 525–531. Gartner, S., Kaplan, H.S., 1980. Long-term culture of human bone marrow cells. Proc. Natl. Acad. Sci. U.S.A. 77, 4756–4759. Gibson, F.M., Scopes, J., Daly, S., Rizzo, S., Ball, S.E., Gordon Smith, E.C., 1995. IL-3 is produced by normal stroma in long-term bone marrow cultures. Br. J. Hematol. 90, 518–525. Greenberger, J.S., 1978. Sensitivity of corticosteroid-dependent insulin-resistant lipogenesis in marrow preadipocytes of obese-diabetic Ždbrdb. mice. Nature 275, 752–754. Hahn, J., Kolb, H.J., Schumm, M., Beisser, K., Ellwart, J., Rieber, P. et al., 1991. Immunological characterization of canine hematopoietic progenitor cells. Ann. Hematol. 63, 223–226. Huss, R., Hong, D.S., McSweeney, P.A., Hoy, C.A., Deeg, H.J., 1995. Differentiation of canine bone marrow cells with hemopoietic characteristics from an adherent stromal cell precursor. Proc. Natl. Acad. Sci. U.S.A. 92, 748–752. Konwalinka, G., Peschel, C., Geissler, D., Boyd, J., Tomaschek, B., Odavic, R. et al., 1983. Myelopoiesis of human bone marrow cells in a micro-agar culture system: comparison of two sources of colony stimulating activity ŽCSA.. Int. J. Cell Cloning 1, 401–411. Konwalinka, G., Peschel, C., Boyd, J., Geissler, D., Ogriseg, M., Odavic, R. et al., 1984. A miniaturized agar culture system for cloning human erythropoietic progenitor cells. Exp. Hematol. 12, 75–79. Kreja, L., Baltschukat, K., Nothdurft, W., 1988. Growth of erythroid burst-forming units ŽBFU-E. in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs. Exp. Hematol. 16, 647–651. Ladiges, W.C., Storb, R., Thomas, E.D., 1990. Canine models of bone marrow transplantation. Lab. Anim. Sci. 40, 11–15. Linenberger, M.L., Abkowitz, J.L., 1992. Studies in feline long-term marrow culture: hematopoiesis on normal and feline leukemia virus infected stromal cells. Blood 80, 651–662. McNiece, I.K., Williams, N.T., Johnson, G.R., Kriegler, A.B., Bradley, T.R., Hodgson, G.S., 1987. Generation of murine hematopoietic precursor cells from macrophage high-proliferative-potential colony-forming cells. Exp. Hematol. 15, 972–977.
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E. Neuner et al.r Veterinary Immunology and Immunopathology 61 (1998) 1–16
Moore, M.A., 1991. Review: Stratton lecture 1990. Clinical implications of positive and negative hematopoietic stem cell regulators. Blood 78, 1–19. Ploemacher, R.E., van der Sluijs, J.P., Voerman, J.S., Brons, N.H., 1989. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763. Schuening, F.G., Storb, R., Meyer, J., Goehle, S., 1989. Long-term culture of canine bone marrow cells. Exp. Hematol. 17, 411–417. Schuening, F.G., Kawahara, K., Miller, A.D., To, R., Goehle, S., Stewart, D. et al., 1991. Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. Blood 78, 2568–2576. Schumm, M., Gunther, W., Kolb, H.J., Kremmer, E., Vogl, C., Wilmanns, W. et al., 1995. Cytokine ¨ requirements for the in vitro expansion of purified canine progenitor cells. Acta Hematol. 93, 153. Shull, R.M., Suggs, S.V., Langley, K.E., Okino, K.H., Jacobsen, F.W., Martin, F.H., 1992. Canine stem cell factor Žc-kit ligand. supports the survival of hematopoietic progenitors in long-term canine marrow culture. Exp. Hematol. 20, 1118–1124. Sutherland, H.J., Eaves, C.J., Eaves, A.C., Dragowska, W., Lansdorp, P.M., 1989. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74, 1563–1570. Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C., Eaves, C.J., 1990. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. U.S.A. 87, 3584–3588. Sutherland, H.J., Eaves, C.J. Freshney, R.I., Pragnell, I.B., Freshney, M.G. ŽEds... Long-term culture of human myeloid cells, 139-62. Culture of Hematopoietic Cells. Wiley-Liss, New York, 1994. Verfaillie, C., Blakolmer, K., McGlave, P., 1990. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J. Exp. Med. 172, 502–509.
Studies on canine bone marrow long-term culture: effect of stem cell factor Evelin Neuner a , Michael Schumm a , Eva-Maria Schneider b, Wolfgang Guenther b, Elisabeth Kremmer a , Christiane Vogl a , c Matthias Buttner , Stefan Thierfelder a , Hans-Jochem Kolb a,b,) ¨ a
Institut fuer Immunologie, GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit, Muenchen, Germany b Institut fuer Klinische Haematologie, GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit, Muenchen, Germany c Bundesforschungsanstalt fur ¨ Viruskrankheiten der Tiere, Tuebingen, Germany Accepted 15 September 1997
Abstract Long-term culture of canine marrow cells allows in vitro studies of the hematopoietic system of the dog and characterization of early progenitor cells. Colonies of fresh marrow cells grew equally good in both agar or methylcellulose supplemented with fetal calf serum, while colonies of long-term cultures required agar-based medium containing human serum. Optimum colony growth was obtained when stem cell factor ŽSCF. and granulocyte-macrophage-colony-stimulating factor ŽGM-CSF. were used as growth stimuli of colony forming units ŽCFU.. Similar results were achieved with several cell culture media. Addition of hydrocortison to long-term cultures improved clonogenic growth of cultured cells. Addition of 2-mercaptoethanol had no effect. Strong differences were observed in long-term culture with different horse serum lots and the addition of fetal calf serum to long-term culture suppressed CFU growth of cultured cells.
Abbreviations: Avg, average; BFU-E, burst forming unit-erythroid; BSA, bovine serum albumine; CD, cluster of differentiation; CFU, colony forming units; CFU-G, colony forming unit-granulocyte; CFU-GEMM, colony forming units-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM, colony forming unitgranulocyte-macrophage; FCS, fetal calf serum; rhu-GM-CSF, recombinant human granulocyte, macrophagecolony-stimulating factor; HS, horse serum; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; LTC-IC, long-term culture-initiating cells; MC, methylcellulose; MEM, minimal essential medium; 2-ME, 2-Mercaptoethanol; MNC, mononuclear cells; PHA-LCM, phythemagglutinin-stimulated dog lymphocyte-conditioned medium; rcan-SCF, recombinant canine stem cell factor; SD, standard deviation ) Corresponding author. GSF-Institut fuer Immunologie, Marchioninistr. 25, 81377 Muenchen, Germany. Tel.: q49 89 7099 327; fax: q49 89 7099 300. 0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 2 6 - 8
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Recharging of cultures with fresh marrow cells on day 7 of culture improved CFU growth only in the following week but had little effect on the outcome. Adding SCF to long-term cultures led to differentiation of more primitive cells and destruction of the stromal layer. Investigation of purified and cultured cell populations was possible when preestablished long-term cultures as stromal layers were used. Loss of long-term culture-initiating ability could be demonstrated in this system with lineage negative marrow cells expanded ex vivo with SCF and GM-CSF. q 1998 Elsevier Science B.V. Keywords: Canine; Long-term culture; Marrow; Hematopoiesis; Stem cell factor
1. Introduction The dog provides an important animal model for studies on transplantation and gene therapy ŽLadiges et al., 1990; Schuening et al., 1991.. The long-term culture-initiating cell has been proven to be the earliest hematopoietic progenitor cell which can be detected in ‘in vitro’ assays and seems to be the equivalent of the pluripotent stem cell with repopulating ability ŽBerardi et al., 1995; Moore, 1991.. Bone marrow long-term culture mimics the hematopoietic system in vitro comprising stromal cells and hematopoietic cells of all differentiation stages. Early progenitor cells reside in close contact to stromal cells and produce differentiating progenitor cells, which are released into the supernatant. Long-term cultures can be maintained for several months as long as very early cells with the capacity of self-renewal continue to produce differentiating descendants. These committed cells can then be determined by their ability to form lineage specific colonies in semisolid medium: BFU-E Žburst forming unit-erythroid., CFU-G Žcolony forming unit-granulocyte., CFU-GM Žcolony forming unit-granulocyte-macrophage., CFU-GEMM Žcolony forming units-granulocyte, erythroid, macrophage, megakaryocyte. ŽAllen and Dexter, 1984; Dexter et al., 1977; Sutherland et al., 1994.. The amount of these colony forming cells after 5–8 weeks of long-term culture has been closely related to the number of long-term culture-initiating cells ŽLTC-IC. in the beginning ŽSutherland et al., 1990.. LTC-IC are the earliest cells to be detected in culture. They represent the in vitro equivalent of the omnipotent stem cell and until now long-term culture is the only ex vivo proof of these cells ŽMoore, 1991.. The long-term culture of marrow cells allows the study of hematopoiesis during culture. Multiple substances, for example, drugs for purging of leukemic cells, cytokines and growth factors may be tested for their action on normal stromal and hematopoietic cells ŽAihara et al., 1990; Fraser et al., 1990; Galvani and Cawley, 1990.. For the investigation of purified hematopoietic cells, a ‘two-step’ long-term culture is favorable. Here, a primary long-term culture is initiated to establish a stromal layer within 2–4 weeks and irradiated to inactivate hematopoiesis. Allogeneic hematopoietic cells separated with monoclonal antibodies and immunomagnetic systems or a cell sorter can be placed on the stromal layer and investigated for their content of early progenitor cells. In this system, cell fractions need not contain stromal cells and even small numbers of purified progenitor cells can be investigated ŽSutherland et al., 1989; Verfaillie et al., 1990..
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Long-term cultures in one- or two-step technique follow the same conditions. Therefore, we had to optimize our system for the one step culture of marrow cells by testing multiple parameters which may influence the microenvironment in culture.
2. Material and methods 2.1. Dogs In our studies, 1- to 5-year-old beagles of both sexes were used as marrow donors. These animals were raised at the GSF-Forschungszentrum fur ¨ Umwelt und Gesundheit. All dogs were healthy, dewormed and vaccinated against distemper, leptospirosis, hepatitis, and parvovirus. 2.2. Comparison of agar- and methylcellulose-based media for determination of colony forming cells Detection of differentiating colonies requires optimum growth conditions. Several components of the culture medium were therefore investigated on fresh marrow cells first. Marrow cells were separated by density gradient centrifugation over Ficoll Ž d s 1.077. and 2 = 10 5 mononuclear cells ŽMNC. were cultured in 2 ml of medium. - Agar-based medium consisted of 0.3% agar ŽDifco Laboratories, Detroit, USA. in Mc Coy’s 5A medium supplemented with 20% heat-inactivated fetal calf serum ŽPansystems, Aidenbach, Germany., 0.75% sodium bicarbonate, 1.25% sodium pyruvate, 0.5% Eagles MEM vitamins, 1% Eagles MEM amino acids, 0.5% nonessential amino acids, 0.5% L-glutamine Žall Gibco, Eggenstein, Germany., 0.5% L-serine Ž21 mgrml. 0.2% L-asparagine Ž10 mgrml, both Merck, Darmstadt, Germany., 100 Urml penicillin and 100 m grml streptomycin ŽGibco., and 20 Urml rhu-erythropoietin ŽBoehringer, Mannheim, Germany., 200 ngrml recombinant human granulocyte, macrophage-colony stimulating factor Žrhu-GM-CSF, Essex, Munich, Germany., 1% hemin ŽSigma, Deisenhofen, Germany. and 20 ngrml recombinant canine stem cell factor Žrcan-SCF, Amgen, Thousand Oaks, USA.. - Methylcellulose-based medium consisted of 1.25% of methylcellulose ŽFluka, Neu-Ulm, Germany. in Iscove’s modified Dulbecco’s medium ŽIMDM, Gibco. supplemented with 20% heat-inactivated fetal calf serum ŽFCS, Pansystems., 1% L-glutamine ŽGibco., 1% penicillin-streptomycin ŽGibco. and 20 Urml rhu-erythropoietin ŽBoehringer., 200 ngrml rhu-GM-CSF ŽEssex., 1% hemin ŽSigma. and 20 ngrml recombinant canine stem cell factor Žrcan-SCF, Amgen.. All cultures were incubated for 14 days at 388C in a humidified atmosphere of 5% CO 2 in air in 35-mm plastic Petri dishes and colonies Ž) 50 cells. were counted thereafter.
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2.3. Comparison of culture supplements and growth stimuli In some experiments, granulocyte-macrophage-colony-stimulating factor ŽGM-CSF. was replaced with either rhu-Interleukin 6 Ž100 Urml. ŽBoehringer., 1% bovine serum albumin ŽMerck., 380 m grml transferrin ŽBehring, Marburg, Germany. or 10% phythemagglutinin-stimulated dog lymphocyte-conditioned medium ŽPHA-LCM.. PHALCM was prepared by culture of peripheral blood leukocytes from 5 young, healthy dogs in the presence of 10 ngrml PHA-M ŽSigma. and 10% normal dog serum for 4 days. 2.4. Determination of colony forming cells from long-term culture No CFU growth was observed with cells from long-term cultures in methylcellulose and FCS. We therefore performed several experiments to ascertain this observation. Cells after long-term culture were cultured in either agar or methylcellulose as described above in the presence of either 20% human AB-serum or fetal calf serum, using rcan-SCF, rhu-GM-CSF and rhu-Epo as growth stimuli. 2.5. Comparison of cell culture media for canine long-term bone marrow culture Marrow cells were centrifuged over Ficoll-Hypaque Ždensity 1.077. at 800 g for 20 min and the low-density cells were collected, washed once in phosphate buffered saline and then twice in phosphate buffered saline containing 10% horse serum ŽSigma. at 600 = g for 10 min, respectively. Mononuclear marrow cells were then cultured in 25-cm2 canted-neck flasks ŽCostar, Bodenheim, Germany. at 2 = 10 6 MNCrml in 10 ml of the following media. Ži. Basal Iscove medium ŽSeromed-Biochrom, Berlin, Germany. supplemented with 20% prescreened heat-inactivated horse serum ŽSigma., 1% L-glutamine Ž200 mM, 100 = , Gibco., 1% penicillin Ž5000 Urml. ŽGibco. and 1% streptomycin Ž5000 m grml. ŽGibco.. Žii. RPMI-1640 medium ŽGibco. containing 20% horse serum, 1% nonessential amino acids, 1% sodium pyruvate, 2% L-glutamine, 1% penicillin and 1% streptomycin. Žiii. McCoy’s 5A medium containing 20% horse serum, 0.75% sodium bicarbonate, 1.25% sodium pyruvate, 0.5% Eagles MEM vitamins, 1% Eagles MEM amino acids, 0.5% nonessential-amino acids, 0.5% L-glutamine, 0.5% L-serine Ž21 mgrml., 0.2% L-asparagine Ž10 mgrml. and 1.5% penicillin-streptomycin. Živ. IMDM Ž340 mosMrkg. according to Gibson et al. Ž1995., containing 12.5% FCS, 12.5% horse serum or 20% horse serum only. 2.6. Addition of media supplements and cytokines to long-term culture Addition of hydrocortisone has been described favorable for the long-term culture of murine ŽGreenberger, 1978., human ŽGartner and Kaplan, 1980. and canine ŽSchuening et al., 1989. marrow cells. We used Hydrocortison-21-phosphate at a concentration of 10y7 molrl in all media unless indicated otherwise.
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2-Mercaptoethanol and a-Thioglycerol is widely used for retarding oxidation in culture media. We investigated the effect of both of these substances on canine long-term culture in parallel at a concentration of 10y4 Mrl. Stem cell factor is described as a very early acting factor in hematopoiesis and therefore possibly advantageous for the long-term culture of progenitor cells ŽBernstein et al., 1991.. We added 10 ngrml rcan-SCF to long-term cultures in weekly intervals at the time of medium change. Cultures without SCF were performed in parallel. 2.7. Comparison of different sera for canine long-term culture 2.7.1. Human serum For human marrow, Sutherland et al. Ž1994. proposed equal parts of fetal calf and horse serum for optimum growth of progenitor cells in long-term culture. We investigated the effect of fetal calf serum in parallel cultures by replacing horse serum to get 0, 5 and 10% of fetal calf serum, respectively. 2.7.2. Canine serum Additional experiments were performed using 20% normal dog serum instead of horse serum. 2.7.3. Horse serum The following horse sera were tested for suitability in canine long-term culture: a1 horse serum lot no. 31P4127 ŽGibco, Gaithersburg, USA., a2 horse serum lot no. 001C ŽSeromed-Biochrom., a3 horse serum lot no. 002C ŽSeromed-Biochrom., a4 horse serum H6762, lot no. 73H0765 ŽSigma., a5 horse serum H9767, lot no. 88F-0428 ŽSigma., a6 fetal equine serum, lot no. F7763 ŽSigma., a7 horse serum H1270, lot no. 43H0691 ŽSigma. and a8 horse serum Hybrimax w , H1263, lot no. 73H0721 ŽSigma.. Long-term cultures were maintained at 378C in a humidified atmosphere of 5% CO 2 in air. At weekly intervals, non-adherent cells and medium were removed from flasks and centrifuged at 600 g for 10 min and fresh medium was added. Half of the used medium was returned into flasks. The non-adherent cell population was assayed for clonogenic growth as described. After 5 weeks, non-adherent cells were harvested and adherent cells suspended by trypsinization. An aliquot of combined non-adherent and adherent cells was then assayed for colony forming units. 2.8. Recharging of canine long-term cultures We studied a marrow ‘boost’ by recharging flasks after 1 week of culture which has been reported as favorable in respect to cell and CFU recovery after long-term culture ŽSchuening et al., 1989.. Marrow was aspirated from the dog used for establishing the stromal layer, separated over Ficoll Ž d s 1.077. and 5 = 10 6 MNC were added to each culture flask.
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2.9. Determination of LTCIC after ex ÕiÕo expansion in stromaless liquid culture The ex vivo expansion of marrow cells by hematopoietic growth factors has been proposed to facilitate bone marrow transplantation. A combination of SCF and GM-CSF has been effective in expanding canine CFU in a stromaless liquid culture system ŽSchumm et al., 1995.. We investigated the effect of ex vivo expansion on LTCIC by long-term culture of marrow cells before and after expansion for 7 days. Mononuclear marrow cells were depleted from differentiated cells with monoclonal antibodies against lineage markers ŽCD5: Dog 15, granulocytes: Dog 17, B-cells: Dog 22 ŽCobbold and Metcalfe, 1994.. and sheep-anti-rat immunomagnetic beads ŽDynal, Oslo, Norway. and cultured for 7 days in Iscove medium supplemented with 20% fetal calf serum, 1% L-glutamine and 1% penicillinrstreptomycin in the presence of 10 ngrml rcan-SCF and 200 ngrml rhu-GM-CSF. The content of LTC-IC before and after liquid culture was determined by seeding cells from liquid culture onto a pre-established, irradiated stromal layer Ž15 Gy., and CFU growth was determined weekly. Control cultures with fresh marrow cells were performed on stromal layer from the same marrow aspiration.
Fig. 1. Clonogenic growth of fresh canine marrow: Comparison of stimuli and media. Clonogenic growth of fresh canine marrow cells relative to CFU growth of marrow cells stimulated with SCF and PHA-LCM in agar culture Žcontrol.. Media consisted of McCoy’s 5A medium supplemented 20% of fetal calf serum, 20 Urml erythropoietin and ingredients as indicated. ŽMC: methylcellulose, BSA: bovine serum albumine, PHA-LCM: PHA-lymphocyte-conditioned medium.. Given is the average"standard deviation ŽSD. of CFU growth relative to control cultures from 3 independent experiments Žcontrol: 105, 137, 438 coloniesr2=10 5 MNC, respectively..
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3. Results 3.1. Clonogenic assays for determination of differentiating cells The highest numbers of CFU were observed in both agar and methylcellulose when recombinant canine stem cell factor Žrcan-SCF. and rhu-GM-CSF were added as growth factors. PHA-conditioned medium was less effective in stimulating CFU growth as well as the addition of bovine serum albumin ŽBSA. with or without human transferrin which enhanced the number of BFU-E but not the total number of colonies ŽFig. 1.. The addition of SCF also resulted in larger colonies. CFU growth differed substantially between fresh and cultured cells. Unlike fresh marrow cells, cells from long-term culture grew only in agar supplemented with human serum. Here, colonies were mainly compact CFU-GM ŽFig. 2., but other types were also observed. Results of the 3 experiments are given in Table 1. 3.2. Cell culture media for bone marrow long-term culture Highest numbers of CFU were achieved when long-term cultures contained RPMI 1640 or basal Iscove medium and both stromal layers were of identical morphology. McCoy’s 5A medium produced an incomplete stromal layer with a large number of free floating cells after one week. The stromal layer did not reach confluency during the culture period of 5 weeks resulting in a lower number of cumulative CFU. Fewer
Fig. 2. Colonies from cells after 5 weeks of long-term culture grown in agar supplemented with 20% of human serum, SCF, GM-CSF and erythropoietin. Both non-adherent and adherent cell fractions were used.
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Table 1 CFU growth of non-adherent cells from long-term culture Total number of clonogenic cellsrflask Methylcellulose
Experiment 1 Experiment 2 Experiment 3
Agar
With human serum
With FCS
With human serum
With FCS
3 0 25 clusters
0 0 0
6480 3593 2485
0 1 0
CFU growth in methylcellulose and agar of non-adherent cells from week 3 of long-term culture initiated with 2=10 7 MNC.
macrophages and more fibroblasts were observed with IMDM Ž340 mosMrkg. accompanied with less CFU growth. In conclusion, all media were able to sustain long-term culture of canine marrow cells, but the highest number of CFU were recovered with basal Iscove medium and RPMI 1640 ŽTable 2.. 3.3. Supplements and sera We investigated the effect of hydrocortisone in 5 independent experiments. In 4 experiments, the addition of hydrocortisone resulted in 4–26 fold higher cumulative CFU growth in long-term culture for 5 weeks. Detailed results are given in Table 3.
Table 2 CFU growth of cells from canine long-term culture using different media Week
Experiment
RPMI-1640
Basal Iscove
McCoy’s 5A
IMDM
1
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
112 3534 6076 128 13 500 990 168 22 434 53 135 263 273 10 500 5670 734 27 691 13 433
75 14 876 6365 36 4995 828 63 56 784 84 104 195 30 16 500 5968 288 36 531 14 140
297 9234 5967 26 328 728 6 76 702 0 11 360 17 125 5368 346 9774 13 125
120 4725 2220 84 120 553 6 17 38 0 14 42 276 5988 933 486 10 864 3786
2
3
4
5
Ý
CFU growth of non-adherent cells Žweeks 1–4., adherent and non-adherent cells Žweek 5. and cumulative number of CFU from long-term culture using different culture media.
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Table 3 Effect of hydrocortisone on canine long-term culture Experiment
Without hydrocortisone coloniesrflask
With hydrocortisone coloniesrflask
CFU index
1 2 3 4 5 Avg"SD
3531 5737 274 110 23 580
4957 26 025 5272 2932 17 834
1,4 4,5 19,2 26,7 0,8 10,5"11.7
Cumulative growth after 5 weeks of long-term culture with or without the addition of hydrocortisone-21-phosphate. CFU index: CFU growth relative to control.
We found no benefit of 2-Mercaptoethanol in 5 experiments. The cumulative number of CFU was 0.75 fold Ž0.57–1.16 fold. lower in cultures with 2-mercaptoethanol Ž2-ME.. Same results were obtained when a-Thioglycerol was added. The morphology of the stromal layer did not differ from control cultures in respect to cellularity and composition. 3.3.1. Fetal calf serum To examine the effect of fetal calf serum on canine long-term cultures we replaced horse serum by fetal calf serum stepwise. Results are shown in Fig. 3. In all experi-
Fig. 3. Effect of fetal calf serum on canine long-term culture. Cumulative CFU growth in long-term cultures for 5 weeks. RPMI 1640 with fetal calf serum ŽFCS. and horse serum ŽHS. as indicated.
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ments, the cumulative number of CFU after 5 weeks of culture decreased with increasing concentration of fetal calf serum. Morphology of the stromal layer changed when fetal calf serum was added: with horse serum macrophages dominated the stromal layer which were stepwise replaced by fibroblasts with increasing concentrations of FCS. 3.3.2. Normal dog serum Heat-inactivated serum from healthy dogs was not able to build a confluent stromal layer, after 2 weeks of culture, all cells were found in the supernatant. Serum lots differ widely in their suitability for long-term cultures ŽDexter et al., 1977; Schuening et al., 1989.. We observed considerable differences between serum lots in developing a stromal layer and producing colonies in culture. All serum lots were tested with the addition of hydrocortisone. Results of 5 experiments are given in Fig. 4. Fetal equine serum was unable to build a stromal layer. Only few cells adhered to the bottom of the flask and within 3 weeks all cells died. Confluency of the stromal layer correlated with the output of CFU: only confluent layers were able to yield high numbers of colonies.
Fig. 4. Effect of serum lot on canine long-term culture. Cumulative CFU growth in long-term culture using different lots of horse serum. Average and standard deviation of 5 independent experiments are given in percent of a reference serum Ža1. tested in advance for suitability. Ža6. Fetal equine serum. ) Significant difference in Wilcoxon test for matched pairs.
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Fig. 5. Recharging of long-term culture with fresh marrow cells at day 7. Weekly CFU yield with or without recharging of canine long-term culture with 5=10 6 autologous marrow cellsrflask at day 7 of culture.
3.4. Influence of culture temperature Several authors proposed lower incubation temperatures for long-term culture of marrow cells ŽCoulombel et al., 1983; Dexter et al., 1977.. Five independent long-term
Fig. 6. Effect of rcan stem cell factor on canine long-term culture. Weekly CFU yield from long-term culture with or without addition of 10 ngrml rcan SCF. AvgqSD of 4 independent experiments.
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Table 4 Long-term culture of cytokine expanded marrow cells Weeks of long-term culture
0 1 2 3 4
Total number of CFUrflask With fresh marrow cells
With cells after stromaless liquid culture
506 1500 12 733 1116
444 25 77 0 0
Clonogenic growth in long-term culture of cells after expansion with SCF and GM-CSF in stromaless culture for 7 days. Mean of 3 experiments.
cultures were performed in parallel at 33 and 378C, respectively, to investigate the influence of incubation temperature. At both temperatures, marrow cultures could be maintained for more than 5 weeks. However, confluency of the layer was achieved within 1 week at 378C and within 3 weeks at 338C. The cumulative number of CFU after 5 weeks was about 1.77 fold higher Ž0.92–2.85 fold. at 378C. 3.5. Marrow recharging after one week Recharging of long-term cultures with marrow cells one week after initiation increased the number of CFUs from culture at week 2 Ž6.6–42 fold higher numbers of CFU. but had no influence thereafter Ž0.8–1.5 fold.. One representative experiment out of 5 is shown in Fig. 5. 3.6. Addition of canine stem cell factor to long-term culture Addition of rcan-SCF to long-term culture enhanced CFU growth of cells from culture within the first 3 weeks Ž1.8–68.6 fold.. However, the stromal layer lost confluency thereafter and the yield of CFUs decreased Ž0–0.6 fold.. The results of 4 experiments are shown in Fig. 6. 3.7. Two step long-term culture for inÕestigation of cells after stromaless liquid culture The expansion of marrow cells in the presence of SCF and GM-CSF was not able to increase the number of LTCIC. CFU growth of expanded marrow cells declined rapidly in long-term cultures in contrast to control cultures with fresh marrow cells. Results of 3 independent experiments are shown in Table 4.
4. Discussion Several protocols exist for short-term clonogenic assays using mostly agar or methylcellulose as matrix ŽDexter et al., 1977; Konwalinka et al., 1983; Sutherland et al., 1994. supplemented with fetal calf serum and growth stimuli like PHA-conditioned
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medium, post-irradiation serum or recombinant cytokines ŽHahn et al., 1991; Konwalinka et al., 1984, 1983; Kreja et al., 1988.. Using fresh marrow cells, we found equally good growth using agar or methylcellulose stimulated with SCF, GM-CSF and erythropoietin. The addition of SCF did not only improve the number of colonies but did also enhance the number of cells per colony. If cultured for more than 14 days, it even stimulated colonies with more than 50 000 cells. These HPP-CFC Žhigh proliferative potential colony forming cells. which are derived from more primitive progenitor cells than CFU-GM ŽMcNiece et al., 1987. were not seen with PHA-LCM. However, cells from long-term culture only grew in the presence of human AB-serum, indicating the need of additional growth factors. All cell culture media which were investigated were suitable for long-term culture. IMDM with elevated osmolarity which has been described for human cultures ŽGibson et al., 1995. was less effective than other media with lower osmolarity. Other media were well suited for canine cells and in our experiments Iscove medium seemed to be more stable than RPMI 1640 or McCoy 5A medium. Dexter et al. Ž1984. found that free corticosteroids are responsible for the qualification of a serum for long-term cultures. Schuening et al. Ž1989., however, proposed that well-suited sera did not need addition of steroids and CFU growth can even be detrimental. In our experiments, CFU-growth could be enhanced by addition of hydrocortisone, but the influence of the serum lot was much higher than the influence of hydrocortisone. Addition of fetal calf serum is widely used in human long-term cultures ŽCoulombel et al., 1983; Sutherland et al., 1994. and Carter et al. Ž1990. reported the use of fetal calf serum in canine long-term cultures. We found dose dependent decrease of CFU production when FCS was added to canine long-term cultures confirming observations of Schuening et al. Ž1989.. When FCS was added to cultures, the morphology of the stromal layer changed: macrophages were replaced by fibroblasts and the stromal layer lost confluency. As a consequence, the number of CFU declined rapidly. The structure of the canine long-term culture differs from other species. Here, a dense fibroblastic layer covers early progenitors which can be detected as dark cobblestones under the layer. Differentiating cells are found on top of the fibroblasts and in the supernatant ŽGartner and Kaplan, 1980; Linenberger and Abkowitz, 1992; Ploemacher et al., 1989.. Canine cells are not covered by fibroblasts and can therefore be lost easily during medium change and typical cobblestone areas can hardly be found ŽFig. 7.. Early progenitor cells survive in the presence of a stromal layer. Therefore, it seems to be useful to first establish a stromal layer and then recharge the culture with a second autologous marrow. Dexter et al. Ž1977. and Schuening et al. Ž1989. found improved CFU production with this procedure. We also observed improved CFU growth especially in week 2, but the CFU output returned to control values at the end of the culture. Obviously, the stromal layer is able to support the survival of LTC-IC from the beginning and additional LTC-IC have little effect. The development of recombinant cytokines offers the possibility to substitute the cell derived growth factors and to improve culture conditions. Stem cell factor is one of the earliest acting cytokines on hematopoietic progenitors and allows the survival of canine cells in stroma-free cultures ŽShull et al., 1992.. Moreover, cell-bound stem cell factor can be detected in a canine stromal cell line which enables long-term culture of canine
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Fig. 7. Stromal layer at day 16 of canine long-term culture in Iscove Medium supplemented with 20% of horse serum.
marrow cells ŽHuss et al., 1995.. In our experiments, stem cell factor obviously induced a transformation of the adherent stromal layer cells into non-adherent cells. As a consequence, the stromal layer disappeared completely towards the end of the culture and the number of CFU declined. This transformation has been reported by Huss et al, who postulated a differentiation of CD34 negative stromal cells into CD34 positive hemopoietic cells ŽHuss et al., 1995.. Ex vivo expansion of marrow cells mostly has been evaluated for the increase of cells or CFU. We could show the detrimental effect of SCFrGM-CSF on LTCIC in liquid culture, an observation reported by other groups for different factor combinations ŽShull et al., 1992.. Long-term culture experiments can give a more precise answer on the marrow repopulating ability of ex vivo expanded marrow than short-term CFU assays. In conclusion, canine marrow cells show special growth requirements after long-term culture which differ from fresh marrow cells. Addition of stem cell factor leads to a decrease of CFU production possibly due to an outgrow of early progenitor cells. The long-term culture system is suitable for the examination of the ex vivo expansion of very early progenitor cells. Acknowledgements We thank Karin Oettrich, Sabine Sagebiel-Kohler, Michael Hagemann, Christine Voss and Andrea von Arco-Zinneberg for excellent technical assistance. The study was supported by the Wilhelm-Sander-Foundation, Neustadt a.d. Donau, Germany.
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References Aihara, M., Sikic, B.I., Blume, K.G., Chao, N.J., 1990. Assessment of purging with multidrug resistance ŽMDR. modulators and VP-16: results of long-term marrow culture. Exp. Hematol. 18, 940–944. Allen, T.D., Dexter, T.M., 1984. The essential cells of the hemopoietic microenvironment. Exp. Hematol. 12, 517–521. Berardi, A.C., Wang, A., Levine, J.D., Lopez, P., Scadden, D.T., 1995. Functional isolation and characterization of human hematopoietic stem cells. Science 267, 104–108. Bernstein, I.D., Andrews, R.G., Zsebo, K.M., 1991. Recombinant human stem cell factor enhances the formation of colonies by CD34q and CD34qlin- cells, and the generation of colony-forming cell progeny from CD34qlin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocytemacrophage colony-stimulating factor. Blood 77, 2316–2321. Carter, R.F., Kruth, S.A., Valli, V.E., Dube, I.D., 1990. Long-term culture of canine marrow: cytogenetic evaluation of purging of lymphoma and leukemia. Exp. Hematol. 18, 995–1001. Cobbold, S., Metcalfe, S., 1994. Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop ŽCLAW.. Tissue Antigens 43, 137–154. Coulombel, L., Eaves, A.C., Eaves, C.J., 1983. Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62, 291–297. Dexter, T.M., Allen, T.D., Lajtha, L.G., 1977. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell Physiol. 91, 335–344. Dexter, T.M., Spooncer, E., Simmons, P., Allen, T.D., 1984. Long-term marrow culture: an overview of techniques and experience. Kroc. Found. Ser. 18, 57–96. Fraser, C.C., Eaves, C.J., Szilvassy, S.J., Humphries, R.K., 1990. Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells. Blood 76, 1071–1076. Galvani, D.W., Cawley, J.C., 1990. The effects of alpha interferon on human long-term bone marrow culture. Leuk. Res. 14, 525–531. Gartner, S., Kaplan, H.S., 1980. Long-term culture of human bone marrow cells. Proc. Natl. Acad. Sci. U.S.A. 77, 4756–4759. Gibson, F.M., Scopes, J., Daly, S., Rizzo, S., Ball, S.E., Gordon Smith, E.C., 1995. IL-3 is produced by normal stroma in long-term bone marrow cultures. Br. J. Hematol. 90, 518–525. Greenberger, J.S., 1978. Sensitivity of corticosteroid-dependent insulin-resistant lipogenesis in marrow preadipocytes of obese-diabetic Ždbrdb. mice. Nature 275, 752–754. Hahn, J., Kolb, H.J., Schumm, M., Beisser, K., Ellwart, J., Rieber, P. et al., 1991. Immunological characterization of canine hematopoietic progenitor cells. Ann. Hematol. 63, 223–226. Huss, R., Hong, D.S., McSweeney, P.A., Hoy, C.A., Deeg, H.J., 1995. Differentiation of canine bone marrow cells with hemopoietic characteristics from an adherent stromal cell precursor. Proc. Natl. Acad. Sci. U.S.A. 92, 748–752. Konwalinka, G., Peschel, C., Geissler, D., Boyd, J., Tomaschek, B., Odavic, R. et al., 1983. Myelopoiesis of human bone marrow cells in a micro-agar culture system: comparison of two sources of colony stimulating activity ŽCSA.. Int. J. Cell Cloning 1, 401–411. Konwalinka, G., Peschel, C., Boyd, J., Geissler, D., Ogriseg, M., Odavic, R. et al., 1984. A miniaturized agar culture system for cloning human erythropoietic progenitor cells. Exp. Hematol. 12, 75–79. Kreja, L., Baltschukat, K., Nothdurft, W., 1988. Growth of erythroid burst-forming units ŽBFU-E. in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs. Exp. Hematol. 16, 647–651. Ladiges, W.C., Storb, R., Thomas, E.D., 1990. Canine models of bone marrow transplantation. Lab. Anim. Sci. 40, 11–15. Linenberger, M.L., Abkowitz, J.L., 1992. Studies in feline long-term marrow culture: hematopoiesis on normal and feline leukemia virus infected stromal cells. Blood 80, 651–662. McNiece, I.K., Williams, N.T., Johnson, G.R., Kriegler, A.B., Bradley, T.R., Hodgson, G.S., 1987. Generation of murine hematopoietic precursor cells from macrophage high-proliferative-potential colony-forming cells. Exp. Hematol. 15, 972–977.
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E. Neuner et al.r Veterinary Immunology and Immunopathology 61 (1998) 1–16
Moore, M.A., 1991. Review: Stratton lecture 1990. Clinical implications of positive and negative hematopoietic stem cell regulators. Blood 78, 1–19. Ploemacher, R.E., van der Sluijs, J.P., Voerman, J.S., Brons, N.H., 1989. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763. Schuening, F.G., Storb, R., Meyer, J., Goehle, S., 1989. Long-term culture of canine bone marrow cells. Exp. Hematol. 17, 411–417. Schuening, F.G., Kawahara, K., Miller, A.D., To, R., Goehle, S., Stewart, D. et al., 1991. Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. Blood 78, 2568–2576. Schumm, M., Gunther, W., Kolb, H.J., Kremmer, E., Vogl, C., Wilmanns, W. et al., 1995. Cytokine ¨ requirements for the in vitro expansion of purified canine progenitor cells. Acta Hematol. 93, 153. Shull, R.M., Suggs, S.V., Langley, K.E., Okino, K.H., Jacobsen, F.W., Martin, F.H., 1992. Canine stem cell factor Žc-kit ligand. supports the survival of hematopoietic progenitors in long-term canine marrow culture. Exp. Hematol. 20, 1118–1124. Sutherland, H.J., Eaves, C.J., Eaves, A.C., Dragowska, W., Lansdorp, P.M., 1989. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74, 1563–1570. Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C., Eaves, C.J., 1990. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. U.S.A. 87, 3584–3588. Sutherland, H.J., Eaves, C.J. Freshney, R.I., Pragnell, I.B., Freshney, M.G. ŽEds... Long-term culture of human myeloid cells, 139-62. Culture of Hematopoietic Cells. Wiley-Liss, New York, 1994. Verfaillie, C., Blakolmer, K., McGlave, P., 1990. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J. Exp. Med. 172, 502–509.