Search tips
Search criteria 


Logo of jexpmedHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Exp Med. 1996 December 1; 184(6): 2301–2310.
PMCID: PMC2196367

Preferential Proliferation of Murine Colony-forming Units in Culture in a Chemically Defined Condition with a Macrophage Colony-stimulating Factor–negative Stromal Cell Clone


The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell–dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220 TER119 commonβ+ IL-2Rγ+ gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2–transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extracellular signals.

Full Text

The Full Text of this article is available as a PDF (4.2M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
1. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haematopoietic stem cells in vitro. J Cell Physiol. 1977;91:335–344. [PubMed]
2. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential stem cell differentiation by purified polypeptides. Nature (Lond) 1988;336:688–690. [PubMed]
3. Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature (Lond) 1988;336:684–686. [PubMed]
4. Tsuji K, Zsebo KM, Ogawa M. Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit. Blood. 1991;78:1223–1229. [PubMed]
5. Mayani H, Little MT, Dragowska W, Thornbury G, Lansdorp PM. Differential effects of the hematopoietic inhibitors MIP-1alpha, TGF-beta, and TNF-alpha on cytokine-induced proliferation of subpopulations of CD34+cells purified from cord blood and fetal liver. Exp Hematol. 1995;23:422–427. [PubMed]
6. Cicuttini FM, Welch KL, Boyd AW. The effect of cytokines on CD34+ Rh-123high and lowprogenitor cells from human umbilical cord blood. Exp Hematol. 1994;22:1244–1251. [PubMed]
7. Shiohara M, Koike K, Nakahata T, Komiyama A. Hematopoietic progenitors and synergism of interferongamma and stem cell factor. Leuk Lymphoma. 1994;14:203–211. [PubMed]
8. Mayani H, Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines. Blood. 1993;82:2664–2672. [PubMed]
9. Verfaillie CF. Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood. 1993;82:2045–2053. [PubMed]
10. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol. 1995;23:461–469. [PubMed]
11. Verfaillie CM, Catanzarro PM, Li WN. Macrophage inflammatory protein 1α, interleukin 3, and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med. 1994;179:643–649. [PMC free article] [PubMed]
12. Migliaccio AR, Visser JW. Proliferation of purified murine hematopoietic stem cells in serum-free cultures stimulated with purified stem-cell-activating factor. Exp Hematol. 1986;14:1043–1048. [PubMed]
13. Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. Functional isolation and characterization of human hematopoietic stem cells. Science (Wash DC) 1995;267:104–108. [PubMed]
14. Greenberger JS, Sakakeeny MA, Humphries RK, Evans CJ, Eckner RJ. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci USA. 1983;80:2931–2935. [PubMed]
15. Scolnick EM, Weeks MO, Shih TY, Ruscetti SK, Dexter TM. Markedly elevated levels of an endogenous src protein in a hematopoietic precursor cell line. Mol Cell Biol. 1981;1:66–74. [PMC free article] [PubMed]
16. Palacios R, Karasuyama H, Rolink A. Ly1+PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo. EMBO (Eur Mol Biol Organ) J. 1987;6:3687–3693. [PubMed]
17. Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science (Wash DC) 1994;265:1098–1101. [PubMed]
18. Chen CA, Okayama H. Calcium phosphatemediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988;6:632–638. [PubMed]
19. Carraway III, K.L., and L.C. Cantley. A neu acquaintance for ErbB3 and ErbB4: a role for receptor heterodimerization in growth signaling. Cell. 1994;78:5–8. [PubMed]
20. Kalthoff H, Roeder C, Gieseking J, Humburg I, Schmiegel W. Inverse regulation of human ERBB2 and epidermal growth factor receptors by tumor necrosis factor alpha. Proc Natl Acad Sci USA. 1993;90:8972–8976. [PubMed]
21. Yamamoto T, Nishida T, Miyajima N, Kawai S, Ooi T, Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the srcgene family. Cell. 1983;35:71–78. [PubMed]
22. Kincade PW, Lee G, Scheid MP, Blum MD. Characterization of murine colony-forming B cells II. Limits to in vitro maturation, Lyb-2 expression, resolution of IgD+subsets, and further population analysis. J Immunol. 1980;124:947–953. [PubMed]
23. Lee G, Namen AE, Gillis S, Ellingsworth LR, Kincade PW. Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-β J Immunol. 1989;142:3875–3883. [PubMed]
24. Yasunaga M, Wang F-H, Kunisada T, Nishikawa S, Nishikawa S-I. Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kitin a fully defined medium. J Exp Med. 1995;182:315–323. [PMC free article] [PubMed]
25. Ikuta K, Kina T, MacNeil I, Uchida N, Peault B, Chein YH, Weissman IL. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell. 1990;62:863–874. [PubMed]
26. Nishikawa S, Kusakabe M, Yoshinaga K, Ogawa M, Hayashi SI, Kunisada T, Era T, Sakakura T, Nishikawa S-I. In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit dependency during melanocyte development. EMBO (Eur Mol Biol Organ) J. 1991;10:2111–2118. [PubMed]
27. Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi SI, Kunisada T, Sudo T, Kina T, Nakauchi H, Nishikawa S-I. Expression and function of c-kitin hemopoietic progenitor cells. J Exp Med. 1991;174:63–71. [PMC free article] [PubMed]
28. Sudo T, Nishikawa S, Ohno N, Akiyama N, Tamakoshi M, Yoshida H, Nishikawa S-I. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proc Natl Acad Sci USA. 1993;90:9125–9129. [PubMed]
29. Sudo T, Nishikawa S, Ogawa M, Kataoka H, Ohno N, Izawa A, Hayashi S-I, Nishikawa S-I. Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M. Oncogene. 1995;11:2469–2476. [PubMed]
30. Taga T, Kishimato T. Signaling mechanisms through cytokine receptors that share signal transducing receptor components. Curr Opin Immunol. 1995;7:17–23. [PubMed]
31. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86:1243–1254. [PubMed]
32. Yin T, Taga T, Tsang ML, Yasukawa K, Kishimoto T, Yang YC. Involvement of IL-6 signal transducer gp130 in IL-11–mediated signal transduction. J Immunol. 1993;151:2555–2561. [PubMed]
33. Saito T, Taga T, Miki D, Futatsugi K, Yawata H, Kishimoto T, Yasukawa K. Preparation of monoclonal antibodies against the IL-6 signal transducer, gp130, that can inhibit IL-6-mediated functions. J Immunol Methods. 1993;163:217–223. [PubMed]
34. Gorman DM, Itoh N, Kitamura T, Schreurs J, Yonehara S, Yahara I, Arai K, Miyajima A. Cloning and expression of a gene encoding an interleukin 3 receptor-like protein: identification of another member of the cytokine receptor gene family. Proc Natl Acad Sci USA. 1990;87:5459–5463. [PubMed]
35. Takaki S, Mita S, Kitamura T, Yonehara S, Yamaguchi N, Tominaga A, Miyajima A, Takatsu K. Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein AIC2B is a component of the high affinity interleukin-5 receptor. EMBO (Eur Mol Biol Organ) J. 1991;10:2833–2838. [PubMed]
36. Kondo M, Takeshita T, Ishii N, Nakamura M, Watanabe S, Arai K, Sugamura K. Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4. Science (Wash DC) 1993;262:1874–1877. [PubMed]
37. Kimura Y, Takeshita T, Kondo M, Ishii N, Nakamura M, Van-Snick J, Sugamura K. Sharing of the IL-2 receptor gamma chain with the functional IL-9 receptor complex. Int Immunol. 1995;7:115–120. [PubMed]
38. Magli MC, Iscove NN, Odartchenko N. Transient nature of early haematopoietic spleen colonies. Nature (Lond) 1982;295:527–529. [PubMed]
39. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science (Wash DC) 1988;241:58–62. [PubMed]
40. Nishikawa S, Nakasato M, Takakura N, Ogawa M, Kodama H, Nishikawa S-I. Stromal cell-dependent bone marrow culture with a nearly protein-free defined medium. Immunol Lett. 1994;40:163–169. [PubMed]
41. Kodama H, Nose M, Niida S, Nishikawa S, Nishikawa S-I. Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp Hematol. 1994;22:979–984. [PubMed]
42. Yoshida H, Hayashi S-I, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S-I. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature (Lond) 1990;345:442–444. [PubMed]
43. Hayman MJ, Meyer S, Martin F, Steinlein P, Beug H. Self-renewal and differentiation of normal avian erythroid progenitor cells: regulatory roles of the TGFα/cErbB and SCF/c-kit receptors. Cell. 1993;74:157–169. [PubMed]
44. Pain B, Woods CM, Saez J, Flickinger T, Raines M, Peyrol S, Moscovici C, Moscovici MG, Kung H-J, Jurdic P, et al. EGF-R as a hematopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal. Cell. 1991;65:37–46. [PubMed]
45. Heimfeld S, Weissman IL. Characterization of several classes of mouse hematopoietic progenitor cells. Curr Top Microbiol Immunol. 1992;177:95–105. [PubMed]
46. Heimfeld S, Weissman IL. Development of mouse hematopoietic lineages. Curr Top Dev Biol. 1991;25:155–175. [PubMed]
47. Morrison SJ, Lagasse E, Weissman IL. Demonstration that THYlosubsets of mouse bone marrow that express high levels of lineage markers are not significant hematopoietic progenitors. Blood. 1994;83:3480–3490. [PubMed]
48. Boettiger, D., S. Anderson, and T.M. Dexter. Effect of src infection on long-term marrow cultures: increased selfrenewal of hemopoietic progenitor cells without leukemia. Cell. 36:763–773.
49. Spooncer E, Heyworth CM, Dunn A, Dexter TM. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation. 1986;31:111–118. [PubMed]
50. Palacios R, Samaridis J. Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro. Mol Cell Biol. 1992;12:518–530. [PMC free article] [PubMed]
51. Palacios R, Samaridis J. Bone marrow clones representing an intermediate stage of development between hematopoietic stem cells and pro-T-lymphocyte or pro-B-lymphocyte progenitors. Blood. 1993;81:1222–1238. [PubMed]

Figures and Tables

Figure 2
Growth kinetics and c-Kit expression of BM cells cultured on OP9 stroma cells with mSFO2 containing EGF and SCF. (A) Unfractionated BM cells (105) from C57BL/6 mice were plated on OP9 stroma cells in mSFO2 containing 30 ng/ml EGF (open circle) ...
Figure 1
Phenotypes of BM cells cultured under various conditions. Unfractionated BM cells (105) from C57BL/6 mouse were plated onto the OP9 stroma cells and cultured in mSFO2 containing 10% FCS (A), 10 ng/ml bFGF, and 100 ng/ml SCF (B), or 30 ng/ml EGF and 100 ...
Figure 3
Growth kinetics of BM cells cultured on the OP9/erbB2 stromal layer and surface phenotype of recovered cells. (A) Unfractionated BM cells (105) derived from C57BL/6 were plated on OP9/erbB2 stroma cells in mSFO2 supplemented with 100 ng/ml SCF. On the ...
Figure 4
Immediate differentiation of cultured c-Kit+Mac-1dull cell in medium-containing serum. Unfractionated BM cells were cultured on OP9/erbB2, as described in the legend to Fig. Fig.3.3. 21 d later, the cells were transferred onto a fresh ...
Table 1
Frequency of Colony-forming Cells

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press