Molecular analysis of the hematopoiesis supporting osteoblastic cell line U2-OS.
OBJECTIVE
Osteoblasts play an important role in regulating hematopoiesis in the bone marrow. Here we show that U2-OS, a widely used osteoblastic cell line derived from an osteosarcoma, has the capacity to support proliferation of human hematopoietic progenitor cells in vitro. In this study, U2-OS cells are characterized at the molecular level to unravel the molecular mechanisms underlying the support of hematopoiesis.


METHODS
U2-OS was analyzed in great detail using RT-PCR and flow cytometry. In addition, a cDNA library was constructed and randomly sequenced to obtain insight in the repertoire of expressed molecules.


RESULTS
A broad panel of growth factors and cytokines is expressed by U2-OS. TGF-beta, GM-CSF, c-kit ligand, and IL-7 are produced constitutively and IL-1beta, IL-6, IL-8, TNF-alpha, IFN-gamma, and MIP1-alpha are upregulated upon stimulation. In addition to those, mRNAs of the CC chemokine LARC and leukemia inhibitory factor were identified. U2-OS cells express high levels of beta1-integrins at the cell surface: VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, and the integrin alphavbeta3. Besides integrins, ALCAM and NCAM are detected on the cell surface of U2-OS. Interestingly, we show that CD34(+) progenitor cells expressing ALCAM are highly proliferative when compared with CD34(+) ALCAM(low) cells, hinting at a role for ALCAM in anchoring progenitor cells to the bone marrow stroma. Interestingly, random sequencing of an U2-OS cDNA library yielded almost 10% of novel cDNAs with a potential role in hematopoiesis. The involvement of these novel molecules in hematopoiesis is an interesting target for future investigations.


CONCLUSIONS
We conclude that U2-OS supports outgrowth of hematopoietic progenitor cells and accordingly expresses adhesion molecules and growth factors and a number of novel, as yet uncharacterized potentially interesting genes.
Nelissen
J M
JM
Department of Tumor Immunology, University Hospital Nijmegen St. Radboud, Nijmegen, The Netherlands.
Torensma
R
R
Pluyter
M
M
Adema
G J
GJ
Raymakers
R A
RA
van Kooyk
Y
Y
Figdor
C G
CG
D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000096:biosynthesis / Q000235:genetics; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D006133:Growth Substances / Q000096:biosynthesis / Q000235:genetics; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D016023:Integrins / Q000096:biosynthesis; D015759:Ionomycin / Q000494:pharmacology; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis; D054367:Receptors, CXCR3; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D019707:Receptors, Chemokine / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020410:Activated-Leukocyte Cell Adhesion Molecule; D015703:Antigens, CD; D018952:Antigens, CD34; D000945:Antigens, Differentiation, T-Lymphocyte; C062279:CD6 antigen; C515187:CXCR3 protein, human; D015815:Cell Adhesion Molecules; D016207:Cytokines; D006133:Growth Substances; D018960:Hyaluronan Receptors; D016023:Integrins; D012333:RNA, Messenger; D054367:Receptors, CXCR3; D011956:Receptors, Cell Surface; D019707:Receptors, Chemokine; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate