Adult mesenchymal stem cells with multilineage differentiation potentially exist in the bone marrow, but have also been isolated from the peripheral blood. The differentiation of stem cells after leaving their niches depends predominately on the local milieu and its new microenvironment, and is facilitated by soluble factors but also by the close cell-cell interaction in a three-dimensional tissue or organ system. We have isolated CD34-negative, mesenchymal stem cell lines from human bone marrow and peripheral blood and generated monoclonal cell populations after immortalization with the SV40 large T-antigen. The cultivation of those adult stem cell clones in an especially designed in vitro environment, including self-constructed glass capillaries with defined growth conditions, leads to the spontaneous establishment of pleomorphic three-dimensional cell aggregates (spheroids) from the monoclonal cell population, which consist of cells with an osteoblast phenotype and areas of mineralization along with well-vascularized tissue areas. Modifications of the culture conditions favored areas of bone-like calcifications. After the transplantation of the at least partly mineralized human spheroids into different murine soft tissue sites but also a dorsal skinfold chamber, no further bone formation could be observed, but angiogenesis and neovessel formation prevailed instead, enabling the transplanted cells and cell aggregates to survive. This study provides evidence that even monoclonal adult human CD34-negative stem cells from the bone marrow as well as peripheral blood can potentially differentiate into different mesenchymal tissues depending on the local milieu and responding to the needs within the microenvironment.

1.
Asahara, T., T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li, B. Witzenbichler, G. Schatteman, J.M. Isner (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964–967.
2.
Baron, M.H. (2001) Molecular regulation of embryonic hematopoiesis and vascular development: a novel pathway. J Hematother Stem Cell Res 10: 587–594.
3.
Bianchi, G., A. Muraglia, A. Daga, G. Corte, R. Cancedda, R. Quarto (2001) Microenvironment and stem cell properties of bone marrow-derived mesenchymal stem cells. Wound Repair Regen 9: 460–466.
4.
Conrad, C., B. Göttgens, S. Kinston, J. Ellwart, R. Huss (2002) GATA transcription in a small rhodamine 123(low)CD34(+) subpopulation of a peripheral blood-derived CD34(–)CD105(+) mesenchymal cell line. Exp Hematol 30: 887–895.
5.
Dennis, J.E., J.P. Carbillet, A.I. Caplan, P. Charbord (2002) The STRO-1+ marrow cell population is multipotential. Cells Tissue Organs 170:73–82.
6.
Festuccia, C., D. Giunciuglio, F. Guerra, I. Villanova, A. Angelucci, P. Manduca, A. Teti, A. Albini, M. Bologna (1999) Osteoblasts modulate secretion of urokinase-type plasminogen activator (uPA) and matrixmetalloproteinase-9 (MMP-9) in human prostate cancer cells promoting migration and matrigel invasion. Oncol Res 11: 17–31.
7.
Gehring, M., A.R.F. Rodaway, G. Göttgens, R.K. Patient, A.R. Green (1998) The SCL gene specifies haemangioblast development from early mesoderm. EMBO J 17: 4029–4045.
8.
Grant, M.B., W.S. May, S. Caballero, G.A.J. Brown, S.M. Guthrie, R.N. Mames, B.J. Byrne, T. Vaught, P.E. Spoerri, A.B. Peck, E.W. Scott (2002) Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 8: 607–612.
9.
Harraz, M., C. Jiao, H.D. Hanlon, R.S. Hartley, G.C. Schatteman (2001) CD34-blood-derived human endothelial cell progenitors. Stem Cells 19: 304–312.
10.
Harris CT, L.F. Cooper (2004) Comparison of bone graft matrices for human mesenchymal stem cell-directed osteogenesis. J Biomed Mater Res 68: 747–755.
11.
Hartlapp, I., R. Abe, R.W. Saeed, T. Peng, W. Voelter, R. Bucala, C.N. Metz (2001) Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo. FASEB J 15: 2215–2224.
12.
Hattori, K., B. Heissig, Y. Wu, S. Dias, R. Tejada, B. Ferris, D.J. Hicklin, Z. Zhu, P. Bohlen, L. Witte, J. Hendrikx, N.R. Hackett, R.G. Crystal, M. Moore, Z. Werb, D. Lyden, S. Rafii (2002) Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1+ stem cells from bone-marrow microenvironment. Nat Med 8: 841–849.
13.
Horwitz, E.M., D.J. Prockop, L.A. Fitzpatrick, W.W.K Koo, P.L. Gordon, M. Neel, M. Sussman, P. Orchard, J.C. Marx, R.E. Pyeritz, M. Brenner (1999) Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5: 111–116.
14.
Huang, X.L., N. Takakura, T. Suda (1999) In vitro effects of angiopoietins and VEGF on hematopoietic and endothelial cells. Biochem Biophys Res Commun 264: 133–138.
15.
Huss, R., D.S. Hong, P.A. McSweeney, C.A. Hoy, H.J. Deeg (1995) Differentiation of canine marrow cells with hematopoietic characteristics from an adherent stromal cell precursor. Proc Natl Acad Sci USA 92: 748–752.
16.
Huss, R. (1997) Functional and ultrastructural studies on the burst of c-kit ligand containing secretory vesicles. Acta Histochem 99: 111–116.
17.
Huss, R. (1998) CD34-negative stem cells as the earliest precursors of hematopoietic progeny. Exp Hematol 26: 1022–1023.
18.
Huss R. (2000) Isolation of primary and immortalized CD34– hematopoietic and mesenchymal stem cells from various sources. Stem Cells 18: 1–9.
19.
Huss, R., C. Lange, E.M. Weissinger, H.J. Kolb, K. Thalmeier (2000) Evidence of peripheral blood-derived, plastic adherent CD34–/low hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem Cells 18: 252–260.
20.
Huss, R., M. Heil, S. Moosmann, T. Ziegelhöffer, S. Sagebiel, S. Seliger, S. Kinston, B. Göttgens (2004) Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL+ multipotent adult progenitor cell (MAP-C) clones from peripheral blood. J Vasc Res 41: 422–431.
21.
Kale, S., S. Biermann, C. Edwards, C. Tarnowski, M. Morris, M.W. Long (2000) Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat Biotechnol 18: 929–930.
22.
Kaushal, S., G.E. Amiel, K. Guleserian, O.M. Shapira, T. Perry, F.W. Sutherland, E. Rabkin, A.M. Moran, F.J. Schoen, A. Atala, S. Soker, J. Bischoff, J.E. Mayer (2001) Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med 7: 1035–1040.
23.
Korff, T., S. Kimmina, G. Martiny-Baron, H.G. Augustin (2001) Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J 15: 447–457.
24.
Lange, C., C. Kaltz, K. Thalmeier, H.J. Kolb, R. Huss (1999) Long-term hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34–, Sca-1+, Thy-1 low, c-kit+ stem cell line. J. Hematother Stem Cell Res 8: 335–342.
25.
Lehr, H.A., M. Leunig, M.D. Menger, D. Nolte, K. Messmer (1993) Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol 143: 1055–1062.
26.
Li, Y., T. Ma, D.A. Kniss, S.T. Yang, L.C. Lasky (2001) Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro stimulation of the marrow microenvironment. J Hematother Stem Cell Res 10: 355–368.
27.
Loges, S., B. Fehse, M.A. Brockmann, K. Lamszus, M. Butzal, M. Guckenbiehl, G. Schuch, S. Ergün, U. Fischer, A.R. Zander, D.K Hossfeld, W. Fiedler, U.M. Gehling (2004) Identification of the adult human hemangioblast. Stem Cells Dev 13: 229–242.
28.
Lutton, A., M. Tjwa, L. Moons, Y. Wu (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 8: 831–840.
29.
Lyden, D., K. Hattori, S. Dias, C. Coast, P. Blaikie, L. Butros, A. Chadburn, B. Heissig, W. Marks, L. Witte, Y. Wu, D.J. Hicklin, Z. Zhu, N.R. Hackett, R.G. Crystal, M.A.S. Moore, K.A. Hajjar, K. Manova, R. Benezra, S. Rafii (2001) Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 7: 1194–1201.
30.
Mackay, A.M., S.C. Beck, J.M. Murphy, F.P. Barry, C.O. Chichester, M.F. Pittenger (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4: 415–428.
31.
Masuda, H., C. Kalka, T. Asahara T (2000) Endothelial progenitor cells for regeneration. Hum Cell 13: 153–160.
32.
Menger, M.D., S. Jaeger, P. Walter, F. Hammersen, K. Messmer (1990) A novel technique for studies on the microvasculature of transplanted islets of Langerhans in vivo. Int J Microcirc Clin Exp 9: 103–117.
33.
Otani, A., K. Kinder, K. Ealt, F.J. Otero, P. Schimmel, M. Friedlander (2002) Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis. Nat Med 8: 1004–1010.
34.
Pelosi, E., Valtieri M., S. Coppola, R. Botta, M. Gabbianelli, V. Lulli, G. Marziali, B. Masella, R. Muller, C. Sgadari, U. Testa, G. Bonanno, C. Peschle (2002) Identification of the hemangioblast in postnatal life. Blood 100: 3203–3208.
35.
Pipp, F., M. Heil, K. Issbrücker, T. Ziegelhoeffer, S. Martin, J. van den Heuvel, H.H. Weich, B. Fernandez, G. Golomb, P. Carmeliet, W. Schaper, M. Clauss (2003) VEGFR-1-selective VEGF homologue PIGF is arteriogenic: Evidence for a monocytemediated mechanism. Circ Res 92: 378–385.
36.
Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D. Marshak (1999) Multilineage potential of adult human mesenchymal stem cells. Science 276: 318–321.
37.
Singer, J.W., A. Keating, J. Cuttner, A.M. Gowan, R. Jacobson, P.D. Killen, J.W. Moohr, V. Najfeld, J. Powell, J. Sanders (1984) Evidence for a stem cell common to hematopoiesis and its in vitro microenvironment: studies of patients with clonal hematopoietic neoplasia. Leuk Res 8: 535–545.
38.
Shi, S., S. Gronthos, S. Chen, A. Reddi, C.M. Counter, P.G. Robey, C.Y. Wang (2002) Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase. Nat Biotechnol 20: 587–591.
39.
Thalmeier, K., P. Meissner, G. Reisbach, L. Hültner, B.T. Mortenson, A. Brechtel, R.A. Oostendorp, P. Dörmer (1996) Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines. Exp Hematol 24: 1–9.
40.
Thalmeier, K., P. Meissner, S. Moosmann, S. Sagebiel, I. Wiest, R. Huss (2001) Mesenchymal differentiation and organ distribution of established human stromal cell lines in NOD/SCID mice. Acta Haematol 105: 150–165.
41.
Vollmar, B., M.W. Laschke, R. Rohan, J. Koenig, M.D. Menger (2001) In vivo imaging of physiological angiogenesis from immature to preovulatory ovarian follicles. Am J Pathol 159: 1661–1670.
42.
Zhang, S.C., M. Wernig, I.D. Duncan, O. Brüstle, J.A. Thomson (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19: 1129–1133.
43.
Ziegler, B.L., M. Valtieri, G.A. Porada, R. De Maria, R. Muller, B. Masella, M. Gabbianelli, I. Casella, E. Pelosi, T. Bock, E.D. Zanjani, C. Peschle (1999) KDR receptor: a key marker defining hematopoietic stem cells. Science 28: 1553–1558.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.