In vitro Expansion von hämatopoetischen Stamm- und Progenitorzellen durch Komponenten der Stammzellnische

A successful in vitro expansion of hematopoietic stem- and progenitor cells (HPC) is important for reliable cord blood stem cell transplantations with low cell numbers. For in vitro cultivation of HPC, there are no safe protocols existing which provide the ability to control self-renewal and differentiation. In this study, analyses of cell divisions and immunophenotype demonstrated that co-culture with mesenchymal stromal cells (MSC) especially stimulates proliferation of the primitive HPC subset (CD34+ CD133+ CD38-) and also maintenance of a more primitive immunophenotype. Without co-culture, the cells rapidly undergo maturation. In particular, early MSC passages were suitable for culture-expansion of HPC. The expression of specific adhesion proteins (N-Cadherine and VCAM1) and extracellular matrix binding-proteins (CD44 and ITGB1) was specifically inhibited by siRNA knockdown resulting in a higher proportion of quiescent HPC as well as maintenance of primitive immunophenotype. Subsequently, the effect of addition of 5 previously described cytokines (SCF, TPO, FGF, ANGPTL5 and IGFBP2) on proliferation and immunophenotype of HPC was analyzed in 20 different combinations with and without MSC-co-culture. Addition of SCF, TPO and FGF enhanced expansion of HPC without and with MSC-co-culture. Furthermore, transplantation experiments in a murine model showed an enhancement of hematopoietic reconstitution with co-cultured HPC. As a next step, factors were investigated that might regulate and control regeneration of hematopoiesis after chemotherapy. Serum was obtained from patients before and at different time points after autologous stem cell transplantation. 10% of serum was added to culture medium to test its influence on proliferation and immunophenotype of HPC cultured with and without MSC. Serum isolated 4 to 11 days after transplantation significantly enhanced proliferation, colony-stimulating activity and maintenance of primitive immunophenotype. This stimulating effect was hardly detectable with serum isolated more than 14 days after transplantation. Chemokine arrays were used to analyze the chemokine composition of serum samples. Among 174 chemokines, 8 (including 3 PDGF isoforms) were significantly down-regulated during hematopoietic stimulation on day 8, only concentration of monocyte-chemotactic protein 1 (MCP1) was increased. In conclusion, these results verify that co-culture with MSC enhances HPC-proliferation together with maintenance of stem cell function. This effect is further promoted by suitable chemokines. In addition, the hematopoietic reconstitution seems to be controlled through systemic mechanisms of regulation. Therefore, the results of this study further contribute to a better understanding of the regulative and controlling mechanisms in the hematopoietic stem cell niche.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2011

Author dc:creator

• Walenda, Thomas

Contributors dc:contributor

• Wagner, Wolfgang

• info:eu-repo/classification/ddc/570
• Hämatopoese
• Blutstammzelle
• Cytologie
• Zelldetermination
• Zelldifferenzierung
• Zellteilung
• Zelltransplantation
• Zellproteine
• Leukämie
• Biowissenschaften, Biologie
• hematopoietic stem cells
• mesenchymal stem cells
• stem cell niche
• in vitro expansion
• leukemia

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114528%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A52292