1. Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification.
- Author
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Kokkaliaris KD, Drew E, Endele M, Loeffler D, Hoppe PS, Hilsenbeck O, Schauberger B, Hinzen C, Skylaki S, Theodorou M, Kieslinger M, Lemischka I, Moore K, and Schroeder T
- Subjects
- Animals, Cell Culture Techniques, Cell Line, Cell Survival drug effects, Cell Survival genetics, Chondroitin Sulfate Proteoglycans genetics, Chondroitin Sulfate Proteoglycans pharmacology, Extracellular Matrix Proteins genetics, Extracellular Matrix Proteins pharmacology, Hematopoietic Stem Cells cytology, Male, Mice, Mice, Transgenic, Stromal Cells cytology, Stromal Cells metabolism, Time Factors, Chondroitin Sulfate Proteoglycans metabolism, Extracellular Matrix Proteins metabolism, Hematopoietic Stem Cells metabolism
- Abstract
The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo., (© 2016 by The American Society of Hematology.)
- Published
- 2016
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