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Hematopoiesis
Secreted factors from mouse embryonic fibroblasts maintain repopulating function of single cultured hematopoietic stem cells
Romero Marquez,1,2* Franziska Hettler,1* Renate Hausinger,1 Christina Schreck,1 Theresa Landspersky,1 Lynette Henkel,3 Corinne Angerpointner,3 Ihsan E. Demir,2 Matthias Schiemann,3 Florian Bassermann,1,4 Katharina S. Götze,1,4 Rouzanna Istvánffy1,2# and Robert A.J. Oostendorp1#
1Technical University of Munich, Klinikum Rechts der Isar, Clinic and Polyclinic for Internal Medicine III, Munich; 2Technical University of Munich, Klinikum rechts der Isar, Department of Surgery, Munich; 3Technical University of Munich, Flow Cytometry Unit of the Technical University Munich, Institute for Medical Microbiology, Immunology and Hygiene (CyTUM-MIH), Munich and 4German Cancer Consortium (DKTK), Heidelberg, Germany
*SRM and FH contributed equally as co-first authors. #RI and RAJO contributed equally as co-senior authors.
ABSTRACT
Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mecha- nisms. We previously demonstrated that proliferation of murine hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells.1 Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypic identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth fac- tors, conditioned medium from mouse embryonic fibroblasts increased the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, with both two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supported self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust, alternative, serum-free source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.
Introduction
A major challenge in hematology is to decipher through which mechanisms and conditions hematopoietic stem cells (HSC) are maintained in the bone marrow. An additional promise of the efforts to meet this challenge is the identification of niche factors, which could be utilized to supplement protocols to expand murine and human HSC ex vivo. Although many investigators have described possible mediators of HSC maintenance in vivo, the precise factors or their combinations are still to be elucidated in detail. Also, whereas in most culture protocols, the expansion of hematopoietic colony-forming cells can readily be achieved, main- taining or expanding HSC with long-term repopulation (LTR) capacity in numbers relevant for clinical applications is a subject of intense investigation.2 Hence, there is a continued need to understand how HSC are maintained by the bone marrow niche and to determine which factors are involved.
Ferrata Storti Foundation
Haematologica 2021 Volume 106(10):2633-2640
Correspondence:
ROBERT A.J. OOSTENDORP
robert.oostendorp@tum.de
Received: February 6, 2020. Accepted: November 10, 2020. Pre-published: November 19, 2020.
https://doi.org/10.3324/haematol.2020.249102 ©2021 Ferrata Storti Foundation
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