An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHematopoietic stem cells (HSCs) maintain the ability to self-renew and differentiate within their in vivo microenvironment, the bone marrow (BM). From a clinical perspective, HSCs are important because they can generate the full blood cell repertoire upon transplantation () and are therefore critical determinants of clinical BM transplant success. Additionally, in combination with gene therapy approaches HSCs also offer the significant potential to treat a range of inherited hematological disorders. However, our ability to maintain and expand HSCs outside of their in vivo microenvironment is currently limited.The current protocols for ex vivo expansion of HSCs can be broadly divided into two groups, based on their use of cell-intrinsic or cell-extrinsic factors (). Cell-intrinsic factors include exogenous expression transcription factors such as HoxB4 (), and chromatin remodeling factors such as Bmi1 (). Such approaches have to date required genetic modification that limits their direct translational application. By contrast, cell-extrinsic factors such as cytokines are simply added to the culture media and act on unmodified HSCs.Cytokines and other extrinsic factors are present in the specialized BM microenvironments, the so-called BM niche, and are thought to be involved in migration, quiescence, and differentiation of HSCs (). Many different cell types have been proposed as the candidate for the BM niche, including osteoblasts (, ), endothelial cells (), chemokine ligand 12 (CXCL12)-abundant reticular cells (), mesenchymal stem cells (), and non-myelinating Schwann glial cells (, ).BM niche cells are thought to secrete numerous factors such as stem cell factor (SCF) () and thrombopoietin (TPO) (), which are generally necessary for HSC maintenance. These cytokines have long been added to culture media to investigate HSC proliferation and reconstitution ability. However, there are concerns about data reproducibility between laboratories, with such discrepancies often being ascribed to differences in experimental culture conditions.HSCs have been widely analyzed using liquid or methylcellulose culture in the presence of fetal bovine serum (FBS). FBS contains myriad of growth factors, adhesion molecules, and other components, and also protects cells from rapid changes in pH. However, because of the high degree of unknown factors, FBS is now often replaced with serum-free medium containing BSA fraction V (BSA-FV; the fifth ethanol fraction in the original purification process of plasma proteins) () for in vitro HSC culture. BSA-based serum-free cultures have been well established for pluripotent stem cells. However, stable in vitro expansion of HSCs remains difficult and non-reproducible. This is at least in part due to the use of different batches (lots) of BSA-FV by different laboratories.To address these issues, we tested 15 different lots of commercially available BSA-FV; each exhibited different abilities to maintain HSCs and unique protein profiles. To identify the best molecular candidates for HSC maintenance in BSA-FV, we developed a fully defined culture system using all-recombinant proteins. Using this approach, we provide evidence that HSC maintenance is strongly supported by two factors in BSA-FV, interleukin-1α (IL-1α) and hemopexin (HPX). Further investigation found that HPX reduced HSC intracellular reactive oxygen species (ROS) levels and that HPX was present on non-myelinating Schwann cells, a constituent of HSC niche in BM. These findings highlight the utility of all-recombinant protein-based systematic analysis for ex vivo HSC self-renewal and differentiation, and identification of bona fide growth factors that contribute to these cell-fate decisions.