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Secreted MEF factors maintain HSC cultures
survival, or the maintenance of LSK cells in culture or repopulating activity of growing clones. This suggests that although NGF and Col1 improve HSC survival in culture, the two factors do not cooperate or synergize with other MEF-CM factors in maintaining or expanding HSC with repopulating ability.
MEF-CM has previously been used to maintain murine embryonic stem cells.9 A proteomic study of serum-free MEF-CM showed a considerable amount of different colla- gens – types α1 (I, II, and IV), α2 (I), pro-collagens (type Va2), and collagen remodeling enzymes (MMP-2 and -3, PCOLCE, and TIMP2),18 suggesting that perhaps adding more collagen could be counter-productive. The effects of different concentrations of soluble collagen on the produc- tion of short- and long-term repopulating cells have, as far as we know, not been explored. However, NGF was not detected in this study of MEF-CM,18 suggesting a low con- centration of this cytokine or that other factors in the MEF- CM can substitute for NGF. Thus, it is likely that in combi- nation with MEF-CM, the precise concentrations of NGF- like activity and Col1 are critical in collaborating with other soluble factors to promote survival and self-renewal. These results indicate that precise titration of growth factors and collagen concentrations and assessment of collaborative and synergistic interactions with other stromal factors in the MEF-CM are needed, as noted in other studies.19,20
In order to use MEF-CM in protocols for maintaining or expandingHSC,theMEFandtheirCMneedtobedefined precisely, particularly to minimize batch-to-batch variation. Indeed, although MEF have been considered homogeneous, it has been noted that within the morphologically indistin- guishable fibroblasts, cells with different potentials may be present.21 Yet, our study shows that MEF-CM prepared from fresh MEF or previously frozen samples of MEF showed minimal variation in their support of proliferation and survival of HSC. The precise form in which the sup- portive factors are presented to the HSC remains to be established. We harvest serum-free medium from MEF monolayers incubated with SFM. Thus, our MEF-CM will not only include secreted factors, but also extracellular matrix, matricellular proteins, and extracellular vesicles, which may carry mRNA and regulatory micro-RNA. Indeed, the matricellular protein CCN2 has been shown to be important for HSC self-renewal,14 and purified extracel- lular vesicles from different sources have been used to sup- port growth of both murine22 and human HSC.23,24 Thus, it will be of interest in future studies to dissect the identity of HSC-supportive factors in the different structures included in the CM.
Our study defines a novel source of stromal cells capable of maintaining repopulating HSC in vitro under non-contact conditions. In previous studies, we used different cell lines to filter cell-type-specific factors and determine stromal fac- tors required for HSC in co-cultures.6 These efforts yielded several new mediators of HSC self-renewal under stress conditions in vivo.3,16,25 Although these studies are helping to dissect molecular mechanisms in the stromal cell-mediated regulation of HSC, only the UG26-1B6 and EL08-1D2 (with extra addition of WNT5A) cell lines were consistently shown to maintain HSC under non-contact conditions.1,6,7 Thus, together with these stromal lines, MEF can be used to better define soluble factors critical for maintaining repopu- lating activity in vitro, perhaps by combined analyses as we have previously described for HSC-supportive stromal cells
requiring direct contact with HSC.8 Such knowledge will be critical to improve protocols aimed at maintaining or expanding HSC in vitro.
Current knowledge of such expansion protocols have shown that precise titration of SCF, IL-1120 and throm- bopoietin,26-28 are critical for successful maintenance of LTR-HSC activity. Moreover, since variations in batches of bovine serum albumin (BSA) in SFM have been shown to affect culture outcomes,29 replacement of BSA with hemopexin29 or polyvinyl alcohol may lead to more con- sistent results in HSC cultures.26 In addition, new approaches are aiming to replace cytokines by pharmaco- logical substances directly modulating critical signaling pathways to inhibit HSC differentiation, such as MEK inhibitors,30 arylhydrocarbon receptor antagonists,31,32 or epigenetic regulators.33 Using these cytokines and sub- stances, expansions of murine LTR-HSC numbers by an estimated 10-fold in 10 days29 and 236- to 899-fold in 28 days26 have been described. Although we did not quantify the number of HSC in our experiments, we could assume a moderate expansion of 3-fold after 5 days from our transplantation experiments. If we further assume that the expanding pool of differentiating cells would not affect the number of LTR-HSC generated,34 culture in MEF-CM 2GF could result in 9-fold expansion after 10 days and over 200-fold expansion after 25 days. Thus, even moderate improvements in expansion suggest that combining factors isolated from MEF-CM with BSA replacement agents or pharmacological inhibitors may be viable strategies to improve HSC expansion protocols.
In summary, our results show that cultures of HSC in E13.5 MEF-CM 2GF support induction and propagation of cell division, as well as survival of individual HSC. More importantly, MEF-CM supports self-renewal of LTR-HSC in culture. Since MEF are easily generated, renewable and can be genetically modified, MEF and their CM represent promising tools in defining novel secreted stromal factors for promoting survival, cell division and self-renewal of HSC, which may help to improve in vitro expansion proto- cols and HSC engraftment in vivo. Furthermore, such studies may facilitate the understanding of the mechanistic basis of how stromal cells maintain HSC in the niche.
Disclosures
No conflicts of interest to disclose.
Contributions
FH, SRM, RH, TS and CS performed experiments; FH, SRM and RI collected and analyzed data; LH, CA and MS sorted cells; KSG, IED and FB provided infrastructure and contributed critical reagents, materials or analytic tools; RI and RAJO designed research; FH, SRM, RI and RAJO wrote the manuscript.
Acknowledgments
We thank the animal caretakers of the Center for Preclinical Research (ZPF, TranslaTUM, Klinikum rechts der Isar) for excel- lent animal care.
Funding
This work was supported by the German Research Council (Deutsche Forschungsgemeinschaft: DFG) (SFB 1243 [project A09]; OO 8/16, and FOR 2033 [project B3]) and the German "José Carreras Leukemia" Foundation (DJCLS) through funds to KSG (R14/18).
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