A biofunctional microcavity platform to dissect autocrine and paracrine signals and to control fate decisions of hematopoietic stem and progenitor cells

摘要

Homeostasis of hematopoietic stem and progenitors cells (HSPC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSPC over the whole mammalian lifetime. The still limited understanding of signaling pathways hinders ex vivo HSPC expansion and differentiation as well as tight control of HSPC engraftment in clinical transplantation. Previously, we demonstrated cell-sized micropattems to maintain a quiescent, non-differentiated state of human HSPC in vitro. We now considerably expanded micropatterned substrates to a combinatorial microcavity platform involving biohybrid hydrogels besides surface-functjonalized silicone materials. We used a new micropatteming approach to imprint cell-sized features in the poly(ethylene glycol) based hydrogels additionally containing biofunctional glycosaminoglycan and adhesion peptide features. The extensive analysis of a 7-day in vitro culture of CD34+ primary human HSPC involved quantification of cell proliferation and differentiation as well as expression analysis of many cytokines involved in HSPC regulation. A partial least square analysis of the multifactorial dataset was used to reveal relevant juxtacrine, paracrine and autocrine signals. Finally, in vivo experiments were performed to functionally validate the in vitro data in a mouse repopulation model. Our results show that the usage of the microcavity platform in combination with a partial least square analysis of a mechanistic model of cell proliferation allows to decipher autocrine versus paracrine signals of soluble factors in HSPC culture, which was not possible up to now in other in vitro and in vivo settings. Autocrine signals of RANTES and paracrine signals of IL12 were found as stimulating factors, while autocrine signals of HGF and paracrine signals of ANG2 gave an inhibitory function. Based on these findings we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSPC in single-cell niches. Furthermore, the in vivo repopulation studies proved the HSPC maintaining effect of single-cell microcavities. We conclude, that the introduced microcavity platform combined with an in-depth analysis provides a new biotechnological tool to entangle convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin. Specifically, it unraveled distinct autocrine and paracrine functions of cytokines in HSPC maintenance.