Mechanistic and metabolic insights into bioengineering the bone marrow niche in vitro

Stem cell balance of proliferation, differentiation and self-renewal, is regulated by the microenvironment in which they reside, termed the stem cell niche (Schofield, 1978). Niche microenvironments provide physical and functional regulatory cues that control fundamental cell intrinsic and extrinsic mechanisms. In adults, the process of haematopoiesis is sustained by a population of haematopoietic stem cells (HSCs) that are found primarily in the bone marrow (BM) (Jagannathan-Bogdan and Zon, 2013). The BM niche also houses populations of mesenchymal stromal and perivascular cells (MSPCs), that are themselves regulated by the niche, and are fundamental cellular constituents in HSC regulation (Pinho and Frenette, 2019). Both HSCs and MSPCs hold enormous clinical potential. HSCs have the ability to reconstitute the entire blood and immune system (Jagannathan-Bogdan and Zon, 2013), whereas MSPCs contain immunosuppression capacity and have the ability to regenerate damaged and diseased tissue (Caplan, 1991; Uccelli et al., 2008). However, there are still important hurdles that must be overcome before the potential of these cells are fully realised. The regenerative capacity of these stem cells is quickly lost upon ex vivo culture, meaning achieving clinically relevant numbers of cells is challenging (Dalby et al., 2018; Zon, 2008). Although BM MSPCs (such as nestin+ MSPCs) contain HSC support activity, their ability to maintain HSCs ex vivo is only modest due to loss of expression of these niche factors in culture (Kunisaki et al., 2013; Nakahara et al., 2019). The absence of sustained self-renewal or maintenance of the stem cell phenotype could be related to the lack of integration of biophysical and biochemical cues required for stem cell regulation, provided by the native BM niche microenvironment in vivo. This has led to a focus on biomaterial and engineering strategies that aim to recapitulate BM niche properties in vitro (Müller et al., 2014). It is envisaged that bioengineered artificial niches will offer protocols for ex vivo expansion and maintenance of HSCs without the need for high risk protocols (e.g. genetic manipulations (Nakahara et al., 2019)), but also platforms on which to study the fundamental mechanisms that control self-renewal in both HSCs and MSPCs in the niche. In this thesis, biomaterial strategies were employed to mimic aspects of the BM niche microenvironment. First retention of HSC support activity in a population of MSPCs was investigated and the metabolic mechanisms that may support this phenotype were probed. The ability of the system to support HSC maintenance in vitro was then assessed. Poly(ethyl acrylate) (PEA) is a polymer that causes spontaneous unfolding of the extracellular matrix protein (ECM) fibronectin (FN). These physiological-like networks expose key binding sites on the FN molecule, which can be harnessed for cell adhesion and growth factor tethering (Cheng et al., 2018; Llopis-hernández et al., 2016). Noting that low-stiffness matrices support nestin expression in MSPCs (Engler et al., 2006), a key niche marker (Kunisaki et al., 2013; Pinho et al., 2013), low-stiffness collagen hydrogels were introduced into the system. We were able to use this system to promote a population of nestin+ MSPCs that express key HSC support factors and were able to maintain a population of HSCs in vitro. The nestin+ MSPCs utilise hypoxic-like metabolic mechanisms in response to low-stiffness, that may be important in retaining this BM niche-like phenotype in long term in vitro culture.