Los especialistas serían más vulnerables al disturbio

The concept of a specialised HSC microenvironment within BM, termed a niche, is long established (Schofield 1978) and is the subject of ongoing investigation. The niche functions to maintain a quiescent pool of HSCs, which are nevertheless able to self-renew, differentiate, and mobilise in the event of haematopoietic injury. The niche also protects the cells from overstimulation and prevents inappropriate differentiation. Cell-cell interactions, the SNS, and hormonal inputs have all been identified as playing a role in the regulation of haematopoiesis. Cell-extracellular matrix (ECM) interactions, oxygen tension, and calcium ion concentration are also fundamental physical features of the niche (Wang & Wagers 2011). The HSC niche is clearly a very complex environment and many features are not yet fully understood.

Examining this niche is necessary in order to recapitulate a suitable HSC culture environment ex vivo. In adults, HSCs reside in the BM, predominantly in the trabecular rich metaphysis (Ellis et al. 2011). Here, HSCs have been observed in contact with the endosteum (the surface of the bone), and localised around sinusoids (capillaries which supply the marrow), as shown earlier in Figure 1.1

(Kiel et al. 2005). This has given rise to a ‘zoned model’ of the haematopoietic niche (Figure 1.5, Suárez-Álvarez et al. 2012). It is hypothesised that the endosteum houses quiescent HSCs, with the more active cycling HSCs located around the sinusoid (Ehninger & Trumpp 2011). However, with the recent use of advanced techniques for imaging HSCs in situ or in large areas of tissue, it has been suggested that this model is over-simplified: that in the trabecular bone the endosteum and vasculature are in close contact and so HSCs may be interacting with both suggested niche components simultaneously (Ugarte & Forsberg 2013; Nombela-Arrieta et al. 2013; Ellis et al. 2011). Kunisaki et al. 2013 imaged BM from both long bones and the sternum, finding evidence for the sinusoid being a proliferative niche, but also identifying a putative arteriolar niche. Arterioles, exhibiting characteristic smooth muscle, nerves, and matrix, were associated with highly nestin+ _{perivascular cells, which were also more quiescent and produced}

more HSC-supportive factors than those localised to sinusoids. Quiescent HSCs localised to these arterioles.

Figure 1.5 The ‘zoned model’ of the HSC niche.

HSCs are hypothesised to reside in two separate zones in the bone marrow: the sinusoidal niche and the endosteal niche, migrating between the two. Nestin+ _{MSCs are important for} HSC support in both zones. Adapted from Ehninger & Trumpp 2011.

The precise location of HSCs relative to endosteum or elements of the vasculature (sinusoids and arterioles), and thus the nature of cellular signals they receive, may determine whether the cells proliferate or are quiescent. Different areas of the BM or cellular components may serve distinct functions depending on the stage of

Chapter 1 33 haematopoiesis, with each lineage progenitor residing in its own specific microenvironment (Wang & Wagers 2011; Ding & Morrison 2013).

1.4.1 Cellular support of HSCs

Several different cell types have a proposed role in HSC regulation; including MSCs, pericytes, ECs, and OBs. The observation that most HSCs are located adjacent to bone in the endosteum initially placed osteoblasts as the main supporting cell. However, later genetic evidence called this into question, concluding that any effect of osteoblasts is indirect (Morrison & Scadden 2014) as the endosteum is in such close proximity to the vasculature. Other cell types have also been implicated, including C-X-C motif chemokine 12 (CXCL12)-abundant reticular (CaR) cells, which functionally overlap in part with MSCs and are located both perivascularly and endosteally (Sugiyama et al. 2006). However, the extent to which stromal cell subsets are distinct has not yet been established (Wang & Wagers 2011).

More recently, the emphasis has been on nestin+ _{MSCs, a subset of CaR cells, as}

the main HSC support. They are enriched in several important HSC maintenance factors, localise to perivascular regions, and are closely associated with the SNS. As osteoprogenitors they have features of both the osteoblastic and the endothelial niches, replacing the idea of osteoblasts themselves maintaining HSCs, and giving a ‘master’ role to the MSC which can coordinate many other cell types (Méndez-Ferrer et al. 2010). These cells are themselves maintained as quiescent by the niche and establish gradients of secreted cell products to control HSCs (Bianco 2011).

Although OBs may not be direct regulators of HSCs, they do have several unique functions. Firstly, they produce the matrix protein osteopontin (OPN) at the endosteum, to which HSCs can bind to via β1 integrins, an interaction which promotes HSC quiescence (Janeczek et al. 2015). They also produce thrombopoietin (TPO), and angiopoietin-1 (Ang-1) which are both important for HSC regulation. However, deletion of the adhesion protein N-cadherin has no effect on proliferation or differentiation of HSCs, showing that direct cell-cell interactions are not necessary for the role of OBs in the niche (Janeczek et al. 2015). A further complication arises from the fact that identification of cells of

the osteoblastic lineage is not straightforward due to a lack of available cell- surface markers. Furthermore, the endosteal lining of the marrow is made up of active osteoblasts and quiescent bone lining cells, the latter outnumbering the former. The positions of these cells in the osteoblastic hierarchy remains to be distinguished, and they may have different roles in the HSC niche. In fact, the main member of the OB lineage in HSC regulation may be an immature skeletal stem cell or pre-osteoblast, which have both been shown to produce HSC-relevant cytokines and can be found in perivascular locations (Askmyr et al. 2009).

ECs form the network of vasculature in the trabeculum. Although this is mostly comprised of sinusoids (Kunisaki et al. 2013), several other vessel types are also present. As well as providing a mechanical barrier for blood cells and platelets, ECs release angiocrine signals that participate in HSC development and regulation, and contribute to the sinusoidal HSC niche. In particular, HSCs require stem cell factor (SCF) expressed by ECs, but not SCF expressed by OBs or nestin+ _{cells (Ding}

et al. 2012). Regenerated sinusoidal ECs are required for successful engraftment of HSCs following myeloablative therapy (Hooper et al. 2009). Furthermore, the integrity of blood vessels may have an effect on HSCs: HSCs close to sinusoids have higher ROS levels than arteriolar HSCs, and retention is higher at arterioles. Homing and endothelial transmigration are exclusive to sinusoids as they are highly permeable (Itkin et al. 2016). Hence, differences between ECs may contribute to the heterogeneity of HSC niches.