The gold standard for determining efficient HSPC homing

The gold standard for determining efficient HSPC homing to bone marrow niches is their subsequent hematological reconstitution following transplantation in humans (Dahlberg et al., 2011; Ramirez et al., 2012), or, as surrogates, in immunodeficient mice or non-human primates (Goessling et al., 2011; Larochelle et al., 2012). Similar models are used to assess the efficacy of mobilizing agents (Hoggatt and Pelus, 2011; Bonig and Papayannopoulou, 2013). However, surrogate assays are time consuming and costly and do not discriminate between direct effects on HSPCs nor indirect mechanisms mediated by bone marrow niche elements. An initial homing/migration assay ex vivo, which reduces animal usage and allows refinement of pre-transplant protocols would make screening of expansion/manipulation/mobilization protocols more efficient and provide essential insights into mechanisms. Although current transwell migration end-point assays (Toetsch et al., 2009) measure cell migration towards CXCL12, these simply give a percentage of ampa migrating across a membrane towards a stimulus. We have developed a novel reproducible in vitro homing/migration assay using 3D μ-slide chemotaxis chambers from Ibidi GmbH and timelapse microscopy to track individual human HSPCs and have used CXCL12 as a paradigm. This allows the user to compare chemotactic and chemokinetic effects of a stimulus on HSPC homing/migration, as well as observing individual cells during the migratory process. Importantly, using this new methodology, we have definitively demonstrated, in contrast to previous assumptions that chemokines alter both chemotactic and chemokinetic responses simultaneously (Entschladen et al., 2005), that CXCL12 regulates human umbilical cord blood (UCB) CD133+ HSPC chemotaxis but not chemokinesis. Furthermore, we use this assay to compare the chemotactic and chemokinetic responses of UCB CD133+ cells after an 8day expansion on a cell-free nanofiber scaffold in defined cytokine cocktails.
Materials and methods
Results
Discussion The key advantages of this novel timelapse assay over existing transwell assays include the ability i) to measure the chemotactic and chemokinetic responses of individual non-adherent progenitor cells for periods comparable to published in vivo homing assays (18–24h) (Larochelle et al., 2012), ii) to distinguish chemotactic from chemokinetic responses by individual cells to stimuli over time, parameters which are difficult to define in standard transwell assays (Toetsch et al., 2009), iii) to directly compare the strength or response to different or competing stimuli in a single assay, iv) to assess whether stimuli act directly on HSPCs without the complication of niche influences observed in vivo (Shiozawa and Taichman, 2010; Bianco, 2011; Calderón and Boehm, 2012; Park et al., 2012; Psaila et al., 2012), v) to assess small molecule inhibitor effects on individual HSPCs in this multiwell platform for multiple cell migration parameters, and vi) to assess migratory responses of HSPCs in a 3D rather than a 2D environment. A disadvantage is that the substantial data generated in this new assay required manual tracking. When we used Wimasis semi-automated tracking (Khoo et al., 2011), the Wimasis algorithm was unable to follow cells for the duration of the experiment and therefore requires further development. Using identical CD133+ cells to initiate each assay, the number of human UCB CD133+ cells migrating in the 3D μ-slide chemotaxis assay was 3–5 fold higher than those migrating in the transwell assay (25.9+9.2% means+SEM for n=3 independent experiments). Since the transwell migration results are consistent with those described previously (Aiuti et al., 1997; Kim and Broxmeyer, 1998; Kollet et al., 2002; Forde et al., 2007), the differences may be explained by 3D migration in collagen I versus 2D migration on fibronectin, different endpoint times and in situ tracking of migrating cells rather than the need to recover cells prior to analyses. Renkawitz and Sixt (2010) have described plasticity in cell migratory mechanisms in vitro when 3D is compared with 2D environments. Using the 3D assay, we demonstrated increasing cell migration towards CXCL12 over 18 to 22h of between 67% and 97% of the cells, indicating that these cells do not stop responding to CXCL12 in the 3D environment within the 4–6hour timeframe used for the 2D transwell assay. In developing this new assay, the weak adhesion of human UCB HSPCs to ECM coated surfaces did not, in our hands, provide an optimal platform for use of the Ibidi 2D μ slide chemotaxis assay. When we attempted to reproduce the 2D μ-slide chemotaxis assay of Grassinger et al. (2009), where the cells were not encapsulated in a matrix, we found that the cells were not easily retained in the observation chamber when the reservoirs were filled. While HSPCs are known to bind to fibronectin through α4β1 and α5β1 integrins (Ulyanova et al., 2011 and references therein), they do not express significant levels of the collagen receptors, α1β1, α2β1, α3β1, α10β1, and α11β1 (Humphries et al., 2006) and hence this may also affect the mechanics of each assay.