Importantly using CXCL CXCR interactions as a paradigm for

Importantly using CXCL12/CXCR4 interactions as a paradigm for other factors and receptors, we demonstrated that CXCL12 is required for chemotaxis, but not chemokinesis of human UCB CD133+ HSPCs in the absence of niche elements, and that the CXCR4 antagonist, AMD3100, inhibits chemokine sensing but not the ability of these ampa to move randomly. This disputes previous suggestions by Entschladen et al. (2005) that chemotaxis does not occur without increasing chemokinesis, and provides a means to study in much more detail the effect of chemokines such as CXCL12 on inducing chemotaxis and/or chemokinesis in leukemic cell lines and primary leukemic stem/progenitor cells (Zepeda-Moreno et al., 2012). Other studies examining the chemotactic and chemokinetic movement of other cell types, such as the HT-1080 carcinoma cell line and human umbilical vein endothelial cells (HUVECs) towards an FCS gradient (Zengel et al., 2011), have demonstrated using Ibidi chemotaxis slides that chemokinetic migration as measured by cell velocity and accumulated distance can be influenced by the presence of a stimulus. We also examined the effects of short term (8day) expansion of human UCB CD133+ cells on a cell free nanofiber scaffold in the presence of cytokines on their chemotactic and chemokinetic responses to CXCL12. Using identical human UCB CD133+ cells to initiate the cultures, a greater and consistent expansion of CD133+CD34+ cells was also seen after 8days in our cultures with the addition of IGFBP2 and Angptl5 to SCF, Flt-3 ligand, IL-6 and TPO than without (averaging 73 versus 52 fold respectively). Drake et al. (2011) using Angptl5 and IGFBP2 together with SCF, TPO and FGF-1 in StemSpan media without a nanofiber scaffold, demonstrated on average a 21 fold increase in CD133+CD34+ cells after 10days and a much more variable response between expansion rates than observed here, suggesting that the Nanex scaffold and media used here could be enhancing expansion by greater than 2–3 fold over a shorter period of time. Although Drake et al. (2011) showed that CD133+CD34+ cell number correlates with repopulating ability in NSG mice, we have yet to demonstrate this. However, when we isolated the CD133+ cells from our expansion cultures and tested their chemotactic response to CXCL12 in the 3D μ-slide chemotaxis assay, we found a non-significant trend towards enhanced chemotaxis but not chemokinesis with the addition of IGBP2 and Angptl5, with these cells continuing to express CXCR4. The 8day culture did not compromise the chemotactic ability of the expanded CD133+ cells compared to our other experiments where CD133+ cells were cultured for 24h in SCF, Flt-3 ligand, IL-6 and TPO.
Conclusions The following are the Supplementary data related to this article.
Acknowledgments This work was supported by research funding from the NHS Blood and Transplant (EEP, SMW), the Technology Strategy Board (SMW) and the National Institutes of Health Research (EEP, SMW). This report presents independent research commissioned by the National Institutes for Health Research (NIHR) under its Programme Grants Scheme (RP-PG-0310-1003). The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We would like to thank Ibidi GmbH for providing prototypes of the 3D μ-slide chemotaxis while they were being developed and for permission to use or reproduce images in Fig. 1. We would also like to acknowledge Arteriocyte Inc. and their distributor, Cambridge Biosciences, for the provision of samples of Nanex plates and media. We thank Mrs. S. Britt and Mrs. J. Walton for collecting the cord blood units. Authorship contributions
Disclosure of conflicts of interest
Introduction Bone marrow stromal cells (BMSCs) consist of a heterogeneous population of cells that provide the structural and functional support for hematopoietic cells (Bianco et al., 2001; Sacchetti et al, 2007). These cells have fibroblast-like morphology, express CD29, CD44, CD73, CD90, CD105 and CD166, and are negative for CD14, CD34, and CD45 (Boxall and Jones, 2012). Additionally, BSMC populations contain a sub-population of stem cells with the ability to differentiate into cartilage, bone, stroma that supports hematopoiesis, and marrow adipocytes (Spencer et al., 2011).