Acknowledgments: the authors would like to thank Drs. Shirish Yande, RL Marathe, Prakash Daithankar and Dilip Ghaisas for clinical samples. We would also like to thank Ashwini Atre for image acquisition on confocal microscope, and Hemangini, Pratibha and Swapnil for sample acquisition on flow cytometers. The authors wish to thank the anonymous reviewers for their critical review of the manuscript. Funding: this work was support- ed by a grant from the Department of Biotechnology, Government of India, New Delhi, India. (BT/PR10055/MED/31/14/2007) . Manuscript received on June 24, 2011. Revised version arrived on October 10, 2011. Manuscript accepted on October 17, 2011. Correspondence: Vaijayanti P. Kale, Stem Cell Laboratory, National Centre for Cell Science, University of Pune Campus, Ganeshkhind, Pune 411007, India. Phone: international +91.20.25708077. Fax: international +91.20.25692259. E-mail: vpkale@nccs.res.in/vai- jayanti.kale@gmail.com The online version of this article has a Supplementary Appendix. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells Monika B. Sharma, Lalita S. Limaye, and Vaijayanti P. Kale Stem Cell Laboratory, National Center for Cell Science, Ganeshkhind, Pune, India Background A culture system that closely recapitulates marrow physiology is essential to study the niche- mediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro. Design and Methods Hydrogel-based cultures of human placenta-derived mesenchymal stromal cells were estab- lished to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adher- ent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34 + cells were co-cultured with them. The output hematopoietic cells were characterized by vari- ous stem cell-specific phenotypic and functional parameters. Results The hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superi- or phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attrib- uted to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties. Conclusions Our data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology. Key words: functional niche, 3D-cultures, hydrogel, mesenchymal stromal cells, bone marrow, hematopoietic stem cells, hypoxia-gradient. Citation: Sharma MB, Limaye LS, and Kale VP. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica 2012;97(5):651-660. doi:10.3324/haematol.2011.050500 ©2012 Ferrata Storti Foundation. This is an open-access paper. ABSTRACT haematologica | 2012; 97(5) ARTICLES AND BRIEF REPORTS 651 Hematopoiesis