title: Perturbation of single hematopoietic stem cell fates in artificial niches.
abstract: Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet are in limited supply as in culture this self-renewal capacity is lost. Using an approach at the interface of stem cell biology and bioengineering, here we describe a novel platform of hydrogel microwell arrays for assessing the effects of either secreted or tethered proteins characteristic of the in vivo microenvironment, or niche, on HSC fate in vitro. Time-lapse microscopic analyses of single cells were crucial to overcoming inevitable heterogeneity of FACS-enriched HSCs. A reduction in proliferation kinetics or an increase in asynchronous division of single HSCs in microwells in response to specific proteins (Wnt3a and N-Cadherin) correlated well with subsequent serial long-term blood reconstitution in mice in vivo. Single cells that divided once in the presence of a given protein were capable of in vivo reconstitution, providing evidence of self-renewal divisions of HSCs in vitro. These results validate the hydrogel microwell platform as a broadly applicable paradigm for dissecting the regulatory role of specific signals within a complex stem cell niche.
authors.affiliation: Department of Microbiology and Immunology, BioX and Stem Cell Institute, Stanford University School of Medicine, CA 94305, USA.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Lutolf
authors.forename: Regis
authors.initials: R
authors.lastname: Doyonnas
authors.forename: Karen
authors.initials: K
authors.lastname: Havenstrite
authors.forename: Kassie
authors.initials: K
authors.lastname: Koleckar
authors.forename: Helen M
authors.initials: HM
authors.lastname: Blau
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002469:Cell Separation / Q000295:instrumentation*; D002478:Cells, Cultured; D004867:Equipment Design; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis / Q000295:instrumentation*