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.

Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives

“…However, importance of N-cadherin for HSC-niche interactions was later questioned (Kiel et al, 2007), thus rising substantial controversy. In an elegant in vitro study Lutolf et al (2009) have shown that N-cadherin, as well as Wnt3a, are the only proteins among those tested that were capable of supporting self-renewal divisions of HSCs in vitro. N-cadherin expression was also shown to be important for maintenance of long-term repopulating cells in culture (Hosokawa et al, 2010).…”

Integration column: microwell arrays for mammalian cell culture

“…72 HSC behavior was analyzed in vitro by live-cell microscopy in combination with in vivo transplantation assays. To 'deconstruct' and mimic HSC niches, PEG hydrogel microwells were selectively functionalized at the bottom with regulatory proteins using a combination of soft lithography and microcontact printing (Fig.…”

“…6). 72 Using automated time-lapse microscopy, the kinetic proliferation profiles of single cells were quantified, for example, as the distribution of numbers of HSC progeny generated per microwell as a function of time (Fig. 6).…”

In situ cell manipulation through enzymatic hydrogel photopatterning

“…The application of such artificial ECMs (aECMs), and in particular their fabrication with well-defined physicochemical bulk properties, has also enabled researchers to gain insights into the biology of crosstalk between cells and microenvironmental components (for example, refs [3][4][5]. However, the control over bulk gel properties is often not sufficient to elicit a desired morphogenetic response and to ultimately form a functional tissue de novo.…”

“…3f). ProteinA contains five high-affinity binding sites (K a = 10 8 per mole) for the Fc-region of human, mouse and rabbit immunoglobulins, and thus allows for easy, site-selective immobilization of Fc-tagged proteins 4 , many of which are commercially available. To this end, we modified ProteinA with caged Q-peptides and enzymatically patterned it within hydrogels.…”