Perturbation of single hematopoietic stem cell fates in artificial niches

Department of Microbiology and Immunology, BioX and Stem Cell Institute, Stanford University School of Medicine, CA 94305, USA.
Integrative Biology (Impact Factor: 3.76). 01/2009; 1(1):59-69. DOI: 10.1039/b815718a
Source: PubMed


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.

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Available from: Helen M Blau, Aug 19, 2015
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    • "Of additional interest is the observation that the time taken for mitogenically stimulated ESLAM cells to complete a first mitosis is positively associated with the likelihood that at least one of their two daughter cells will retain HSC functionality. This is consistent with previous evidence that longer cell-cycle transit times correlate with the most primitive HSCs (Dykstra et al., 2006; Lutolf et al., 2009; Yamazaki et al., 2009). Such associations suggest the possibility that cell-cycle control, like retention of GM differentiation potential, may be mechanistically linked to DSR competence in adult mouse BM HSCs. "
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    • "As these matrices were able to alter biophysical properties in a non-invasive manner, they were used to investigate the progression of biophysical changes associated with muscle fibrosis or disease (Engler et al., 2004). Moreover, Lutolf et al. demonstrated that PEG hydrogels were suitable for single-stem cell clonal assays and resistant to non-specific cell adhesion mediated by protein adsorption (Lutolf et al., 2009a). However, further studies are necessary to define exactly all the components that constitute the microenvironment of the SCs and the molecular steps that regulate the transition between SCs quiescence and proliferation. "
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    • "Stem cell niches are 3-D microenvironments composed of hydrated , crosslinked networks of extracellular matrix (ECM) proteins , sugars and cytokines [17]. Therefore, materials should physically mimic the architecture of the natural niche and also be able to localize biological cues such as ECM proteins and growth factors to establish an artificial niche [17] [18] [19] [20]. The self-assembling peptide RADA16-I (AcN-RADARADARADARADA-CONH 2 ) can undergo spontaneous assembly into well-ordered interwoven nanofibers in water and rapidly form hydrogel with $10 nm fiber diameter, 5–200 nm pore size and >99% water content under physiological conditions [21] [22] [23], which is similar to the structure of natural ECM. "
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