Proper tissue function and regeneration rely on robust spatial and temporal control of biophysical and biochemical microenvironmental cues through mechanisms that remain poorly understood. Biomaterials are rapidly being developed to display and deliver stem-cell-regulatory signals in a precise and near-physiological fashion, and serve as powerful artificial microenvironments in which to study and instruct stem-cell fate both in culture and in vivo. Further synergism of cell biological and biomaterials technologies promises to have a profound impact on stem-cell biology and provide insights that will advance stem-cell-based clinical approaches to tissue regeneration.

Designing materials to direct stem-cell fate

Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.

Eyes wide open: a critical review of sphere-formation as an assay for stem cells.

Mechanisms controlling the proliferative activity of neural stem and progenitor cells (NSPCs) have a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (Fasn), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of Fasn in mouse NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene previously implicated in lipid metabolism, that we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for Fasn to fuel lipogenesis. Thus, we identify here a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation.

https://www.hifo.uzh.ch/research/jessberger/publications/Knobloch2013Nature.pdf

Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis

To understand the regulatory role of niches in maintaining stem-cell fate, multifactorial in vitro models are required. These systems should enable analysis of biochemical and biophysical niche effectors in a combinatorial fashion and in the context of a physiologically relevant cell-culture substrate. We report a microengineered platform comprised of soft hydrogel microwell arrays with modular stiffness (shear moduli of 1-50 kPa) in which individual microwells can be functionalized with combinations of proteins spotted by robotic technology. To validate the platform, we tested the effect of cell-cell interactions on adipogenic differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of substrate stiffness on osteogenic MSC differentiation. We also identified artificial niches supporting extensive self-renewal of nonadherent mouse neural stem cells (NSCs). Using this method, it is possible to probe the effect of key microenvironmental perturbations on the fate of any stem cell type in single cells and in high throughput.

Artificial niche microarrays for probing single stem cell fate in high throughput

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSC) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere 'merging' (Singec et al., Nature Methods, 2006; Jessberger et al., Stem Cells, 2007). Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared to conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to elucidate better the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdf/10.1634/stemcells.2008-0498

Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays

Conventional in situ hydrogel micropatterning techniques work successfully for relatively stiff hydrogels, but they often result in locally damaged surfaces upon demolding in the case of soft and fragile polymer networks formed at low precursor concentration. To overcome this limitation, we have developed a facile, versatile method, termed soft embossing, for the topographical micropatterning of fragile chemically cross-linked polymer hydrogels. Soft embossing is based on the imprinting of a PDMStemplate into a gel surface that is only partially cross-linked and thus contains free functional groups that continue to be consumed to confine the microstructure irreversibly upon complete cross-linking. Here we identify and optimize the parameters that control the soft embossing process and show that this method allows the fabrication of desired topographies with good fidelity. Finally, one of the produced gel micropatterns, an array of microwells, was successfully utilized for culturing and analyzing live single hematopoietic stem cells. Confining the stem cells to their microwells allowed for efficient quantification of their growth potential during in vitro culturing.

Monika Limacher

Papers

Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays

Micropatterning of hydrogels by soft embossing.