Impact of topographic stimuli on the biology of mesenchymal and induced pluripotent stem cells

Mechanical stimuli are an integrant component of the microenvironment controlling the differentiation and self-renewal of stem cells; nonetheless, it is still not fully understood how such cues can regulate stem cell functions and drive their fate. In this thesis we investigated the impact of various types of groove-ridge substrates on the behavior of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs). To this purpose, we employed different micro- and nanofabrication approaches to generate defined patterns on the surface of a plastic polymer. We then assessed how such topographic cues modulate various aspects of the biology of MSCs and iPSCs through the use of confocal microscopy, image analysis tools and genome wide gene expression profiles. Our results show that both micro- and nanogrooved biomaterials induced the elongation of MSCs, affected the size and number of focal adhesions and guided their migration. Furthermore, specific substrates were able to favor the differentiation of MSCs toward adipogenic or osteogenic lineage; however, the overall impact of topography on gene expression and proliferation was only moderate. With regards to iPSCs, nanostructured materials elicited an increase in the aspect ratio of both single cells and colonies by guiding the orientation of actin fibers and the polarity of cell division. Morphological changes of iPSCs were reflected in the spatial distribution of pluripotency markers, moreover elongated colonies showed increased differentiation upon BMP4 treatment. Finally, we elucidated the role that mechanotransducers like YAP and TAZ play in mediating these phenotypes and described novel shuttling dynamics and molecular interactions of such transcription factors. Taken together these findings shed new light on how surface topography controls crucial molecular processes and orchestrates the overall architecture of cells and tissues. In conclusion our results open new perspectives for the use of tailored biomaterials to support the directed differentiation of stem cells

Epigenetic Rejuvenation of Mesenchymal Stromal Cells Derived from Induced Pluripotent Stem Cells

SummaryStandardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging

3D non-woven polyvinylidene fluoride scaffolds : fibre cross section and texturizing patterns have impact on growth of mesenchymal stromal cells

<div><p>Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.</p></div

Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages

Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 μm ridges increased adipogenic differentiation whereas 2 μm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces – e.g. by multi beam laser interference in sub-micrometer scale – do not induce differentiation of MSCs per se, but support their directed differentiation

Surface Topography Guides Morphology and Spatial Patterning of Induced Pluripotent Stem Cell Colonies

The relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs

MSC growth on TCP and in 3D-PVDF non-wovens.

<p>SEM pictures showing MSC morphology on TCP and in PVDF non-wovens: MSCs span over non-woven pores forming large confluent cell layers. Photos at higher magnifications show that cells align along the fibres and accumulate at fibre intersections. Some individual cells are exemplarily depicted by red dotted lines and PVDF fibres are marked with F.</p

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells

International audienceCulture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types

Fibre Characteristics.

<p>*Mean single fibre fineness [µm] was calculated for the different fibre profiles by deviation of the ideally round fibre profile.</p

Manufacturing steps of three-dimensional PVDF non-wovens.

<p>Round shaped (f24 0.4 L/D 2, left), trilobal (Y24 250×552 L/D 2, middle), and snowflake (f24 L/D 2, right) spinnerets (A). Schematic overview of the fabrication process of non-wovens made of PVDF fibres (B). SEM pictures of round (left), trilobal (middle) and snowflake (right) non-wovens cross-sections (C). Fibre texturizing of fibres knitted with 10 (left), 16 (middle), or 28 (right) needles/inch (D). Round scaffolds are punched out of the non-woven fabrics (E).</p

Characterisation of non-wovens properties.

<p>Characterisation of non-wovens properties.</p