Emergence of patterned stem cell differentiation within multicellular structures

Abstract

The ability of stem cells to differentiate into specified lineages in the appropriate locations is vital to morphogenesis and adult tissue regeneration. Although soluble signals are important regulators of patterned differentiation, here we show that gradients of mechanical forces can also drive patterning of lineages. In the presence of soluble factors permitting osteogenic and adipogenic differentiation, human mesenchymal stem cells at the edge of multicellular islands differentiate into the osteogenic lineage, whereas those in the center became adipocytes. Interestingly, changing the shape of the multicellular sheet modulated the locations of osteogenic versus adipogenic differentiation. Measuring traction forces revealed gradients of stress that preceded and mirrored the patterns of differentiation, where regions of high stress resulted in osteogenesis, whereas stem cells in regions of low stress differentiated to adipocytes. Inhibiting cytoskeletal tension suppressed the relative degree of osteogenesis versus adipogenesis, and this spatial patterning of differentiation was also present in three-dimensional multicellular clusters. These findings demonstrate a role for mechanical forces in linking multicellular organization to spatial differentials of cell differentiation, and they represent an important guiding principle in tissue patterning that could be exploited in stem cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

Patterned segregation of lineages occurs within multicellular aggregates. (A) Schematic showing substrate preparation by microcontact printing. (B) Phase image of MSCs on 1mm circular patterns at day 0. (C–F) 1mm circular MSC aggregates stained for fat droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media (C), growth media (D), adipogenic media alone (E) or osteogenic media alone (F). (G) Concentric circles were placed over circular aggregates to divide them into four zones of equal area. Nuclei were labeled with DAPI to determine cell location. (H) Bar graph showing fraction of each cell type within each zone after 14 days in mixed media. (I) Bar graph showing fraction of each cell type within each zone determined by assaying for transcription factors after 5 days in mixed media. * indicates p<0.05 for all comparisons of the same cell type across different zones or different cell types within the same zone except for those marked with #. All Scale bars indicate 250 μm.

Figure 2

Partitioning of differentiation scales with size (A) Circular MSC aggregates with diameters 250, 500, 750, and 1000 μm stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media. (B) Bar graphs show fraction of each cell type within each zone for circular aggregates of all four sizes. All scale bars indicate 250 μm.

Figure 3

Geometry determines spatial patterning of differentiation. (A–H) MSC aggregates in the shape of a square (A), rectangles (B), an ellipse (C), a half-ellipse (D), an offset annulus (E), an elliptical annulus (F), and sinusoidal bands (G,H) stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media. Red arrows indicate adipogenesis at concave edges and blue arrows indicate osteogenesis at convex edges. All Scale bars indicate 250 μm.

Figure 4

Geometry determines spatial distribution of cytoskeletal tension. (A) Phase images of MSCs on mPADs in the shape of a sinusoidal band. Middle panel shows a traction force vector map superimposed on the image. Right two panels show close-ups of the convex and concave edges. Scale bars are 50 μm. (B) Frequency distribution of forces along the convex and concave edge. * indicates p<0.05. Dashed lines show means.

Figure 5

Cytoskeletal tension is necessary for patterned differentiation. (A–D) Circular MSC aggregates stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media without inhibitor (A) or in the presence of 50 μM blebbistatin (B), 10 μM Y-27632 (C) or 10 μM ML-7 (D). (E–H) Bar graphs showing fraction of each cell type within each zone. * indicates p<0.05 when compared to corresponding mean in the untreated condition (E). Scale bar indicates 250 μm.

Figure 6

3D multicellular structures undergo partitioning of differentiation. (A) Schematic showing method to create three-dimensional multicellular structures of MSCs. (B) Phase image of MSCs in three-dimensional structures at day 0. (C) Phase image of a three-dimensional MSC structure stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media. (D,E) Longitudinal and cross sections of MSC multicellular structures. All scale bars indicate 250 μm.

Figure 7

3D multicellular structures undergo partitioning of differentiation that can be modulated by cytoskeletal tension. (A,B) Cross sections of 3D MSC structures stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media in the absence or presence of 50 μM blebbistatin. (C,D) Cross sections of 3D MSC structures produced with either GFP or ROCKΔ3-infected cells stained for oil droplets (red) and alkaline phosphatase (blue) after 14 days in mixed media. (E–H) Bar graphs showing fraction of each cell type within each zone. In (E), * indicates p<0.05 for all comparisons of the same cell type across different zones or different cell types within the same zone except for those marked with #. In (F) and (H), * indicates p<0.05 when compared to corresponding mean in their respective controls ((E) and (G)). Scale bar indicates 100 μm.