Geometric control of human stem cell morphology and differentiation

Abstract

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function.

Fig. 1

Experimental design for studying spatial patterns of stem cell function. (A) Micro-fabrication and micro-contact printing for patterning of human adipose-derived stem cells (hASCs). PDMS (polydimethylsiloxane) elastomeric stamps were casted with pre-polymers onto a negative photoresist mold, which was formed with UV (ultraviolet) crosslinking through a mask containing desired small features (rectangles and rings of different sizes). An adhesive self-assembly monolayer (SAM) octadecanethiol was transferred via PDMS stamp onto gold-coated glass slides, which were then sequentially subject to a non-adhesive ethylene glycol-terminated SAM HS-(CH₂)₁₁-EG₃ and extracellular matrix protein, fibronectin. (B) Cell proliferation, osteogenic and adipogenic differentiation in the corresponding culture medium were examined by BrdU assay, Alkaline phosphatase (ALP) and Nile red staining, respectively.

Fig. 2

Localization of stem cell proliferation and differentiation. (A) Combined fluorescence images of stem cell proliferation on ring and rectangular patterns in growth medium for 1 day (Green: Bromodeoxyuridine (BrdU); Blue: nuclei); (B) Bright field images of Alkaline phosphatase staining (with Fast Blue dye) of human adipose derived stem cells (hASCs) after 3 days incubation in osteogenic medium; (C) fluorescence images of Nile red staining of lipid droplets inside hASCs after cultured in adipogenic medium for 4 days. Scale bars: 100 μm.

Fig. 3

Cell differentiation maps and cell morphology. (A) Image processing for the distribution of alkaline phosphatase (ALP) activity of stem cells cultured for 3 days on a ring pattern in osteogenic medium. High AKP activity is shown by purple in bright field images (A1), black in thresholded images (A2), and the dark regions in overlaid images (A3). The probability of cell differentiation was calculated at each pixel by averaging multiple binary images (A2) (n=40–80) and the frequency maps were then generated as shown for ALP expression (B 1–2) in stem cells in osteogenic medium, and for lipid droplets in adipogenic medium (B3–4). (C) Phase contrast images of cells on micropatterns. Cells at the outer regions of rings and the corners of rectangles are shown with a larger projection area, while cells at inner rings and regions close to short edges of rectangles are smaller and narrower. (D) Morphological analysis of cells on rings (inner diameter: 500 μm; width: 200 μm) as a function of the radial position. Cell differentiation colocalized with the small size and large aspect ratio of the cultured cells (*p < 0.05). Scale bars: 200 μm.

Fig. 4

Dependence of stem cell differentiation frequency maps on pattern geometry. (A) ALP activity of human adipose-derived stem cells (hASCs) in osteogenic differentiation medium for 3 days. Frequency maps (1–4) of ALP activity are shown for stem cells on rectangles measuring 1000 × 200 μm, 1000 × 100 μm, 500 × 200 μm, and 500 × 100 μm; the corresponding average ALP activity along the length of each pattern is shown in 5–8. (B) ALP activity of hASCs on rings. Stem cell ALP activities on ring patterns with different sizes are shown in the frequency maps (from left to right 1–4: inner diameter D=1000 μm, width W=200 μm; D=1000 μm, W=100 μm; D=500 μm, W=200 μm; and D=500 μm, W = 100 μm), and the corresponding ALP activity profile across the radius is shown in 5–8. Double-ended arrows indicate the patterned cell-adhesive regions. (# significantly different from the peak ALP activity near two ends in the same rectangle, * significantly from other groups, and ** significantly different from the ring with an inner diameter of 500 μm and width of 200 μm; p < 0.05).

Fig. 5

The role of cytoskeleton. Disruption of cytoskeletal tension diminished the patterns of geometrically controlled osteogenic differentiation of hASCs. The frequency maps (top) of ALP activities are shown for stem cells on ring patterns (inner radius: 500 μm and width: 200 μm) after 3 days incubation (from left to right) in osteogenic medium (control) and in osteogenic medium with addition of 10 μM Blebbistatin, 10 μM ML-7, or 2 μ MY-27632. The corresponding cell ALP activities across the radius are shown below the frequency maps. (* significantly different from other groups in the peak ALP activity; p < 0.05).