Frequent mechanical stress suppresses proliferation of mesenchymal stem cells from human bone marrow without loss of multipotency

Abstract

Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not only to biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such "dynamic in vitro niche" can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage.

Figure 1. Schematic illustration of stimulus responsive substrates.

Chemical structure of the pH sensitive triblock copolymer (PDPA-PMPC-PDPA) and the changes within the micellar structure induced by the pH change. pH titration between 7.2 and 8.0 enables one to reversibly switch the elastic modulus by a factor of 20.

Figure 2. Morphological response of bone marrow-derived, human mesenchymal stem cells (hMSCs) on PDPA₅₀-PMPC₂₅₀-PDPA50 copolymer gels.

Phase contrast images of hMSCs at t = 10 d on (a) stiff (E = 40 kPa) and (b) soft (E = 2 kPa) substrates. Fluorescence images of phalloidin-labeled actin (green) at t = 10 d on (c) stiff (E = 40 kPa) and (d) soft (E = 2 kPa) substrates. The order parameters <S> of actin cytoskeletons can be extracted from the pixel orientation maps calculated from the original images are presented as insets.

Figure 3. hMSCs sustains multipotency after 20 d independent from substrate stiffness.

(a) Flow of experiments: Type A; substrate elasticity was always kept “stiff” (E = 40 kPa) for 20 d, Type B; substrate elasticity was always kept “soft” (E = 2 kPa) for 20 d, Type C; substrate elasticity was switched from “soft” to “stiff” at t = 10 d, Type D; substrate elasticity was switched from “stiff” to “soft” at t = 10 d. (b) Fluorescence images of hMSCs always cultured on stiff substrates (Type A) and soft substrates (Type B). Labels: (b1) anti-STRO-1, (b2) Oil Red O (ORO), and (b3) Alizarin Red S (ARS).

Figure 4. Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28 d.

Prior to induction hMSCs were cultured for 20 days on hydrogel substrates as illustrated in Fig. 3a. Significance levels p < 0.05 by Wilcoxon-test.

Figure 5. Morphological phenotypes of hMSCs.

(a–d) Plot of circularity γ vs. aspect ratio AR for Type A–Type D hMSCs at t = 20 d. Representative fluorescence images (actin: green, nucleus: blue) are presented in insets. Each data set represents from n > 30 cells. Morphological populations were grouped by two ellipses characteristic for Type A hMSCs (89%) and Type B hMSCs (100%). Type A phenotype can represent 70% of Type C hMSCs, while Type B phenotype represents 93% of Type D hMSCs.

Figure 6. Dynamic morphological response of hMSCs to mechanical stresses over time.

(a–d) Change in aspect ratio (AR) of hMSCs vs. time for four types of hMSCs (Type A–Type D). Each histogram was extracted from n > 30 cells. Phase contrast images of representative cells for the four types (Type A–D) at t = 20 d are presented as insets. The cell contour was highlighted in yellow.

Figure 7. Impact of the frequency of mechanical stress f⁻¹ on hMSC proliferation.

(a–d) Dual staining with DAPI (blue) and anti-BrdU (magenta) of hMSCs (t = 20 d) experiencing: (a) no change (Type A), (b) f⁻¹ = 10 d (Type C), (c) f⁻¹ = 2 d. (d) hMSCs cultured on plastic dishes (control). (e) Fractions of anti-BrdU positive (proliferating) cells χ plotted as function of duration of a mechanical step f⁻¹ at t = 10 d (blue) and 20 d (red) exhibiting a non-linear relationship between χ and f⁻¹. Each data point represents mean values ± SD for n > 30 cells.