Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer

Abstract

A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

Figure 1

Morphology of nuclei and F-actin, and cell nuclear parameters of human mesenchymal stem cells (hMSCs) grown on conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF) on day 14. (a) Morphology of nuclei (blue) and F-actin (red) of hMSCs. The cells exhibited slightly elongated nuclei and finer F-actin fibers in the two 2% O₂ samples (HN and HF), whereas cultures from the two 20% O₂ samples displayed significantly more elongated nuclei. White arrows indicate the direction of nanogratings on the surface of PDMS. (b) Morphology of F-actin of hMSC layers at day 14 at conditions of NN, NF, HN, and HF in a low magnification. Uniform cell layers were formed at the two 2% O₂ samples (HN and HF), whereas the NN samples displayed a patchy structure. (c) The distribution of the cell nuclear alignment angles in the four conditions. The percentage of aligned cells with angles <10&Ring; was significantly higher in HN samples than NN samples, and the distribution of alignment angles was also much narrower in the HN condition than in the NN condition. (d) Cell nuclear morphology in the four conditions after 14 days of culture. The nuclei of hMSCs grown under the two 2% O₂ conditions were more elongated than their counterparts in the normal oxygen cultures. Values are means ± SD for six samples of each substrate (*P < 0.05).

Figure 2

Morphology of connexin-43 (green) at day 14 in hMSCs at conditions of NN, NF, HN and HF. The connexin-43 secreted by hMSCs cultured in the two 20% O₂ conditions formed clusters on both nanopatterned (NN) and flat surfaces (NF), whereas only small spots uniformly distributed in the cultures at 2% O₂ conditions (HN, HF). The F-actin was conunterstained with phalloidin (red). Yellow arrows indicate the connexin-43 clusters in the samples. White arrows indicate the direction of nanogratings on the surface of poly(dimethylsiloxan).

Figure 3

Morphology and western blotting of fibronectin. (a) Morphology of fibronectin (green) at day 7 in human mesenchymal stem cells (hMSCs) at conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF). The F-actin was conunterstained with phalloidin (red). Yellow arrows indicate cell clusters. White arrows indicate the direction of nanogratings on the surface of poly(dimethylsiloxan). (b) Western blotting of fibronectin at four different conditions at days 4 and 7. (c) Quantitative analysis of fibronectin expression in hMSCs cultured in the four conditions. The expression of fibronectin was normalized with the expression of endogenous control β-actin. The fibronectin secretion at days 4 and 7 was significantly higher in the two hypoxic samples (HN and HF) than the other two normoxic samples (NN and NF).

Figure 4

Extracellular matrix (ECM) morphology (green) at day 14 in the conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF). Abundant fibronectin and laminin were secreted in all the samples. There were no apparent distinctions in the intensity of stains in the four cultures. The two 20% O₂ cultures appeared to express collagen I and collagen IV at significantly lower levels than those seen in the two 2% O₂ cultures. The cell nuclei was conunterstained with 4′,6-diamidino-2-phenylindole (blue). White arrows indicate the direction of nanogratings on the surface of poly(dimethylsiloxan).

Figure 5

Cell proliferation and viability of hMSCs in the four conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF). (a) The percentage of cells in proliferation status (S and M phases) of the cell cycle over the 21-day culture period, obtained from flow cytometry–based propidium iodide (PI) cell cycle assay (supporting data). The total percentage of proliferating cells on the two nanopatterned surfaces was significantly lower than those on the two flat surfaces at day 7. There were no significant difference among all the samples after day 7. (b) Cell viability by flow cytometry–based Annexin-V/PI apoptosis assay at days 7 and 21. Significantly higher percentage of dead cells appeared in the 2% O₂ samples, whereas fewer cells were in necrotic apoptotic status at day 7. At day 21 the NN samples displayed the lowest percentage of viable cells, while the HF samples gave the highest percentage of viable cells.

Figure 6

Stemness of human mesenchymal stem cells (hMSCs) in the four conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF). (a) Morphology of colonies formed by the cells from the four culture conditions. The cells were harvested after a predetermined time, and then further cultured for 14 days at a low seeding density. The colonies were stained with crystal violet. (b) The colony-forming ability of hMSCs. A significant difference appeared between the 2% O₂ and 20% O₂ cultures starting from day 4. (c) The RNA expression levels of stem cell genes Oct-4, Rex-1, and Sox-2, in hMSCs over the 21-day culture period. The expression of genes was normalized with the expression of endogenous control β-actin. Human embryonic stem cell sample was used as a positive control. Culture under 2% O₂ condition enhanced all of the mRNA expression levels in hMSCs grown on both aligned pattern and flat surfaces.

Figure 7

Multipotency of human mesenchymal stem cells (hMSCs) after 21-day culture in the four conditions of nanopatterned surface at 20% O₂ (NN), flat surface at 20% O₂ (NF), nanopatterned surface at 2% O₂ (HN), and flat surface at 2% O₂ (HF). (a) Adipocyte differentiation showed large lipid droplet clusters in the HN and HF constructs compared to the NN and NF samples. (b) Von Kossa staining indicated mineralization (dark regions) of the extracellular matrix (ECM) secreted by the cells. Significant calcium deposition was displayed within the ECM in the HN and HF cultures, while very little mineralization was displayed in the NN and NF samples. Samples were induced in adipoinductive and osteoinductive media for 21 days.