A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications. Introduction Human mesenchymal stem cells Rabbit Polyclonal to ADRA2A (hMSCs) can differentiate into multiple cell lineages, serving as an excellent cell source for regenerative medicine.1,2,3,4,5 Among different forms of applying hMSCs to engineer tissues, a scaffold-free approach is particularly attractive. It avoids any foreign-body response to the scaffold and other complications arising from the by-products of scaffold biodegradation.6,7,8 A micromass pellet culture of hMSCs to generate cartilaginous tissue exemplifies the appeal of this approach.9 A free-standing MSC sheet comprising only cells and their deposited extracellular matrix (ECM) is another prominent example for the regeneration of scarred myocardium10 and bone tissues.11 Although CHR-6494 IC50 cell sheets alone are restricted in clinical application by their insufficient mechanical strength, three-dimensional tissue structure may be created by utilizing laminar cellular assemblies.12 In addition, fragments of MSC sheet can serve as cell delivery vehicle by providing a favorable ECM environment to retain the transplanted cells and improve the efficacy of therapeutic cell transplantation via direct intramyocardial13 or intramuscular14 injection. Although the multilineage differentiation capability allows hMSC sheets to reconstruct complex tissues, even more attractive would be a uniform cell sheet with aligned hMSCs in a relatively undifferentiated CHR-6494 IC50 state. Cellular organization, in many cases alignment, provides functional competence to many tissue types. We have previously fabricated an hMSC sheet from aligned, electrospun thermosensitive chitosan fibers.15 We have also studied the CHR-6494 IC50 alignment of hMSC on nanogratings fabricated by soft lithography and nanoimprinting, and established that nanopatterns exert a more pronounced effect than micropatterns in aligning cells.16,17 To form an aligned hMSC sheet, the first crucial step would be to grow hMSCs into confluency with a high degree of alignment. We frequently observe hMSCs forming clusters when cultured on a flat surface, consistent with reports in the literature.18 On nanogratings, the hMSCs have an even greater tendency to grow into an uneven patchy layer. A desirable cell sheet should comprise cells forming tight junctions with each other and secrete plenty of ECM proteins to hold the cell sheet together.6,19,20 A nonuniform or patchy structure could make the cell sheet vulnerable to tearing during handling, in addition to compromising the quality of the engineered tissue. Another complication of culturing hMSCs on nanopatterns is the differentiation driven by nanotopographical cues. Nanostructures stimulate hMSCs to differentiate along the neuronal, myogenic, and osteogenic lineages in a proliferative, nondifferentiation medium, while decrease their proliferation.15,17,21 To fully exploit the cell sheet engineering concept with hMSCs, it is highly desirable to form an aligned, confluent hMSC layer while keeping the cells in a relatively undifferentiated state. We propose to achieve this by culturing hMSCs under physiologically relevant oxygen tension and on substrates with nanogratings. Low-oxygen tension is a native physiological condition of the hMSC niche.22 It maintains the undifferentiated state of hMSCs, stimulates hMSC proliferation, and upregulates the secretion of ECM proteins in both two- and three-dimensional cultures.18,22 Low-oxygen tension, when in a suitable range (1C3%), also increases cell motility > 0.05) and elongation factor (> 0.05) between the nanopatterned (HN) and flat (HF) surfaces (Figure 1d). In contrast, cells grown at the normoxic condition displayed a more elongated nuclear morphology than their low-oxygen counterparts. The nuclear factor E of hMSCs from NN condition was 2.7 compared to 1.0 for HN (< 0.01) (Figure 1d) (a spherical nucleus would have an = 0 and RN = 1; RN > 1 indicates a less than spherical shape). The RN of hMSCs from normoxic conditions was both significantly higher than those from hypoxic conditions (< 0.01), indicating a less rounded cell nuclear shape. In contrast, no significant differences were observed between the nanopatterned (NN) and flat (NF) surfaces under normoxic condition (Figure 1d). Gap junction communication and ECM secretion The connexin-43 secreted by cells cultured in conventional normoxic condition formed clusters on both flat (NF) and nanopatterned (NN) surfaces, whereas only distinct, small spots were observed in the 2% O2 conditions (Figure 2). Fibronectin, an important ECM protein responsible for cell adhesion was examined at the early culture period. Immunofluorescent staining at day 7 showed that more abundant fibronectin was secreted in the two hypoxic samples than in the two normoxic samples (Figure 3a). The counterstaining of F-actin illustrated that large gaps.