Chondrocyte Differentiation of Human Endometrial Gland‐Derived MSCs in Layered Cell Sheets

Abstract

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy.

Optical coherence microscopy of living cells and bioengineered tissue dynamics in high-resolution cross-section

“…Those cells were then seeded on a 35‐mm polystyrene culture dish (Corning, NY) and cultured at 37°C until observation by OCM. NIH3T3 cell sheets and BAEC sheets were fabricated by using a 35‐mm thermoresponsive culture dish (UpCell ® dish) (CellSeed, Tokyo, Japan), and the fabrication of 3‐D tissues by layering the cell sheets was performed as described in previous reports . Recently, we developed a rapid fabrication system for cell sheet‐tissues by centrifugation, which can reduce the fabrication time by approximately two‐thirds compared to the conventional method .…”

“…After incubation, the magnet was removed, and a cell sheet containing metal‐labeled cells was harvested by decreasing the temperature (Supporting Information, Figure 1A), and was then reattached onto a 35‐mm polystyrene culture dish. For observing the 3‐D migration of metal‐labeled cells, a nonlabeled cell sheet was then layered onto the metal‐labeled cell sheet (Supporting Information, Figure 1B) as described in previous reports . Finally, the fabricated cell sheet‐tissue was cultured at 37°C before observation by OCM.…”

“…NIH3T3 cell sheets and BAEC sheets were fabricated by using a 35-mm thermoresponsive culture dish (UpCell V R dish) (CellSeed, Tokyo, Japan), and the fabrication of 3-D tissues by layering the cell sheets was performed as described in previous reports. 1,[5][6][7]19,23 Recently, we developed a rapid fabrication system for cell sheet-tissues by centrifugation, which can reduce the fabrication time by approximately two-thirds compared to the conventional method. 24 Thus, in this study, each cell sheet was rapidly attached by centrifugation (12-34g) for 3 min onto a culture surface after reattachment, or onto another cell sheet after layering, as described in the previous report.…”

“…A microscope is an essential tool in the field of cell biology. The histological observation of three‐dimensional (3‐D) tissues using microscopy provides cross‐sectional information, for example, thickness, cell density, cell viability, and cell differentiation . However, the fabrication of a paraffin section or a cryosection for observation terminates the life of cells and tissues.…”

“…The histological observation of three-dimensional (3-D) tissues using microscopy provides cross-sectional information, for example, thickness, cell density, cell viability, and cell differentiation. [1][2][3][4][5][6][7] However, the fabrication of a paraffin section or a cryosection for observation terminates the life of cells and tissues. A confocal microscope is a valuable tool for cross-sectional analyses of cultured cells and biological tissues.…”