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Chondrocyte differentiation of human endometrial gland-derived MSCs in layered cell sheets.

Sekine W, Haraguchi Y, Shimizu T, Yamato M, Umezawa A, Okano T - ScientificWorldJournal (2013)

Bottom Line: 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 addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

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.

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Related in: MedlinePlus

Histological observation of layered human endometrial gland-derived mesenchymal stem cells (hEMSC) sheets after a 24 h cultivation on porous membranes. All microphotographs are the hematoxylin and eosin stained cross-sections of the cell sheets ((a) single-layered cell sheet; (b) double-layered cell sheet; (c) triple-layered cell sheet; (d) quadruple-layered cell sheet; (e) quintuple-layered cell sheet; (f) sextuple-layered cell sheet). Enlarged photographs showed chondrocyte-like cells. Independent three experiments were performed and those experiments showed similar results. The representative photographs were shown in the figure.
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fig1: Histological observation of layered human endometrial gland-derived mesenchymal stem cells (hEMSC) sheets after a 24 h cultivation on porous membranes. All microphotographs are the hematoxylin and eosin stained cross-sections of the cell sheets ((a) single-layered cell sheet; (b) double-layered cell sheet; (c) triple-layered cell sheet; (d) quadruple-layered cell sheet; (e) quintuple-layered cell sheet; (f) sextuple-layered cell sheet). Enlarged photographs showed chondrocyte-like cells. Independent three experiments were performed and those experiments showed similar results. The representative photographs were shown in the figure.

Mentions: hEMSC sheets were layered into 3D tissues onto a porous membrane, and the differentiation kinetics of the stem cells within the cell sheet constructs were histologically examined. At 24 h after the cultivation of layered hEMSC sheets, the constructs were observed cross-sectionally. Because, after detaching from temperature-responsive culture dishes, a cell sheet shrunk horizontally due to the cytoskeletal tensile reorganization, a single-layer cell sheet consisted of several cell-layers, and the thickness of the cell sheet became more than 25 μm (Figure 1). With increasing the number of stratified cell sheets, the thicknesses of the layered cell sheets increased as shown in Figure 1. The thicknesses of more than triple-layered cell sheets were more than 100 μm, and those cell layer tissues were clearly cell dense. Some characteristic cells were found within the multilayered cell-sheet constructs and resembled differentiated chondrocytes (Figure 1). Many chondrocyte-like cells were found within more than quadruple-layered cell-sheet constructs (Figures 1 and 2). After the cell sheet layering, hEMSCs might differentiate into chondrocytes very rapidly (24-h cultivation) within the multilayered cell sheet constructs. On the other hand, in single-, double-, triple-layered cell-sheet constructs, similar cell structures were observed (Figure 1).


Chondrocyte differentiation of human endometrial gland-derived MSCs in layered cell sheets.

Sekine W, Haraguchi Y, Shimizu T, Yamato M, Umezawa A, Okano T - ScientificWorldJournal (2013)

Histological observation of layered human endometrial gland-derived mesenchymal stem cells (hEMSC) sheets after a 24 h cultivation on porous membranes. All microphotographs are the hematoxylin and eosin stained cross-sections of the cell sheets ((a) single-layered cell sheet; (b) double-layered cell sheet; (c) triple-layered cell sheet; (d) quadruple-layered cell sheet; (e) quintuple-layered cell sheet; (f) sextuple-layered cell sheet). Enlarged photographs showed chondrocyte-like cells. Independent three experiments were performed and those experiments showed similar results. The representative photographs were shown in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3852803&req=5

fig1: Histological observation of layered human endometrial gland-derived mesenchymal stem cells (hEMSC) sheets after a 24 h cultivation on porous membranes. All microphotographs are the hematoxylin and eosin stained cross-sections of the cell sheets ((a) single-layered cell sheet; (b) double-layered cell sheet; (c) triple-layered cell sheet; (d) quadruple-layered cell sheet; (e) quintuple-layered cell sheet; (f) sextuple-layered cell sheet). Enlarged photographs showed chondrocyte-like cells. Independent three experiments were performed and those experiments showed similar results. The representative photographs were shown in the figure.
Mentions: hEMSC sheets were layered into 3D tissues onto a porous membrane, and the differentiation kinetics of the stem cells within the cell sheet constructs were histologically examined. At 24 h after the cultivation of layered hEMSC sheets, the constructs were observed cross-sectionally. Because, after detaching from temperature-responsive culture dishes, a cell sheet shrunk horizontally due to the cytoskeletal tensile reorganization, a single-layer cell sheet consisted of several cell-layers, and the thickness of the cell sheet became more than 25 μm (Figure 1). With increasing the number of stratified cell sheets, the thicknesses of the layered cell sheets increased as shown in Figure 1. The thicknesses of more than triple-layered cell sheets were more than 100 μm, and those cell layer tissues were clearly cell dense. Some characteristic cells were found within the multilayered cell-sheet constructs and resembled differentiated chondrocytes (Figure 1). Many chondrocyte-like cells were found within more than quadruple-layered cell-sheet constructs (Figures 1 and 2). After the cell sheet layering, hEMSCs might differentiate into chondrocytes very rapidly (24-h cultivation) within the multilayered cell sheet constructs. On the other hand, in single-, double-, triple-layered cell-sheet constructs, similar cell structures were observed (Figure 1).

Bottom Line: 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 addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

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.

Show MeSH
Related in: MedlinePlus