Volume 115, Issue 3, Pages 605-617 (September 1998) Identification of distinct molecular phenotypes in cultured gastrointestinal smooth muscle cells  Jacqueline Brittingham, Christopher Phiel, Wendy C. Trzyna, Vijayalakshmi Gabbeta, Kirk M. McHugh  Gastroenterology  Volume 115, Issue 3, Pages 605-617 (September 1998) DOI: 10.1016/S0016-5085(98)70140-4 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Phase-contrast light micrographs of cultured ISMCs plated at low density. (A) Day 1–cultured ISMCs show a broad, spread shape with numerous mitotic figures present. (B) Day 3–cultured ISMCs show a similar shape as day 1 cells with an increase in cell-to-cell contact. (C) Day 5–cultured ISMCs show a more fusiform cellular shape with regions of cells aligned in parallel. (D) Day 7–cultured ISMCs appear subconfluent and show a fusiform shape with regions of cells aligned in close, parallel orientation. (E) Day 10–cultured ISMCs show a regular fusiform shape with overlapping cell boundaries forming broad sheets of cells aligned in close, parallel orientation. (F) Day 21–cultured ISMCs show a hills-and-valley shape (H and V) with numerous areas of cellular retraction within the valleys. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Northern blot analysis of cultured ISMCs plated on plastic at low density/10% FBS probed with the γ-smooth muscle, α-smooth muscle, and β-cytoplasmic and γ-cytoplasmic isoactins at isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days 1, 3, 5, 7, and 10 of culture. (B) Relative expression of γ-smooth muscle (●), α-smooth muscle (○), β-cytoplasmic (□), and γ-cytoplasmic (■) isoactins in isolated muscle strips, immediately after enzyme dissociation (0), and at days 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression (not shown). Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Northern blot analysis of cultured ISMCs plated on plastic at low density/10% FBS probed with the γ-smooth muscle, α-smooth muscle, and β-cytoplasmic and γ-cytoplasmic isoactins at isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days 1, 3, 5, 7, and 10 of culture. (B) Relative expression of γ-smooth muscle (●), α-smooth muscle (○), β-cytoplasmic (□), and γ-cytoplasmic (■) isoactins in isolated muscle strips, immediately after enzyme dissociation (0), and at days 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression (not shown). Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Northern blot analysis of cultured ISMCs plated at high density (HD)/10% FBS, high density/0.5% FBS, low density (LD)/10% FBS, and low density/0.5% FBS probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (○) and α-smooth muscle (●) isoactins in cultured ISMCs plated on plastic at (B) low density/10% FBS, (C) high density/10% FBS, (E) high density/0.5% FBS, and (D) low density/0.5% FBS in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Northern blot analysis of cultured ISMCs plated at high density (HD)/10% FBS, high density/0.5% FBS, low density (LD)/10% FBS, and low density/0.5% FBS probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (○) and α-smooth muscle (●) isoactins in cultured ISMCs plated on plastic at (B) low density/10% FBS, (C) high density/10% FBS, (E) high density/0.5% FBS, and (D) low density/0.5% FBS in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Northern blot analysis of cultured ISMCs plated at high density (HD)/10% FBS, high density/0.5% FBS, low density (LD)/10% FBS, and low density/0.5% FBS probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (○) and α-smooth muscle (●) isoactins in cultured ISMCs plated on plastic at (B) low density/10% FBS, (C) high density/10% FBS, (E) high density/0.5% FBS, and (D) low density/0.5% FBS in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Northern blot analysis of cultured ISMCs plated at high density (HD)/10% FBS, high density/0.5% FBS, low density (LD)/10% FBS, and low density/0.5% FBS probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (○) and α-smooth muscle (●) isoactins in cultured ISMCs plated on plastic at (B) low density/10% FBS, (C) high density/10% FBS, (E) high density/0.5% FBS, and (D) low density/0.5% FBS in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Northern blot analysis of cultured ISMCs plated at high density (HD)/10% FBS, high density/0.5% FBS, low density (LD)/10% FBS, and low density/0.5% FBS probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (○) and α-smooth muscle (●) isoactins in cultured ISMCs plated on plastic at (B) low density/10% FBS, (C) high density/10% FBS, (E) high density/0.5% FBS, and (D) low density/0.5% FBS in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Northern blot analysis of cultured ISMCs plated at low density/10% FBS on fibronectin, laminin, and collagen type IV probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (●) and α-smooth muscle (○) isoactins in cultured ISMCs plated at low density/10% FBS on (B) plastic, (C) fibronectin, (D) laminin, and (E) collagen type IV in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Northern blot analysis of cultured ISMCs plated at low density/10% FBS on fibronectin, laminin, and collagen type IV probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (●) and α-smooth muscle (○) isoactins in cultured ISMCs plated at low density/10% FBS on (B) plastic, (C) fibronectin, (D) laminin, and (E) collagen type IV in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Northern blot analysis of cultured ISMCs plated at low density/10% FBS on fibronectin, laminin, and collagen type IV probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (●) and α-smooth muscle (○) isoactins in cultured ISMCs plated at low density/10% FBS on (B) plastic, (C) fibronectin, (D) laminin, and (E) collagen type IV in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Northern blot analysis of cultured ISMCs plated at low density/10% FBS on fibronectin, laminin, and collagen type IV probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (●) and α-smooth muscle (○) isoactins in cultured ISMCs plated at low density/10% FBS on (B) plastic, (C) fibronectin, (D) laminin, and (E) collagen type IV in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Northern blot analysis of cultured ISMCs plated at low density/10% FBS on fibronectin, laminin, and collagen type IV probed with γ-smooth muscle (γ-SMA) and α-smooth muscle (α-SMA) isoactins on days (D) 1, 3, 5, 7, and 10 of culture. Quantitation values have been normalized to GAPDH expression. Relative expression of γ-smooth muscle (●) and α-smooth muscle (○) isoactins in cultured ISMCs plated at low density/10% FBS on (B) plastic, (C) fibronectin, (D) laminin, and (E) collagen type IV in isolated muscle strips (MS), immediately after enzyme dissociation (0), and at days (D) 1, 3, 5, 7, and 10 of culture. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Proposed model of rat gastrointestinal smooth muscle development in vivo at embryonic days 9 (E9), 11 (E11), 15 (E15), and adult showing mucosa (MU), mesenchyme (ME), serosa (SE), primitive circular smooth muscle layer (PCM), primitive longitudinal smooth muscle layer (PLM), primitive muscularis mucosa (PMM), circular smooth muscle layer (CM), longitudinal smooth muscle layer (LM), and muscularis mucosa (MM). (B) Cellular phenotypes principally observed at these developmental time points with the corresponding phenotypes observed at day 1 (D1) and day 10 (D10) of culture marked. Gene products known to be expressed in the various smooth muscle cell phenotypes are highlighted in boxes. Gastroenterology 1998 115, 605-617DOI: (10.1016/S0016-5085(98)70140-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions