Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature

Abstract

Fibroblasts and smooth muscle cells (FSMCs) are principal cell types of connective and adventitial tissues that participate in the development, physiology and pathology of internal organs, with incompletely defined cellular origins. Here, we identify and prospectively isolate from the mesothelium a mouse cell lineage that is committed to FSMCs. The mesothelium is an epithelial monolayer covering the vertebrate thoracic and abdominal cavities and internal organs. Time-lapse imaging and transplantation experiments reveal robust generation of FSMCs from the mesothelium. By targeting mesothelin (MSLN), a surface marker expressed on mesothelial cells, we identify and isolate precursors capable of clonally generating FSMCs. Using a genetic lineage tracing approach, we show that embryonic and adult mesothelium represents a common lineage to trunk FSMCs, and trunk vasculature, with minimal contributions from neural crest, or circulating cells. The isolation of FSMC precursors enables the examination of multiple aspects of smooth muscle and fibroblast biology as well as the prospective isolation of these precursors for potential regenerative medicine purposes.

Figure 1

Derivation of FSMCs from cultured mesothelium. Liver (a–a‴), spleen (b–b‴), kidney (c–c‴), lung (d–d‴), intestine (e–e‴), mesentery (f–f‴), diaphragm (g–g‴). Bright field (a–g), nuclear DAPI staining (a′–g′, h–n). Mesothelium-derived cells express α-SMA (a″–g″, h–h″), Vimentin (i–i″), FSP1 (j–j″), CD90 (k–k″), Collagen type I (l–l″), Collagen type IV (m–m″) and Fibronectin (n–n″). Merged images (a‴-g‴, h″⁻n″, DAPI is red, αSMA is green except h″, l″, m″). Original magnifications ×20 (a–n).

Figure 2

Derivation of FSMCs from transplantation of mesothelium in vivo. Representative images of the abdominal cavity, from mice three months post transplantation of mesothelium. Lower digestive system (a–c), liver (d) and peritoneum (e). Sections through the lower digestive system (f, g) and peritoneum (h) showing FSMCs and cell foci along a vessel opening (g, white arrowheads). Immunohistochemistry of Vimentin (i–i‴) and FSP1 (j–j‴), showing co-localization of the FSMCs markers with graft-derived GFP⁺ cells. Original magnifications: ×10 (a, b, f, i, j), ×20 (c–e, g, h).

Figure 3

Flow cytometry, in-vitro clonal analysis and differentiation of MSLN⁺Lin⁻ cells. (a) X-axis represents MSLN expression, Y-axis represents side scatter. A population of cells characterized by MSLN⁺Lin⁻ is present within the heart (I), lung (II), liver (III), peritoneal wall (IV), kidney (V), and thymus (VI). (b) MSLN⁺Lin⁻ cells express a surface profile associated with a mesenchymal nature. Blue is IgG control, red is antibody. (c) Mixed culture of MSLN⁺Lin⁻ cells from GFP, RFP and CFP-expressing mice shows that emerging FSMC foci are derived from expansions of single precursors (monoclonal). (d–i) Cultured MSLN⁺Lin⁻ cells adopt a mesenchymal nature and express MSLN (d), Vimentin (e), FSP1 (f), Type I Collagen (g), Type IV Collagen (h) and αSMA (i) proteins. DAPI (d′–i′). Merged (d″–i″, DAPI in blue, antibody in red). Original magnifications: ×20 (c–i). The overlay of green/red colors within the merged images in g–i, results from spectral overlap of multiple staining per cultured well.

Figure 4

Genetic lineage tracing of fibroblasts within internal organs. (a) Scheme illustrating the transgenic strategy. The CreER^T2IRES-lacZ-PGK-neo cassette (pCLN) was introduced into a cassette harboring 6.3-kilobase of the mouse Mesothelin gene (pMesothelin). The Mesothelin-CreER^T2-IRES-lacZ construct was subsequently transfected into mouse embryonic stem cells. Selected clones were then injected into C57BL/6 blastocysts following the standard protocol to generate chimeras, and by mating these, MSLN^CLN mice. (b–d) βgal staining on sections from MSLN^CLN mice, following 2 days post tamoxifen injection. Staining is present within the mesothelium covering the lungs (b, red arrowheads), thymus (c, red arrowheads), and lower digestive system (d, red arrowheads). (e–j) MSLN^CLN mice were injected with tamoxifen at e10.5 and analyzed at e17.5. βgal staining is present within fibroblasts in the lower digestive system (e, f, red arrowheads), mesentery (g, red arrowheads), thymus (h, red arrowheads), parathyroid gland (i, red arrowheads) and liver (j, red arrowheads). Original magnifications: ×4 (c), ×20 (b, d–j),

Figure 5

Genetic lineage tracing of smooth muscle within internal organs, and its vasculature. βgal (a–j, in green) staining is present within the muscular layer of the duodenum (a, b), colon (c) and stomach (d), urinary bladder (e) and ureter (f). βgal staining within blood vessels of the mesentery (g), lungs (h), liver (i, red arrowhead) and thymus (j, red arrowhead). Note βgal staining within lung mesothelium (h, red arrowhead). Co-expression of βgal with αSMA protein in the duodenum (k–k‴), urinary bladder (l–l‴), pulmonary vasculature (m–m‴), mesentery vasculature (n–n‴, red arrowheads), renal vasculature (o–o‴, red arrowhead) and liver vasculature (p–p‴, red arrowheads). Nuclear DAPI staining (k–p), βgal staining (k′–p′), αSMA staining (k″–p″), merged images (k‴–p‴, βgal staining in green, αSMA staining in red). Dotted lines outline the smooth muscle layer within the urinary bladder (l–l‴) and pulmonary vasculature (m–m‴). Original magnifications: ×10 (a–p).

Figure 6

Polyclonal origins for smooth muscle revealed by clonal analysis of tetrachimeric mice. Sections through jejunum (a–a″), colon (b–b″, c–c″), duodenum (d–d″), cecum (e–e″), cardiovascular (f–f″) and pulmonary artery (g–g″). Nuclear DAPI staining (a–g), tetrachimera image (a′–g′), merged image (a″–g″). Original magnifications: x4 (f), x10 (a–e, g). The dotted lines in each figure (a–e) are between the epithelial base of the intestine and the overlying serosal layer, including the mesothelium and the underlying, mainly circumferential smooth muscle. In many places, e.g. in b, one can see the clones extending from mesentery to serosal mesothelium; a subset of the overlying mesothelial clone is continuous with a larger patch of smooth muscle, while other mesothelial cells have different color smooth muscle under them. As noted previously (26), the epithelial crypts are always a single color, and up to dozens of adjacent crypts are derived from a single intestinal stem cell.

Figure 7

Polyclonal contributions of MSLN⁺ precursors to smooth muscle during the 1^st postnatal month. Sections through the intestine (a–a″), urinary bladder (b–b″), liver (c–c″), heart atrium (d–d″), thymus (e–e″) and lungs (f–f″) from MSLN^CLN transgenic mice. Significant contribution of MSLN⁺ precursors is evident in the smooth muscle layers of the GI-tract and urinary bladder, as well as trunk vasculature. Sections through the intestine (g–g″), stomach (h–h″), urinary bladder (i–i″), mesenteric vasculature (j–j″) and renal vasculature (k–k″) from MSLN^CLNR26^VT2/GK3 transgenic mice. Multiple MSLN⁺ precursors contribute cumulatively to the postnatal growth of smooth muscle. Original magnifications: ×10 (a–k).

Figure 8

Developmental restriction of MSLN⁺ precursors to FSMCs following sub-lethal irradiation. MSLN^CLN transgenic mice received tamoxifen followed by a single dose of whole-body irradiation at 650 rads. Sections through the stomach (a–a″), intestine (b–b″), urinary bladder (c–c″), uterine horns (d–d″), seminal vesicle (e–e″), thymus (f–f″) and lungs (g–g″). Co-staining of beta-galactosidase with αSMA in the intestine (h–h″), stomach (i–i″), urinary bladder (j–j″), seminal vesicle (k–k″) and renal vasculature (l–l″). Original magnifications: ×10 (a–k), x20 (l).