1. Native Umbilical Cord Matrix Stem Cells Express Hepatic Markers and Differentiate Into Hepatocyte-like Cells 
David Campard, Philippe A. Lysy, Mustapha Najimi, Etienne Marc Sokal 
Gastroenterology 
Volume 134, Issue 3, Pages (March 2008)
DOI: /j.gastro
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2. Figure 1
Characterization of UCMSCs. (A) Morphology of UCMSCs at the third day of culture (A1, original magnification 400×) and at the 11th day when cells reached confluence (A2, original magnification 200×). (B) Expansion of UCMSCs from passage 0 to passage 15. (C) Immunocytochemical analysis of expanding UCMSCs for α–smooth muscle actin (red, C1), vimentin (red, C2) and fibronectin (green, C3) (nuclei were stained blue with 4′,6-diamidino-2-phenylindole; original magnification 200×). (D) A representative flow cytometry analysis of UCMSCs at passage 4. Results are means of rate of positive cells (%) for 8 independent experiments ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
Osteogenic and adipogenic differentiation of UCMSCs. Adipogenic potential of UCMSCs was shown by formation of intracytoplasmic lipid droplets of different sizes that were observed by phase-contrast microscopy (A2) and were positively stained with Oil Red O (A4) (original magnification 400×; inset 1000×). UCMSCs cultured in control medium (A1 and A3, respectively) did not exhibit such adipocytic features. Osteogenic potential of UCMSCs was shown by microscopic observation of bone nodules (B2, original magnification 200×) that incorporated tetracycline (B4, original magnification 100×) and by calcium mineralization as revealed by Alizarin Red (B6, original magnification 100×) and von Kossa stains (B8, original magnification 200×). Respective control cultures of UCMSCs (B1, B3, B5, and B7) did not exhibit such osteocytic features.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Expression of hepatic markers by native UCMSCs. (A) A representative RT-PCR analysis of UCMSCs cultured in expansion medium for late and early hepatic markers compared with human liver cells (HLC). Heterogeneity from culture to culture of UCMSCs was represented by the rate of gene-expressing cultures indicated in the right column. αFP, α-fetoprotein; Cnx-32, connexin 32; HNF-4, hepatocyte nuclear factor 4; PEPCK, phosphoenol pyruvate carboxykinase. (B) Flow cytometry quantification of albumin-, DPPIV- or cytokeratin-positive UCMSCs cultured in expansion medium. Respective isotype control antibody curves are shown in the left panels. Results are means of rate of positive cells (%) for 7–10 independent experiments ± SEM. (C) Immunocytochemical analysis of expanding UCMSCs (n = 4) showed that UCMSCs strongly expressed albumin (red, C1), α-fetoprotein (green, C2), connexin 32 (red, C3), CK-19 (red, C4), and DPPIV (green, C5). Filaments of CK-8 (red, C6) and CK-18 (red, C7) were observed in few cells, while CK-7 (red, C8) and HepPar1 (red, C9) were not expressed. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole (original magnification 200×).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Effect of hepatogenic differentiation protocol on expression of hepatic specific genes. UCMSCs were cultured in differentiation medium with hepatogenic supplements (DM + HS) and were examined for their messenger RNA content for hepatic markers. Gene expression profiles were compared with undifferentiated UCMSCs cultured in differentiation medium without hepatogenic supplements (DM − HS) or human liver cells (HLC). A representative profile is presented, and the rate of gene-expressing cultures for DM − HS and DM + HS conditions is indicated in the 2 right columns. Depending on the culture, expression of a gene was modified or not, suggesting heterogeneity of UCMSCs in response to hepatogenic induction. αFP, α-fetoprotein; Cnx-32, connexin 32; HNF-4, hepatocyte nuclear factor 4; PEPCK, phosphoenol pyruvate carboxykinase.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Effect of hepatogenic differentiation protocol on expression of hepatic specific proteins. Morphology and immunocytochemical analysis of undifferentiated or hepatocytic differentiated UCMSCs cultured in medium without (DM − HS) or with hepatogenic supplements (DM + HS), respectively. Immunocytochemical analysis of differentiated UCMSCs showed maintenance of constitutively expressed hepatic markers compared with UCMSCs cultured in control medium (DM + HS vs DM − HS, positive culture/total of tested culture): albumin (11/11 vs 11/11), α-fetoprotein (αFP; 7/7 vs 6/6), CK-19 (8/8 vs 9/9), and connexin 32 (Cnx32; 6/7 vs 6/7). Small variations have been observed for CK-18 (7/10 vs 5/9) and CK-8 (5/9 vs 4/9), but hepatogenic induction failed to up-regulate mature hepatocyte marker HepPar1 (0/7 vs 0/7). DPPIV was detected in similar levels in both culture conditions (8/8 vs 8/8), but fluorescence spots (white arrowheads) suggest polarization of the protein. Finally, persistence of mesenchymal markers α–smooth muscle actin (αSMA), vimentin, and fibronectin was observed in differentiated UCMSCs (7/7 vs 7/7 for all markers). Original magnification 200× except for DPPIV (100×; inset 200×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

7. Figure 6
Functionality of hepatocyte-like cells derived from UCMSCs. UCMSCs cultured in medium without hepatogenic supplements (A1, control medium) did not display a positive reaction to periodic acid–Schiff staining. However, UCMSCs cultured in hepatogenic differentiation medium were positive, revealing accumulation of glycogen in their cytoplasm (A2). Staining was specific for glycogen because treatment of differentiated UCMSCs with amylase prevented a positive reaction (A3). Efficiency of periodic acid–Schiff staining was tested on fresh hepatocytes as positive control (A4). Original magnification: A1–A3, 200×; A4 and inset in A2, 400×. (B) G6P activity was detected in undifferentiated UCMSCs (B1) and was enhanced after the differentiation process (B2) (n = 4). (C) UCMSCs were cultured in differentiation medium (DM) with (+, n = 5) or without (−, n = 5) hepatogenic supplements (HS) and were stimulated for 24 hours with 0.3 mmol/L NH4Cl before measuring urea concentration in culture supernatants. Urea production was compared with cultures of fresh hepatocytes (HLC) (n = 5). Data are means ± SEM. **.001 ≤ P < .01; *.01 ≤ P < .05. (D) Primary culture of human liver cells (HLC) and undifferentiated (DM − HS) or hepatocytic differentiated UCMSCs (DM + HS) were cultured for 48 hours in medium with or without 1 mmol/L phenobarbital (black and white bars, respectively). Following exposure, catalytic activity of CYP3A4 was determined using 50 μmol/L luciferin substrate (luciferin PFBE). After 3-hour incubation with the substrate, luciferase activity was assessed. Results are expressed as luciferase activity in relative luminescence unit (RLU) and are mean ± SEM of 5 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Undifferentiated UCMSCs engrafted livers of partially hepatectomized SCID mice. One million human UCMSCs were injected into the spleen of hepatectomized SCID mice and were tracked into liver recipients 2 (n = 3), 4 (n = 3), and 6 (n = 4) weeks later by in situ hybridization with human Alu probe. Immunostaining with anti-human mitochondria confirmed engraftment of human UCMSCs into SCID mice (black arrowheads). Immunohistochemical analysis included detection of human albumin, α-fetoprotein (αFP), CK-19, and fibronectin. Small clusters of cells and isolated cells stained expressed albumin in mice that had undergone transplantation. However, only isolated cells stained positive for α-fetoprotein or fibronectin. CK-19 was not detected in livers of mice that had undergone transplantation. PBS-injected mice served as negative controls, and sections of human livers served as positive controls. Original magnification 400× except for anti-mitochondria (530×; inset 1000×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions