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Cited by 356Liver physiology and pathologyMesenchymal stem cell researchHematopoietic Stem Cell Transplantation

The DOI matches its Crossref record; 42 of the 43 references it cites were found in a registry or an index.

Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver

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Suzuki, Atsushi, Zheng, Yun-wen, Kaneko, Shin, Onodera, Masafumi, Fukao, Katashi, Nakauchi, Hiromitsu, Taniguchi, Hideki·2002·The Journal of Cell Biology
DOI 10.1083/jcb.200108066PMC PMC2173576
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Abstract

Using flow cytometry and single cell–based assays, we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing pluripotent stem cells persist in the developing mouse liver and that such cells can be induced to become cells of other organs of endodermal origin under appropriate microenvironment. Manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.

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Figures

Figure 1. Flow cytometric analysis of fetal mouse liver cells. (A) c-Kit− CD45− TER119− cells among ED 11.5, ED 13.5, and ED 15.5 fetal mouse liver cells were fractionated by c-Met and CD49f expression. Sorting gates were then set for c-Met− CD49f−, c-Met− CD49f+/low, c-Met− CD49f+/high, c-Met+ CD49f−, c-Met+ CD49f+/low, and c-Met+ CD49f+/high subpopulations. The percentage of fractionated cells is shown in the upper right. Representative data from six independent experiments are shown. (B) Numbers of H-CFU-C per 3 × 102 cells in each cell subpopulation derived from ED 11.5, ED 13.5, and ED 15.5 fetal mouse livers. This graph shows the average of 18 dishes for each cell subpopulation in six independent experiments (n = 6). *P < 0.001; **P < 0.005; ***P < 0.01.
Figure 1. Flow cytometric analysis of fetal mouse liver cells. (A) c-Kit− CD45− TER119− cells among ED 11.5, ED 13.5, and ED 15.5 fetal mouse liver cells were fractionated by c-Met and CD49f expression. Sorting gates were then set for c-Met− CD49f−, c-Met− CD49f+/low, c-Met− CD49f+/high, c-Met+ CD49f−, c-Met+ CD49f+/low, and c-Met+ CD49f+/high subpopulations. The percentage of fractionated cells is shown in the upper right. Representative data from six independent experiments are shown. (B) Numbers of H-CFU-C per 3 × 102 cells in each cell subpopulation derived from ED 11.5, ED 13.5, and ED 15.5 fetal mouse livers. This graph shows the average of 18 dishes for each cell subpopulation in six independent experiments (n = 6). *P < 0.001; **P < 0.005; ***P < 0.01.CC BY-NC-SA · PubMed Central
Figure 2. In vitro multilineage colony formation from a sorted c-Met + CD49f + /low c-Kit − CD45 − TER119 − cell. (A and B) Single cell culture of c-Met+ CD49f+/low c-Kit− CD45− TER119− cells was performed on laminin-coated 96-well plates for 5 d, and then H-CFU-C colonies were determined. (C) Some H-CFU-C formed even larger colonies when cultured for 21 d. Immunocytochemical staining was conducted after 5 (D–G) or 21 d (H–L) of culture. Cells stained green for single-positive cells marking for albumin or red for cytokeratin 19. After 5 d of culture, most small colonies were composed of cells positive for only albumin (D) or cytokeratin 19 (E). Although most H-CFU-C colonies were formed by cells expressing neither marker (F), some of them included both cells expressing one marker and cells expressing the other (G). After 21 d of culture, most H-CFU-C colonies included both cells expressing albumin and cells expressing cytokeratin 19 (H–J), whereas a few of them were composed of cells that marked only for albumin (K) or for cytokeratin 19 (L). We also observed cells expressing both markers at once (I, arrowhead, yellow). I and J are magnified pictures of the regions surrounded by broken lines in H and I. (M and N) Periodic acid–Schiff staining revealed that most H-CFU-C gave rise to functionally mature hepatocytes, containing abundant glycogen stores, after 21 d of culture. Bars: (A and D–N) 100 μm; (B) 50 μm; (C) 2.5 mm.
Figure 2. In vitro multilineage colony formation from a sorted c-Met + CD49f + /low c-Kit − CD45 − TER119 − cell. (A and B) Single cell culture of c-Met+ CD49f+/low c-Kit− CD45− TER119− cells was performed on laminin-coated 96-well plates for 5 d, and then H-CFU-C colonies were determined. (C) Some H-CFU-C formed even larger colonies when cultured for 21 d. Immunocytochemical staining was conducted after 5 (D–G) or 21 d (H–L) of culture. Cells stained green for single-positive cells marking for albumin or red for cytokeratin 19. After 5 d of culture, most small colonies were composed of cells positive for only albumin (D) or cytokeratin 19 (E). Although most H-CFU-C colonies were formed by cells expressing neither marker (F), some of them included both cells expressing one marker and cells expressing the other (G). After 21 d of culture, most H-CFU-C colonies included both cells expressing albumin and cells expressing cytokeratin 19 (H–J), whereas a few of them were composed of cells that marked only for albumin (K) or for cytokeratin 19 (L). We also observed cells expressing both markers at once (I, arrowhead, yellow). I and J are magnified pictures of the regions surrounded by broken lines in H and I. (M and N) Periodic acid–Schiff staining revealed that most H-CFU-C gave rise to functionally mature hepatocytes, containing abundant glycogen stores, after 21 d of culture. Bars: (A and D–N) 100 μm; (B) 50 μm; (C) 2.5 mm.CC BY-NC-SA · PubMed Central
Figure 3. Multipotency of reclone-sorted progeny derived from a single H-CFU-C. Subcultured cells derived from an H-CFU-C were clone-sorted again, and subjected to single cell culture. (A–C) Resorted cells gave rise to albumin-positive hepatocytes (green) or cytokeratin 19–positive cholangiocytes (red) after 21 d of culture. As upon primary culture, they gave rise to albumin and cytokeratin 19 double positive cells (yellow) and to double negative cells. Duct-like structures formed by cells expressing cytokeratin 19 (A, arrowheads) and binucleate cells (B, arrowheads) interpreted as mature hepatocytes were regularly observed. (D) Ultrastructural study of the progeny of reclone-sorted cells found many ovoid mitochondria and glycogen rosettes in their cytoplasm. Adjacent cells also formed well-defined narrow lumina with numerous microvilli and intracellular tight junctional complexes (arrows). These structures resembled hepatic bile canaliculi. (E and F) Bile duct-like structures formed by neatly aligned cells were morphologically characterized by frequently notched nuclei at the basal aspects, a relatively large nucleus to cytoplasm ratio, numerous short microvilli projecting into luminal regions (F, arrowheads), and close attachment with juxtaluminal junctional complexes between adjacent cells (F, arrows). Bars: (A–C) 100 μm; (D) 2 μm; (E and F) 10 μm.
Figure 3. Multipotency of reclone-sorted progeny derived from a single H-CFU-C. Subcultured cells derived from an H-CFU-C were clone-sorted again, and subjected to single cell culture. (A–C) Resorted cells gave rise to albumin-positive hepatocytes (green) or cytokeratin 19–positive cholangiocytes (red) after 21 d of culture. As upon primary culture, they gave rise to albumin and cytokeratin 19 double positive cells (yellow) and to double negative cells. Duct-like structures formed by cells expressing cytokeratin 19 (A, arrowheads) and binucleate cells (B, arrowheads) interpreted as mature hepatocytes were regularly observed. (D) Ultrastructural study of the progeny of reclone-sorted cells found many ovoid mitochondria and glycogen rosettes in their cytoplasm. Adjacent cells also formed well-defined narrow lumina with numerous microvilli and intracellular tight junctional complexes (arrows). These structures resembled hepatic bile canaliculi. (E and F) Bile duct-like structures formed by neatly aligned cells were morphologically characterized by frequently notched nuclei at the basal aspects, a relatively large nucleus to cytoplasm ratio, numerous short microvilli projecting into luminal regions (F, arrowheads), and close attachment with juxtaluminal junctional complexes between adjacent cells (F, arrows). Bars: (A–C) 100 μm; (D) 2 μm; (E and F) 10 μm.CC BY-NC-SA · PubMed Central
Figure 4. H-CFU-C incorporated BrdU in vivo while retaining the capacity for multilineage differentiation. (A) FACS® analysis of cells that had incorporated BrdU after pulse labeling from ED 12.75 to ED 13.5. Most sorted unfractionated and c-Met+ CD49f+/low c-Kit− CD45− TER119− cells isolated from BrdU-treated mice were BrdU positive. Representative data are shown. (B and C) 10–12 h after c-Met+ CD49f+/low c-Kit− CD45− TER119− cells were plated; most attaching (but not dividing) cells were BrdU positive. (D and E) In sections of BrdU-treated ED 13.5 fetal mouse liver, almost all liver cells were BrdU positive. Bars: (B and C) 25 μm; (D and E) 100 μm.
Figure 4. H-CFU-C incorporated BrdU in vivo while retaining the capacity for multilineage differentiation. (A) FACS® analysis of cells that had incorporated BrdU after pulse labeling from ED 12.75 to ED 13.5. Most sorted unfractionated and c-Met+ CD49f+/low c-Kit− CD45− TER119− cells isolated from BrdU-treated mice were BrdU positive. Representative data are shown. (B and C) 10–12 h after c-Met+ CD49f+/low c-Kit− CD45− TER119− cells were plated; most attaching (but not dividing) cells were BrdU positive. (D and E) In sections of BrdU-treated ED 13.5 fetal mouse liver, almost all liver cells were BrdU positive. Bars: (B and C) 25 μm; (D and E) 100 μm.CC BY-NC-SA · PubMed Central
Figure 5. Clonally expanding H-CFU-C in culture reconstituted liver, pancreas, and intestine in vivo. (A–D) 38 d after transplantation of EGFP-tagged H-CFU-C, both livers and spleens were fixed and sectioned. (A) Many EGFP-positive donor cells are seen engrafted in hepatic cords of periportal zones but not in bile ducts (arrowhead). (B) Overlay image of EGFP, phase picture, and serial section stained by albumin (red) clearly shows that many but not all EGFP-positive donor cells had differentiated into albumin-positive hepatocytes (yellow). A detail is shown at higher magnification in the inset. (C) In the spleens of recipient mice, many bile duct-like structures were formed by keratin- positive donor-derived cholangiocytes. (D) Alcian blue staining (pH 2.5) (Mowry, 1963) also showed that the component cells contained abundant mucin near luminal membranes (counterstain, nuclear fast red). (E) Pancreatic, intestinal, and gastric marker expression were not detected in the sorted c-Met+ CD49f+/low c-Kit− CD45− TER119− cells (lane 1). However, they became detectable in the progeny of a single H-CFU-C after several months of culture (lane 2). (F and G) A H-CFU-C clone isolated from EGFP transgenic fetal mice differentiated into (F) hepatocytes (6 mo posttransplant) or (G) cholangiocytes (2 mo posttransplant) in recipient livers after either carbon tetrachloride or DAPM treatment. (H–M) Differentiation was also seen in cells constituting pancreas (4 mo) and duodenum (2 mo). (H and I) Pancreatic ducts; (J–L) amylase-positive acinar cells (L, yellow); (M) intestinal vili and crypts. Insets in M show EGFP-positive goblet cells stained in alcian blue. (H and J) Hematoxylin eosin stain. PV, portal venule; PD, pancreatic duct; Is, islet. Bars: (A, F, G, and M) 100 μm; (H–L) 50 μm; (D) 2.5 μm.
Figure 5. Clonally expanding H-CFU-C in culture reconstituted liver, pancreas, and intestine in vivo. (A–D) 38 d after transplantation of EGFP-tagged H-CFU-C, both livers and spleens were fixed and sectioned. (A) Many EGFP-positive donor cells are seen engrafted in hepatic cords of periportal zones but not in bile ducts (arrowhead). (B) Overlay image of EGFP, phase picture, and serial section stained by albumin (red) clearly shows that many but not all EGFP-positive donor cells had differentiated into albumin-positive hepatocytes (yellow). A detail is shown at higher magnification in the inset. (C) In the spleens of recipient mice, many bile duct-like structures were formed by keratin- positive donor-derived cholangiocytes. (D) Alcian blue staining (pH 2.5) (Mowry, 1963) also showed that the component cells contained abundant mucin near luminal membranes (counterstain, nuclear fast red). (E) Pancreatic, intestinal, and gastric marker expression were not detected in the sorted c-Met+ CD49f+/low c-Kit− CD45− TER119− cells (lane 1). However, they became detectable in the progeny of a single H-CFU-C after several months of culture (lane 2). (F and G) A H-CFU-C clone isolated from EGFP transgenic fetal mice differentiated into (F) hepatocytes (6 mo posttransplant) or (G) cholangiocytes (2 mo posttransplant) in recipient livers after either carbon tetrachloride or DAPM treatment. (H–M) Differentiation was also seen in cells constituting pancreas (4 mo) and duodenum (2 mo). (H and I) Pancreatic ducts; (J–L) amylase-positive acinar cells (L, yellow); (M) intestinal vili and crypts. Insets in M show EGFP-positive goblet cells stained in alcian blue. (H and J) Hematoxylin eosin stain. PV, portal venule; PD, pancreatic duct; Is, islet. Bars: (A, F, G, and M) 100 μm; (H–L) 50 μm; (D) 2.5 μm.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Suzuki, A., Zheng, Y.-W., Kaneko, S., Onodera, M., Fukao, K., Nakauchi, H., & Taniguchi, H.
Affiliations
University of Tsukuba (JP)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2002-01-07
Volume
156
Issue
1
Pages
173-184
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.200108066
PMC
PMC2173576
OpenAlex
W2121054813
Cited by
356
References
43
Citations by year
2025: 2 · 2024: 3 · 2023: 4 · 2022: 3 · 2021: 1 · 2020: 7 · 2019: 8 · 2018: 10 · 2017: 9 · 2016: 5 · 2015: 4 · 2014: 14 · 2013: 19 · 2012: 15
FWCI
20.45
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
Keywords
hepatic stem cells, pluripotent stem cells, self-renewal, clonal propagation, liver development, hepatocyte differentiation, cholangiocyte differentiation, multilineage differentiation, pancreatic differentiation, intestinal epithelial differentiation, cell transplantation
MeSH terms
Animals, Bile Ducts, Cell Differentiation, Cells, Cultured, Clone Cells, Epithelial Cells, Fetus, Flow Cytometry, Intestines, Liver, Liver Regeneration, Microscopy, Electron, Pancreas, Stem Cells, Biomarkers, Cell Transplantation, Proto-Oncogene Proteins c-kit, Cell Lineage, Proto-Oncogene Proteins c-met, Reverse Transcriptase Polymerase Chain Reaction, Hepatocytes, Mice

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