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

Atsushi Suzuki, Yun-wen Zheng, Shin Kaneko, Masafumi Onodera, Katashi Fukao, Hiromitsu Nakauchi, and Hideki Taniguchi

J Cell Biol 2002 156:173-184.

Published January 7, 2002, doi:10.1083/jcb.200108066

http://jcb.rupress.org/content/156/1/173.full

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 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.