Figure 1.
Stellate cell markers in cultured PSC, UCBSC and muscle fibroblasts.
(A,D,G,J) Freshly isolated PSC cultured for 1 day, (B,E,H,K) clonally expanded UCBSC and (C,F,I,L) muscle fibroblasts from rats were analyzed by phase contrast light microscopy and antibodies against the stellate cell markers (D,E,F) α-SMA, (G,H,I) desmin and (J,K,L) GFAP (red). The isolated PSC were found to be typical stellate cells with respect to their α-SMA, desmin and GFAP expression. The stellate cell markers α-SMA and GFAP were only weakly expressed by UCBSC clones, whereas desmin remained undetectable at protein level. The cell nuclei were stained by DAPI (blue).
Figure 2.
PSC contain retinyl palmitate and express nestin.
(A) HPLC analysis revealed retinyl palmitate (red arrow) in lysates of freshly isolated rat PSC. (B) The isolated PSC were positively labeled with antibodies against nestin (red) at their second day in culture. (C) Nestin-expressing cells were mainly found in pancreatic islets of rats (red). Immunofluorescence staining of insulin (green) was used to verify the presence of nestin+ cells (red) in pancreatic islets. (D) Desmin-expressing cells with branched cell protrusions were observed in pancreatic islets (green). (E) The nestin+ cells (red) of pancreatic islets were also positive for the stellate cell marker desmin (green) as documented by co-staining of the proteins and 3-dimensional (3D) rendering of serial microscopic pictures made by a confocal laser scanning microscope. The co-localization of nestin and desmin was highlighted in white and indicated the presence of PSC in pancreatic islets. (F) Nestin was only detected in few periacinar cells by immunofluorescence staining, (G) whereas ductular cells of the rat pancreas were negative for nestin (red). The cell nuclei were marked by DAPI (blue).
Figure 3.
Quantitative mRNA analysis of stellate cell and stem cell-related factors in PSC.
The expression of the stellate cell markers α-SMA, GFAP, desmin and synemin as well as the stem/progenitor cell-associated proteins CD133, SOX9, GDF3 and slain1 was investigated by qPCR in one day-cultured PSC, PSC clones and UCBSC clones. PSC expressed comparable or higher levels of stem cell-associated factors than UCBSC clones. Three to five independent primary cultures of PSC were used for qPCR analysis. The PSC clones and the UCBSC clones were measured in triplicates. Significant differences and the standard error of mean (±SEM) are indicated [*P<0.05%].
Figure 4.
Analysis of stem/progenitor cell-associated factors in PSC by Western blot.
The expression of CD133 was analyzed in cell membrane fractions of PSC and UCBSC. PSC of three different cell isolations, which were cultured for 7 days (7d), and UCBSC of three different clones were taken for the immunoblot. To indicate cell membrane protein fractions, annexin II served as a control. The notch1 receptor was detected predominantly in freshly isolated PSC cultured for 1 day (1d) and to a lesser extent also in culture-activated PSC (7d) as well as UCBSC. Nuclear localization of β-catenin in PSC and UCBSC indicated active β-catenin-dependet Wnt signaling as investigated by Western blot of nuclear protein fractions. In line with this, the Wnt target gene PITX2 was also detected in both cell types. The stem/progenitor cell-associated numb1/3 isoforms were detected in PSC and UCBSC, whereas numb2/4 remained undetectable in both cell types. Numb2/4 isoforms were found in lysates of whole liver.
Figure 5.
Analysis of stem/progenitor cell-associated factors in PSC by immunofluoresecence.
(A,D,G,J) Freshly isolated PSC, (B,E,H,K) the UCBSC clone 1G11 and (C,F,I,L) muscle fibroblasts from rats were analyzed with antibodies against (A,B,C) CD133, (D,E,F) β-catenin, (G,H,I) PITX2 and (J,K,L) numb by immunofluorescence staining (red). The stem/progenitor cell-associated proteins CD133, PITX2 and numb were restricted to PSC and UCBSC, whereas β-catenin occurred in all cell types. In (D) PSC and (E) UCBSC β-catenin was detected in the cell nucleus, indicating active β-catenin-dependent WNT signaling, (F) whereas fibroblasts displayed β-catenin mainly in the cell membrane. The cell nuclei were stained by DAPI (blue).
Figure 6.
PSC can differentiate into hepatocyte-like cells in vitro.
PSC were expanded for 7 days in culture and subsequently cultured for additional 14 days in (A) IMDM supplemented with 10% FCS (control medium) or (B) IMDM containing cytokines (FGF4, HGF), linoleic acid-albumin and ITS (hepatocyte differentiation medium). (B) Hepatocyte-like cells appeared in primary cultures of PSC during cytokine treatment. (C) Under control conditions primary cultured PSC did not express the hepatocyte marker cytokeratin 18 (CK18) as investigated by immunofluorescence (red), but (D) CK18 synthesis was induced after treatment with hepatocyte differentiation medium for 14 days. (E) The hepatocyte-associated bile salt export pump (BSEP; red) remained undetectable in vimentin-expressing PSC (green) of the control, whereas (F) BSEP and vimentin were detectable in PSC treated with hepatocyte differentiation medium. The cell nuclei were marked by DAPI (blue). (G) Molecular markers of stellate cells (GFAP, α-SMA) as well as of immature and mature hepatocytes (HHEX, CYP7A1, α-fetoprotein, albumin, CX32, HNF1α, HNF4α, HNF6, MRP2) were analyzed by RT-PCR in the control (IMDM and 10% FCS; left column) and cytokine treated PSC (FGF4, HGF, linoleic acid-albumin and ITS; right column) after 14 days of culture. The induction of notch3 and genes typically expressed by liver parenchymal cells indicated cell differentiation. (H) This cell differentiation potential was also preserved by the PSC clones, which displayed a comparable expression pattern after cytokine treatment for 7 days (right column). In the control, clonally expanded PSC were cultured in medium without cytokines (IMDM and 10% FCS; left column). (I) The PSC clone displayed the morphology of myofibroblast-like cells (control), but (J) developed into hepatocyte-like cells after treatment with hepatocyte differentiation medium for 7 days. (K) In this experimental setup, the release of albumin was measured by a rat-specific albumin ELISA. High amounts of albumin were secreted by primary cultured PSC and the PSC clone 2F5 after treatment with hepatocyte differentiation medium, whereas rat albumin was not detected (n.d.) under control conditions or in experimental media without cells (n = 3).
Figure 7.
Contribution of transplanted eGFP+ PSC to liver regeneration in vivo.
(A) eGFP fluorescence (green) in PSC cultured for 7 days. (B) These culture-activated eGFP+ PSC were transplanted into wild type rats that underwent PHX in the presence of 2AAF. The eGFP+ PSC reached the host liver as determined by immunofluorescence staining of eGFP (green) 14 days after transplantation. (C, D) The eGFP+ PSC differentiated into hepatocytes in the host liver as determined by co-localization of eGFP fluorescence (green) and nuclear HNF4α immunofluorescence (red). (E) Their differentiation into hepatocytes was also confirmed by combined immunofluorescence staining of eGFP (green) and cytokeratin 18 (CK18; red). The fate of transplanted eGFP+ PSC was further analyzed by immunohistochemistry through Fast Red staining of eGFP-expressing cells (red). (F, G) The liver from wild type Wistar rats displayed no Fast Red staining after incubation with antibodies against eGFP, (H) but when eGFP+ PSC were transplanted, large areas of the host liver were colored red. This method labeled (I) hepatocytes (yellow arrows) and (J, K) bile duct cells (white arrows). (J) Other cell types in close proximity to bile ducts (black arrow) and liver sinusoids (yellow arrow) showed also eGFP expression as indicated by Fast Red staining. (L) The presence of eGFP-expressing cells in bile ducts of the host liver was further confirmed by combined immunofluorescence of eGFP (green) and CK19 (red). (M) Immunofluorescence staining of HNF4α (red) and FISH of chromosome Y (green) was used to identify hepatocytes in female liver tissue that derived from transplanted male PSC 14 days after PHX in the presence of 2AAF (white arrows). Also non-parenchymal cells with chromosome Y were detected (yellow arrows). (N) Combined FISH of chromosome Y (green) and immunofluorescence staining of panCK (red) revealed that the transplanted PSC had differentiated into duct-forming cholangiocytes. (O, P) Clonally expanded PSC of males were also transplanted into wild type female rats that underwent PHX in the presence of 2AAF. FISH of chromosome Y (green) and immunofluorescence staining of (O) HNF4α (red) or (P) panCK (red) indicated that single cell clones of PSC differentiated into hepatocytes and cholangiocytes (white arrows) as investigated after 14 days of regeneration. (P) Also PSC-derived cells that did not express cytokeratins were found close to bile ducts (yellow arrows). The cell nuclei were marked by DAPI (blue).
Figure 8.
Estimation of PSC-derived cells in the wild type host liver.
(A) In order to quantify the contribution of transplanted eGFP+ PSC to liver regeneration, qPCR of the eGFP DNA was performed 14 days after cell transplantation. Livers from eGFP-expressing and wild type rats served as positive and negative controls for the eGFP gene, respectively. Ten samples of different animals were measured to assess the eGFP expression of liver after PSC transplantation. (B) The engraftment of stellate cells was further verified through qPCR of male-specific SRY DNA after gender-mismatched transplantation of male PSC into female recipient rats (n = 4). In contrast to PSC, muscle fibroblasts were unable to survive in the host liver (n = 4).