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Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells.

Fagoonee S, Famulari ES, Silengo L, Tolosano E, Altruda F - PLoS ONE (2015)

Bottom Line: Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection.This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver.Thus, GPSCs might offer promise for regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biostructures and Bioimages (CNR), Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

ABSTRACT
One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.

No MeSH data available.


Related in: MedlinePlus

Characterisation of Liv2-sorted GPSC-derived hepatocytes.A. Liv2-sorted cells show the typical hepatocyte morphology: binuclear cells with prominent cytoplasm (i, ii) and are positive for glycogen deposits as revealed by PAS staining (iii). PAS staining of non-sorted GPSC-derived hepatocytes are shown as positive control (iv) while mouse embryonic fibroblasts were used as negative control (v) B. Immunostaining of Liv2-sorted cells showed positivity for hepatocyte markers CK18, Dlk1, albumin and E-cadherin. These cells were also positive for Ki67, a proliferation marker. Cells were stained with secondary antibody (IgG-Alexa568) only for negative control. Representative images are shown.
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pone.0136762.g003: Characterisation of Liv2-sorted GPSC-derived hepatocytes.A. Liv2-sorted cells show the typical hepatocyte morphology: binuclear cells with prominent cytoplasm (i, ii) and are positive for glycogen deposits as revealed by PAS staining (iii). PAS staining of non-sorted GPSC-derived hepatocytes are shown as positive control (iv) while mouse embryonic fibroblasts were used as negative control (v) B. Immunostaining of Liv2-sorted cells showed positivity for hepatocyte markers CK18, Dlk1, albumin and E-cadherin. These cells were also positive for Ki67, a proliferation marker. Cells were stained with secondary antibody (IgG-Alexa568) only for negative control. Representative images are shown.

Mentions: Liv2-sorted GPSC-derived hepatocytes were morphologically similar to mouse hepatocytes and were mostly binuclear with prominent cytoplasm (Fig 3Ai and 3Aii). PAS staining showed glycogen deposits in Liv2-sorted GPSC-derived hepatocytes comparable to those of non-sorted GPSC-derived hepatocytes (positive control) (Fig 3Aiii and 3Aiv, respectively). Mouse embryonic fibroblasts, used as negative control, had no glycogen depostis (Fig 3Av). Immunofluorescence analysis showed that these cells were positive for CK18, Dlk1, Albumin and e-cadherin as well as for the proliferation marker Ki67 (Fig 3B). These results indicate that the Liv2-sorted GPSC-derived cells indeed differentiated into hepatocytes as previously reported for unsorted GPSCs.


Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells.

Fagoonee S, Famulari ES, Silengo L, Tolosano E, Altruda F - PLoS ONE (2015)

Characterisation of Liv2-sorted GPSC-derived hepatocytes.A. Liv2-sorted cells show the typical hepatocyte morphology: binuclear cells with prominent cytoplasm (i, ii) and are positive for glycogen deposits as revealed by PAS staining (iii). PAS staining of non-sorted GPSC-derived hepatocytes are shown as positive control (iv) while mouse embryonic fibroblasts were used as negative control (v) B. Immunostaining of Liv2-sorted cells showed positivity for hepatocyte markers CK18, Dlk1, albumin and E-cadherin. These cells were also positive for Ki67, a proliferation marker. Cells were stained with secondary antibody (IgG-Alexa568) only for negative control. Representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556379&req=5

pone.0136762.g003: Characterisation of Liv2-sorted GPSC-derived hepatocytes.A. Liv2-sorted cells show the typical hepatocyte morphology: binuclear cells with prominent cytoplasm (i, ii) and are positive for glycogen deposits as revealed by PAS staining (iii). PAS staining of non-sorted GPSC-derived hepatocytes are shown as positive control (iv) while mouse embryonic fibroblasts were used as negative control (v) B. Immunostaining of Liv2-sorted cells showed positivity for hepatocyte markers CK18, Dlk1, albumin and E-cadherin. These cells were also positive for Ki67, a proliferation marker. Cells were stained with secondary antibody (IgG-Alexa568) only for negative control. Representative images are shown.
Mentions: Liv2-sorted GPSC-derived hepatocytes were morphologically similar to mouse hepatocytes and were mostly binuclear with prominent cytoplasm (Fig 3Ai and 3Aii). PAS staining showed glycogen deposits in Liv2-sorted GPSC-derived hepatocytes comparable to those of non-sorted GPSC-derived hepatocytes (positive control) (Fig 3Aiii and 3Aiv, respectively). Mouse embryonic fibroblasts, used as negative control, had no glycogen depostis (Fig 3Av). Immunofluorescence analysis showed that these cells were positive for CK18, Dlk1, Albumin and e-cadherin as well as for the proliferation marker Ki67 (Fig 3B). These results indicate that the Liv2-sorted GPSC-derived cells indeed differentiated into hepatocytes as previously reported for unsorted GPSCs.

Bottom Line: Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection.This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver.Thus, GPSCs might offer promise for regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biostructures and Bioimages (CNR), Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

ABSTRACT
One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.

No MeSH data available.


Related in: MedlinePlus