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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.


Analysis of hepatocyte-specific gene expression in Liv2-sorted GPSC-derived hepatocytes.A. RT-PCR analysis of liver-specific gene expression. RNA samples were extracted from undifferentiated GPSCs cells (0 day), differentiating EBs (7 days), Liv2-sorted cells (21 days) and adult liver. Actin served as an internal standard. Data shown are representative of 3 independent experiments. B. qRT-PCR was also used to assess the expression of Dlk1, Cyp7a1, CK18 and Oct4 in Liv2-sorted GPSCs during hepatocyte differentiation (n = 3). The relative quantity (RQ) with respect to gene expression in undifferentiated GPSCs (differentiation Day 0) is shown and values have been normalized to 18S expression (n = 3). Abbreviations: ALB, albumin; AFP, alpha fetoprotein; TAT, tyrosine amino transferase; Hp, haptoglobin; Dlk1, Delta-like 1 homolog; Cyp7a1, cytochrome P450 7a1; CK18, cytokeratin 18.
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pone.0136762.g002: Analysis of hepatocyte-specific gene expression in Liv2-sorted GPSC-derived hepatocytes.A. RT-PCR analysis of liver-specific gene expression. RNA samples were extracted from undifferentiated GPSCs cells (0 day), differentiating EBs (7 days), Liv2-sorted cells (21 days) and adult liver. Actin served as an internal standard. Data shown are representative of 3 independent experiments. B. qRT-PCR was also used to assess the expression of Dlk1, Cyp7a1, CK18 and Oct4 in Liv2-sorted GPSCs during hepatocyte differentiation (n = 3). The relative quantity (RQ) with respect to gene expression in undifferentiated GPSCs (differentiation Day 0) is shown and values have been normalized to 18S expression (n = 3). Abbreviations: ALB, albumin; AFP, alpha fetoprotein; TAT, tyrosine amino transferase; Hp, haptoglobin; Dlk1, Delta-like 1 homolog; Cyp7a1, cytochrome P450 7a1; CK18, cytokeratin 18.

Mentions: Next, we wanted to verify whether Liv-2 sorting did not affect the properties of the previously described GPSC-derived hepatocytes[16]. Liv2-sorted GPSC-derived hepatocytes were tested for their ability to express hepatocyte-specific genes. At Day 21 of differentiation, Liv2-sorted GPSC-derived hepatocytes expressed alpha fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT), haptoglobin (Hp) compared to the undifferentiated GPSCs or at differentiation Day 7 (Fig 2A). Moreover, these cells expressed delta-like homolog 1 (Dlk1), cytokeratin 18 (CK18), the liver-specific cytochrome P7a1 (Cyp7a1) (Fig 2B). The expression of the pluripotency marker, octamer-binding transcription factor 4 (Oct4), was absent in Liv2-sorted hepatocytes compared to undifferentiated GPSCs. Interestingly, Liv2-sorted GPSCs also express the Hfe (hemochromatosis) gene as from differentiation Day 14 (S2 Fig)


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)

Analysis of hepatocyte-specific gene expression in Liv2-sorted GPSC-derived hepatocytes.A. RT-PCR analysis of liver-specific gene expression. RNA samples were extracted from undifferentiated GPSCs cells (0 day), differentiating EBs (7 days), Liv2-sorted cells (21 days) and adult liver. Actin served as an internal standard. Data shown are representative of 3 independent experiments. B. qRT-PCR was also used to assess the expression of Dlk1, Cyp7a1, CK18 and Oct4 in Liv2-sorted GPSCs during hepatocyte differentiation (n = 3). The relative quantity (RQ) with respect to gene expression in undifferentiated GPSCs (differentiation Day 0) is shown and values have been normalized to 18S expression (n = 3). Abbreviations: ALB, albumin; AFP, alpha fetoprotein; TAT, tyrosine amino transferase; Hp, haptoglobin; Dlk1, Delta-like 1 homolog; Cyp7a1, cytochrome P450 7a1; CK18, cytokeratin 18.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556379&req=5

pone.0136762.g002: Analysis of hepatocyte-specific gene expression in Liv2-sorted GPSC-derived hepatocytes.A. RT-PCR analysis of liver-specific gene expression. RNA samples were extracted from undifferentiated GPSCs cells (0 day), differentiating EBs (7 days), Liv2-sorted cells (21 days) and adult liver. Actin served as an internal standard. Data shown are representative of 3 independent experiments. B. qRT-PCR was also used to assess the expression of Dlk1, Cyp7a1, CK18 and Oct4 in Liv2-sorted GPSCs during hepatocyte differentiation (n = 3). The relative quantity (RQ) with respect to gene expression in undifferentiated GPSCs (differentiation Day 0) is shown and values have been normalized to 18S expression (n = 3). Abbreviations: ALB, albumin; AFP, alpha fetoprotein; TAT, tyrosine amino transferase; Hp, haptoglobin; Dlk1, Delta-like 1 homolog; Cyp7a1, cytochrome P450 7a1; CK18, cytokeratin 18.
Mentions: Next, we wanted to verify whether Liv-2 sorting did not affect the properties of the previously described GPSC-derived hepatocytes[16]. Liv2-sorted GPSC-derived hepatocytes were tested for their ability to express hepatocyte-specific genes. At Day 21 of differentiation, Liv2-sorted GPSC-derived hepatocytes expressed alpha fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT), haptoglobin (Hp) compared to the undifferentiated GPSCs or at differentiation Day 7 (Fig 2A). Moreover, these cells expressed delta-like homolog 1 (Dlk1), cytokeratin 18 (CK18), the liver-specific cytochrome P7a1 (Cyp7a1) (Fig 2B). The expression of the pluripotency marker, octamer-binding transcription factor 4 (Oct4), was absent in Liv2-sorted hepatocytes compared to undifferentiated GPSCs. Interestingly, Liv2-sorted GPSCs also express the Hfe (hemochromatosis) gene as from differentiation Day 14 (S2 Fig)

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.