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In JoVE (1)
Other Publications (15)
- Hepatology (Baltimore, Md.)
- Stem Cells (Dayton, Ohio)
- Hepatology (Baltimore, Md.)
- Hepatology (Baltimore, Md.)
- Stem Cells (Dayton, Ohio)
- Hepatology (Baltimore, Md.)
- Hepatology (Baltimore, Md.)
- Hepatology (Baltimore, Md.)
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Articles by C. Bart Rountree in JoVE
JoVE General
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
Other articles by C. Bart Rountree on PubMed
Bone Marrow Fails to Differentiate into Liver Epithelium During Murine Development and Regeneration
Recent reports have provided conflicting conclusions regarding the role for bone marrow (BM)-derived cells in the regeneration of liver. Our aim was to investigate the potential of BM to contribute to liver epithelium using different BM transplant models designed to explore differentiation during normal liver development and regeneration after toxic injury. BM cells from transgenic green fluorescent protein (GFP) mice were injected into neonatal and adult immunodeficient and neonatal immune-competent mice. Three distinct models of liver injury were employed to test the contribution of marrow to the regeneration of hepatocytes, cholangiocytes, and oval cells in immune-deficient adult animals after neonatal transplant. Immunohistochemistry was combined with flow cytometry (FACS) and reverse transcription (RT)-PCR to increase the sensitivity and specificity of the analyses. Although GFP+ marrow-derived cells were observed in the livers of all transplanted animals, immunohistochemistry failed to demonstrate any marrow derived hepatocytes or cholangiocytes. FACS confirmed that GFP+ marrow-derived cells in the liver maintained expression of CD45, a leukocyte marker. Gene expression studies of GFP+ cells isolated by FACS failed to demonstrate expression of liver specific genes in these marrow-derived cells. CONCLUSION: Through highly sensitive and specific analyses, we were unable to demonstrate any evidence of transdifferentiation of BM-derived cells into epithelial hepatic tissue during the period of rapid growth in the neonatal period. Furthermore, although increased migration of hematopoietic cells to the liver occurred after toxic injury, these cells did not contribute directly to the replacement of hepatocytes, cholangiocytes, or oval cells.
A CD133-expressing Murine Liver Oval Cell Population with Bilineage Potential
Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.
Fibroblast Growth Factor 10 is Critical for Liver Growth During Embryogenesis and Controls Hepatoblast Survival Via Beta-catenin Activation
Fibroblast growth factor (FGF) signaling and beta-catenin activation have been shown to be crucial for early embryonic liver development. This study determined the significance of FGF10-mediated signaling in a murine embryonic liver progenitor cell population as well as its relation to beta-catenin activation. We observed that Fgf10(-/-) and Fgfr2b(-/-) mouse embryonic livers are smaller than wild-type livers; Fgf10(-/-) livers exhibit diminished proliferation of hepatoblasts. A comparison of beta-galactosidase activity as a readout of Fgf10 expression in Fgf10(+/LacZ) mice and of beta-catenin activation in TOPGAL mice, demonstrated peak Fgf10 expression from E9 to E13.5 coinciding with peak beta-catenin activation. Flow cytometric isolation and marker gene expression analysis of LacZ(+) cells from E13.5 Fgf10(+/LacZ) and TOPGAL livers, respectively, revealed that Fgf10 expression and beta-catenin signaling occur distinctly in stellate/myofibroblastic cells and hepatoblasts, respectively. Moreover, hepatoblasts express Fgfr2b, which strongly suggests they can respond to recombinant FGF10 produced by stellate cells. Fgfr2b(-/-)/TOPGAL(+/+) embryonic livers displayed less beta-galactosidase activity than livers of Fgfr2b(+/+)/TOPGAL(+/+) littermates. In addition, cultures of whole liver explants in Matrigel or cell in suspension from E12.5 TOPGAL(+/+)mice displayed a marked increase in beta-galactosidase activity and cell survival upon treatment with recombinant FGF10, indicating that FGFR (most likely FGFR2B) activation is upstream of beta-catenin signaling and promote hepatoblast survival. CONCLUSION: Embryonic stellate/myofibroblastic cells promote beta-catenin activation in and survival of hepatoblasts via FGF10-mediated signaling. We suggest a role for stellate/myofibroblastic FGF10 within the liver stem cell niche in supporting the proliferating hepatoblast.
Expansion of Liver Cancer Stem Cells During Aging in Methionine Adenosyltransferase 1A-deficient Mice
Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the biosynthesis of S-adenosylmethionine. Hepatic MAT activity falls in chronic liver diseases, and mice lacking Mat1a are predisposed to liver injury and develop hepatocellular carcinoma (HCC) spontaneously by 18 months. The current work examined the hypothesis that liver cancer stem cells contribute to HCC in this model. Livers from 6- and 18-month-old Mat1a-knockout (KO) mice and their wild-type (WT) littermates were fractionated and isolated by flow cytometry. CD45- nonparenchymal (NP) cells were cultured using liver stem cell conditions. Cells were analyzed by real-time PCR and fluorescent immunohistochemistry (FIHC). Tumor formation was assessed by injecting 1 x 10(6) CD133+CD49f+ cells intraperitoneally into immune-deficient mice. The proportion of CD49f+ and CD133+ cells in the CD45-NP fraction increased 4.5- to 5.5-fold from 6 to 18 months in KO mice but not in their WT littermates. Compared to CD49f- cells from old KO mice, CD49f+ cells from the same animals had a markedly increased expression of several oncogenes. CD133+ cells with CD49f coexpression were selected in vitro and exhibited rapid growth, with the expression of biliary cytokeratins, alpha-fetoprotein, and c-Met by FIHC. Clonal expansion of single CD133+CD49f+ cells revealed maintenance of bipotency. After CD133+CD49f+ cells were injected into immune-deficient mice, 3 of the 8 mice developed tumors of liver epithelial cells after 6-8 weeks. Conclusion: Mat1a(-/-) mice have expansion of liver stem cells as they age. These cells have increased expression of several oncogenes and are tumorigenic in vivo. This is the first demonstration of adult liver stem cells possessing tumorigenic potential without the use of a carcinogen or manipulation of tumor-suppressor or oncogene expression.
Expansion of CD133-expressing Liver Cancer Stem Cells in Liver-specific Phosphatase and Tensin Homolog Deleted on Chromosome 10-deleted Mice
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that regulates mitogenic signaling pathways, and deficiency of PTEN results in cell proliferation, survival, and malignancy. Murine liver-specific Pten deletion models develop liver malignancy by 12 months of age. Using this model, we describe a population of CD133+ liver cancer stem cells isolated during the chronic injury phase of disease progression and before primary carcinoma formation. We performed immunohistochemistry and flow cytometry isolation using livers from 3- and 6-month-old Pten(loxP/loxP); Alb-Cre+ mice (mutants) and controls. CD133+CD45- nonparenchymal (NP) cells were analyzed for gene expression profile and protein levels. Single CD133+CD45- oval cells were isolated for clonal expansion and tumor analysis. Cultured and freshly isolated liver CD133+CD45- and CD133-CD45- NP cells were injected into immune-deficient and immune-competent mice. In mutant mice, the NP fraction increased in CD133+CD45- cells in 3- and 6-month-old Pten-deleted animals compared with controls. Clone lines expanded from single CD133+CD45- cells demonstrated consistent liver progenitor cell phenotype, with bilineage gene expression of hepatocyte and cholangiocyte markers. CD133+ cells from expanded clone lines formed robust tumors in immune-deficient and immune-competent mice. Furthermore, freshly isolated CD133+CD45- NP liver cells from 6-month-old mutants formed tumors in vivo, and CD133-CD45- NP cells did not. Consistent with a cancer stem cell phenotype, CD133+ cells demonstrate resistance to chemotherapy agents compared with CD133- cells. CD133+CD45- nonparenchymal cells from chronic injury Pten(loxP/loxP); Alb-Cre+ mice represent a bipotent liver progenitor cell population with cancer stem cell phenotype.
CD133+ Liver Cancer Stem Cells from Methionine Adenosyl Transferase 1A-deficient Mice Demonstrate Resistance to Transforming Growth Factor (TGF)-beta-induced Apoptosis
Methionine adenosyltransferase (MAT) is an essential enzyme required for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133(+)CD45(-) oval cells isolated from 16-month-old Mat1a(-/-) mice represent a liver cancer stem cell population. The transforming growth factor beta (TGF-beta) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF-beta. CD133(+)CD45(-) oval cells were isolated from premalignant 16-month-old Mat1a(-/-) mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133(+) cells in vitro, and three of five clone lines produced tumors. Although TGF-beta1 inhibited cell growth equally in CD133(-) and CD133(+) cells from each clone line, the CD133(+) population demonstrated significant resistance to TGF-beta-induced apoptosis compared with CD133(-) cells. Furthermore, CD133(+) cells demonstrated a substantial increase in mitogen-activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extracellular signal-regulated kinase levels before and after TGF-beta stimulation. MAPK inhibition using mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF-beta-induced apoptosis in CD133(+) cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF-beta in CD133(-) cells. CONCLUSION: CD133(+) liver cancer stem cells exhibit relative resistance to TGF-beta-induced apoptosis. One mechanism of resistance to TGF-beta-induced apoptosis in CD133(+) cancer stem cells is an activated mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.
Epigenetic Regulation of Cancer Stem Cell Marker CD133 by Transforming Growth Factor-beta
Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD133+ liver cancer cells are characterized by resistance to chemotherapy, self-renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor beta (TGFbeta), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFbeta1 is capable of up-regulating CD133 expression specifically within the Huh7 HCC cell line in a time- and dose-dependent manner. Most important, TGFbeta1-induced CD133+ Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFbeta1-induced CD133 expression. Within CD133- Huh7 cells, TGFbeta1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3beta, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD133- Huh7 cells was inhibited by TGFbeta1. DNMT3beta inhibition by TGFbeta1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFbeta1 treatment led to significant demethylation in CD133 promoter-1 in CD133- Huh7 cells. CONCLUSION: TGFbeta1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3beta expression and subsequent demethylation of promoter-1. TGFbeta1-induced CD133+ Huh7 cells are tumorigenic. The mechanism by which TGFbeta induces CD133 expression is partially dependent on the Smads pathway.
Epithelial-to-mesenchymal Transition of Murine Liver Tumor Cells Promotes Invasion
Epithelial-to-mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 x 10(6) liver cells isolated from Pten(loxp/loxp)/Alb-Cre(+) mice, expanded from a single CD133(+)CD45(-) cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real-time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT-associated gene expression of Snail1, Zeb1, and Zeb2; and down-regulated E-cadherin. P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten-deleted tumors. CONCLUSION: EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed-forward mechanism.
Expansion of Hepatic Tumor Progenitor Cells in Pten-null Mice Requires Liver Injury and is Reversed by Loss of AKT2
The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor cells. We investigated how the loss of PTEN activates liver progenitor cells and induces tumorigenesis.
Clinical Application for the Preservation of Phospho-proteins Through In-situ Tissue Stabilization
Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications.
Stem Cells in Drug Discovery, Regenerative Medicine and Cancer
A report on the Stem Cells World Congress held in San Diego, USA, 24-25 January 2011.
C-Met Represents a Potential Therapeutic Target for Personalized Treatment in Hepatocellular Carcinoma
c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. Conclusion: c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.
Snail1 Induces Epithelial-to-mesenchymal Transition and Tumor Initiating Stem Cell Characteristics
Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.
Stem Cells in Liver Diseases and Cancer: Recent Advances on the Path to New Therapies
Stem cells have potential for therapy of liver diseases, but may also be involved in the formation of liver cancer. Recently, the American Association for the Study of Liver Diseases Henry M. and Lillian Stratton Basic Research Single Topic Conference "Stem Cells in Liver Diseases and Cancer: Discovery and Promise" brought together a diverse group of investigators to define the status of research on stem cells and cancer stem cells in the liver and identify problems and solutions on the path to clinical translation. This report summarizes the outcomes of the conference and provides an update on recent research advances. Progress in liver stem cell research includes isolation of primary liver progenitor cells (LPCs), directed hepatocyte differentiation of primary LPCs and pluripotent stem cells, findings of transdifferentiation, disease-specific considerations for establishing a therapeutically effective cell mass, and disease modeling in cell culture. Tumor-initiating stem-like cells (TISCs) that emerge during chronic liver injury share the expression of signaling pathways, including those organized around transforming growth factor beta and β-catenin, and surface markers with normal LPCs. Recent investigations of the role of TISCs in hepatocellular carcinoma have provided insight into the transcriptional and post-transcriptional regulation of hepatocarcinogenesis. Targeted chemotherapies for TISC are in development as a means to overcome cellular resistance and mechanisms driving disease progression in liver cancer.
Regression of Established Hepatocellular Carcinoma is Induced by Chemoimmunotherapy in an Orthotopic Murine Model
The high rate of mortality and frequent incidence of recurrence associated with hepatocellular carcinoma (HCC) reveal the need for new therapeutic approaches. In this study we evaluated the efficacy of a novel chemoimmunotherapeutic strategy to control HCC and investigated the underlying mechanism that increased the antitumor immune response. We developed a novel orthotopic mouse model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice. These MTD2-derived hepatocytes form Tag-expressing HCC tumors specifically within the liver. This approach provides a platform to test therapeutic strategies and antigen-specific immune-directed therapy in an immunocompetent murine model. Using this model we tested the efficacy of a combination of oral sunitinib, a small molecule multitargeted receptor tyrosine kinase (RTK) inhibitor, and adoptive transfer of tumor antigen-specific CD8(+) T cells to eliminate HCC. Sunitinib treatment alone promoted a transient reduction in tumor size. Sunitinib treatment combined with adoptive transfer of tumor antigen-specific CD8(+) T cells led to elimination of established tumors without recurrence. In vitro studies revealed that HCC growth was inhibited through suppression of STAT3 signaling. In addition, sunitinib treatment of tumor-bearing mice was associated with suppression of STAT3 and a block in T-cell tolerance. CONCLUSION: These findings indicate that sunitinib inhibits HCC tumor growth directly through the STAT3 pathway and prevents tumor antigen-specific CD8(+) T-cell tolerance, thus defining a synergistic chemoimmunotherapeutic approach for HCC.
