Faculty Bibliography

Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor-derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56-70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% +/- 3.5% versus 2.6% +/- 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. CONCLUSION: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells.

Precancerous lesions that may be detected in chronically diseased, usually cirrhotic livers, include: clusters of hepatocytes with atypia and increased proliferative rate (dysplastic foci) that usually represent an incidental finding in biopsy or resection specimens; and grossly evident lesions (dysplastic nodules) that may be detected on radiologic examination. There are two types of small hepatocellular carcinoma (HCC) (defined as HCC that measures less than 2 cm): early HCC, which is well-differentiated and has indistinct margins; and distinctly nodular small HCC, which is well- or moderately differentiated, and is usually surrounded by a fibrous capsule. Precise diagnosis of precancerous and early cancerous lesions by imaging methods is often difficult or impossible. Detection of a dysplastic lesion in a biopsy specimen is a marker of increased risk for HCC development, and warrants increased surveillance. High-grade dysplastic nodules and small HCCs should be treated by local ablation, surgical resection, or liver transplantation.

The mechanisms for in vivo production of bone marrow-derived hepatocytes (BMDHs) remain largely unclear. We investigated whether granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of hematopoietic cells increases the phenomenon. Recurrent liver injury in mice expressing green fluorescent protein (EGFP) in all hematopoietic-derived cells was produced by 3 months of carbon tetrachloride (CCL4) injections. Histologically, there were necrotic foci with histiocyte-rich infiltrates, but little oval cell proliferation. Subsequently, some animals were mobilized with G-CSF for 1, 2, or 3 weeks. Animals were sacrificed 1 month after growth factor treatment. BMDH percentages were lower than previously reported, though G-CSF mobilization significantly augmented BMDH production in injured livers. BMDHs originating from in vivo fusion were evaluated by transplanting female EGFP+ cells into male mice. Binucleated, EGFP+ hepatocytes with one Y chromosome, indicating fusion, were identified. In conclusion, (1) mobilization of hematopoietic cells increases BMDH production and (2) as with the FAH-null model, the first model demonstrating hematopoietic/hepatocyte fusion, recurring CCl4-induced injury has macrophage-rich infiltrates, a blunted oval cell response, and a predominantly in vivo fusion process for circulating cell engraftment into the liver. These findings open the possibility of using hematopoietic growth factors to treat nonhematopoietic degenerative diseases.

This essay will address areas of liver stem/progenitor cell studies in which consensus has emerged and in which controversy still prevails over consensus, but it will also highlight important themes that inevitably should be a focus of liver stem/progenitor cell investigations in coming years. Thus concepts regarding cell plasticity, the existence of a physiological/anatomic stem cell niche, and whether intrahepatic liver stem/progenitor cells comprise true stem cells or progenitor cells (or both) will be approached in some detail.

OBJECTIVE: Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. METHODS: In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin(-/+), c-kit(-/+), Sca-1(-/+)) from GFP(+) (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). RESULTS: The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP(+)/CD45(-)) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP(+)/CD45(-) cells were also cytokeratin(+). Mobilization further increased GFP(+)/CD45(-) cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP(+)/CD45(-) in radiation-injured mice transplanted with Lin(-), c-kit(+), or Sca-1(+) marrow cells. Lin(+), c-kit(-), and Sca-1(-) subpopulations did not significantly engraft the lung. CONCLUSIONS: We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell-producing marrow cell has the phenotype of a hematopoietic stem cell.

Recent insights regarding stem cells, repression and de-repression of gene expression, and the application of Complexity Theory to cell and molecular biology require a re-evaluation of many long-held dogmas regarding the nature of the human body in health and disease. Greater than expected cell plasticity, trafficking of cells between organs, 'cellular uncertainty', stochasticity of cell origins and fates, and a reconsideration of Cell Doctrine itself all logically follow from these observations and conceptual approaches. In this paper, these themes will be considered and some implications for the investigative pathologist will be explored.

Lung injury is a common cause of death and disability. Stem cell-related therapies are widely viewed as offering promise for people suffering from various types of pulmonary diseases, and gender-mismatched bone marrow transplant recipients serve as natural populations in which to study the role of bone marrow-derived stem cells in recovery from pulmonary injury. We evaluated the extent of lung repopulation by type II pneumocyte descendents of adult bone marrow-derived stem cells in allogeneic hematopoietic cell transplant recipients. Recut sections were obtained from five lung biopsy specimens and autopsy lung tissues from four female recipients of transplanted mobilized peripheral blood stem cells or bone marrow from male donors. Sequential immunohistochemistry and fluorescence in situ hybridization was performed on each section to evaluate for Y-chromosome-containing type II pneumocytes. A single Y-chromosome-containing type II pneumocyte was found in one lung biopsy from one hematopoietic cell transplant recipient. After adjustment for the effects of incomplete nuclear sampling, this pneumocyte represented 1.75% of all type II pneumocytes in the biopsy sample. There was no evidence of polyploidy to suggest cell-to-cell fusion. No donor-derived type II pneumocytes were found in samples from the other three patients. In conclusion, repopulation by bone marrow-derived stem cells or their progeny occurs at a low frequency in the lungs of hematopoietic cell transplant recipients. Conversely, proliferation by local stem cell populations appears to be more important for recovery from alveolar injury.

Active telomerase is present in the majority of malignant human tumors, including most cases of hepatocellular carcinoma (HCC). Telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, has been found to be expressed in HCCs, dysplastic (precancerous) nodules (DNs), and regenerative nodules arising in cirrhosis. In a study reported in this issue of the journal, hTERT mRNA levels were assessed by quantitative real-time RT-PCR in various nodular lesions dissected from liver specimens of patients with chronic hepatitis B. High levels of hTERT mRNA were present in HCCs, high-grade DNs, and occasional low-grade DNs, whereas low levels were found in normal livers, livers with chronic hepatitis B (with or without cirrhosis), large regenerative nodules, and most low-grade DNs. Therefore, quantitative assessment of hTERT mRNA may provide a useful adjunct to histopathologic evaluation of large hepatic nodules. Indeed, emerging data from gene expression analyses of DNs and HCCs suggest that hTERT can be included in sets of select genes that provide "molecular signatures" with utility in the diagnosis and management of nodular hepatic lesions. Most importantly, tackling the mechanisms of telomerase activation may provide new means of therapy for HCC and other cancers.