Michael Oertel

Bone marrow progenitors are not the source of expanding oval cells in injured liver

Liver progenitor/oval cells differentiate into hepatocytes and biliary epithelial cells, repopulating the liver when the regenerative capacity of hepatocytes is impaired. Recent studies have shown that hematopoietic bone marrow (BM) stem/progenitor cells can give rise to hepatocytes in diseased/damaged liver. One study has reported that BM cells can transdifferentiate into liver progenitor/oval cells, but it has not been proven that the latter can repopulate the liver. To answer this question, we have lethally irradiated female DPP4− mutant F344 rats and transplanted them with 50 million wild‐type male F344 BM cells. One month after transplantation, the recipient BM was reconstituted with male hematopoietic cells, determined by quantitative polymerase chain reaction using primers for Y chromosome–specific sry gene. In addition, DPP4+ cells, single or in clusters and predominantly in the periportal region, were detected in all liver sections of recipient rats. Animals were subjected to the following three different liver injury protocols for activation and expansion of oval cells: D‐galactosamine, retrorsine/partial hepatectomy (Rs/PH), and 2‐acetylaminofluorene/partial hepatectomy (2‐AAF/PH). In all three models, prominent expansion and accumulation of cytokeratin 19–positive (CK‐19+) oval cells was observed. However, most of the DPP4+ clusters dispersed over time, and their total number decreased. Very few oval cells (less than 1%) showed double DPP4/CK‐19 labeling. None of the small hepatocytic clusters in the Rs/PH or 2‐AAF/PH model were comprised of DPP4+ cells. These data demonstrate that the sources of oval cells and small hepatocytes in the injured liver are endogenous liver progenitors and that they do not arise through transdifferentiation from BM cells.

Liver stem cells and prospects for liver reconstitution by transplanted cells

Although it was proposed almost 60 years ago that the adult mammalian liver contains hepatic stem cells, this issue remains controversial. Part of the problem is that no specific marker gene unique to the adult hepatic stem cell has yet been identified, and regeneration of the liver after acute injury is achieved through proliferation of adult hepatocytes and does not require activation or proliferation of stem cells. Also, there are differences in the expected properties of stem versus progenitor cells, and we attempt to use specific criteria to distinguish between these cell types. We review the evidence for each of these cell types in the adult versus embryonic/fetal liver, where tissue‐specific stem cells are known to exist and to be involved in organ development. This review is limited to studies directed toward identification of hepatic epithelial stem cells and does not address the controversial issue of whether stem cells derived from the bone marrow have hepatocytic potential, a topic that has been covered extensively in other recent reviews. (Hepatology 2006;43:S89–S98.)

Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver

BACKGROUND & AIMS Previously, we showed high-level, long-term liver replacement after transplantation of unfractionated embryonic day (ED) 14 fetal liver stem/progenitor cells (FLSPC). However, for clinical applications, it will be essential to transplant highly enriched cells, while maintaining high repopulation potential. METHODS Dlk-1, a member of the delta-like family of cell surface transmembrane proteins, is highly expressed in human and rodent fetal liver. Dlk-1(+) cells, isolated from ED14 fetal liver using immunomagnetic beads, were examined for their hepatic gene expression profile and characteristic properties in vitro and their proliferative and differentiation potential in vivo after transplantation into normal adult rat liver. RESULTS Rat ED14 FLSPC were purified to 95% homogeneity and exhibited cell culture and gene expression characteristics expected for hepatic stem/progenitor cells. Rat ED14 FLSPC are alpha-fetoprotein(+)/cytokeratin-19(+) or alpha-fetoprotein(+)/cytokeratin-19(-) and contain all of the normal liver repopulation capacity found in fetal liver. Hematopoietic stem cells, a major component in crude fetal liver cell preparations that engraft in other organs, such as bone marrow, spleen, and lung, are totally removed by Dlk-1 selection, and Dlk-1 purified FLSPC repopulate only the liver. CONCLUSIONS This is the first study reporting purification of hepatic stem/progenitor cells from fetal liver that are fully capable of repopulating the normal adult liver. This represents a major advance toward developing protocols that will be essential for clinical application of liver cell transplantation therapy.

Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity.

Epithelial cells in embryonic day (ED) 12.5 murine fetal liver were separated from hematopoietic cell populations using fluorescence‐activated cell sorting (FACS) and were characterized by immunocytochemistry using a broad set of antibodies specific for epithelial cells (α‐fetoprotein [AFP], albumin [ALB], pancytokeratin [PanCK], Liv2, E‐cadherin, Dlk), hematopoietic/endothelial cells (Ter119, CD45, CD31), and stem/progenitor cells (c‐Kit, CD34, Sca‐1). AFP+/ALB+ cells represented approximately 2.5% of total cells and were positive for the epithelial‐specific surface markers Liv2, E‐cadherin, and Dlk, but were clearly separated and distinct from hematopoietic cells (Ter119+/CD45+). Fetal liver epithelial cells (AFP+/E‐cadherin+) were Sca‐1+ but showed no expression of hematopoietic stem cell markers c‐Kit and CD34. These cells were enriched by FACS sorting for E‐cadherin to a purity of 95% as defined by co‐expression of AFP and PanCK. Purified fetal liver epithelial cells formed clusters in cell culture and differentiated along the hepatocytic lineage in the presence of dexamethasone, expressing glucose‐6‐phosphatase (G6P) and tyrosine amino transferase. Wild‐type ED12.5 murine fetal liver cells were transplanted into adult dipeptidyl peptidase IV knockout mice and differentiated into mature hepatocytes expressing ALB, G6P, and glycogen, indicating normal biochemical function. Transplanted cells became fully incorporated into the hepatic parenchymal cords and showed up to 80% liver repopulation at 2 to 6 months after cell transplantation. In conclusion, we isolated and highly purified a population of epithelial cells from the ED12.5 mouse fetal liver that are clearly separate from hematopoietic cells and differentiate into mature, functional hepatocytes in vivo with the capacity for efficient liver repopulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;.)

Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells.

In recent years, there has been substantial progress in transplanting cells into the liver with the ultimate goal of restoring liver mass and function in both inherited and acquired liver diseases. The basis for considering that this might be feasible is that the liver is a highly regenerative organ. After massive liver injury or surgical removal of two-thirds or more of the liver tissue, the organ can restore its mass with completely normal morphologic structure and function. It has also been found under highly selective conditions that transplanted hepatocytes can fully repopulate the liver and cure a metabolic disorder or deficiency state. Fetal liver cells can also substantially repopulate the normal liver, and it is hoped in the future that effective repopulation will be achievable with cultured cells or cell lines, pluripotent stem cells from other somatic tissues, embryonic stem cells, or induced pluripotent stem cells, which can now be generated in vitro by a variety of methods. The purpose of this review is to present the major systems that have been used for liver repopulation, the variables involved in obtaining successful repopulation and what has been achieved in these various systems to date with different cell types.

Properties of cryopreserved fetal liver stem/progenitor cells that exhibit long-term repopulation of the normal rat liver.

We have previously achieved a high level of long‐term liver replacement by transplanting freshly isolated embryonic day (ED) 14 rat fetal liver stem/progenitor cells (FLSPCs). However, for most clinical applications, it will be necessary to use cryopreserved cells that can effectively repopulate the host organ. In the present study, we report the growth and gene expression properties in culture of rat FLSPCs cryopreserved for up to 20 months and the ability of cryopreserved FLSPCs to repopulate the normal adult rat liver. After thawing and placement in culture, cryopreserved FLSPCs exhibited a high proliferation rate: 49.7% Ki‐67‐positive on day 1 and 34.7% Ki‐67‐positive on day 5. The majority of cells were also positive for both α‐fetoprotein and cytokeratin‐19 (potentially bipotent) on day 5. More than 80% of cultured cells expressed albumin, the asialoglycoprotein receptor, and UDP‐glucuronosyltransferase (unique hepatocyte‐specific functions). Expression of glucose‐6‐phosphatase, carbamyl phosphate synthetase 1, hepatocyte nuclear factor 4α, tyrosine aminotransferase, and oncostatin M receptor mRNAs was initially negative, but all were expressed on day 5 in culture. After transplantation into the normal adult rat liver, cryopreserved FLSPCs proliferated continuously, regenerated both hepatocytes and bile ducts, and produced up to 15.1% (mean, 12.0% ± 2.0%) replacement of total liver mass at 6 months after cell transplantation. These results were obtained in a normal liver background under nonselective conditions. This study is the first to show a high level of long‐term liver replacement with cryopreserved fetal liver cells, an essential requirement for future clinical applications.

Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes

Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long‐term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV+ F344 rats were transplanted into DPPIV− rats with thioacetamide (TAA)‐induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse‐transcription polymerase chain reaction (RT‐PCR), and immunohistochemistry. After chronic TAA administration, DPPIV− F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk‐1+, AFP+, CD133+, Sox‐9+, FoxJ1+) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long‐term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet‐derived growth factor receptor β, desmin, vimentin, tissue inhibitor of metalloproteinase‐1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells. Conclusion: This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects. (Hepatology 2014;58:284–295)

Fetal liver cell transplantation as a potential alternative to whole liver transplantation

Because organ shortage is the fundamental limitation of whole liver transplantation, novel therapeutic options, especially the possibility of restoring liver function through cell transplantation, are urgently needed to treat end-stage liver diseases. Groundbreaking in vivo studies have shown that transplanted hepatocytes are capable of repopulating the rodent liver. The two best studied models are the urokinase plasminogen activator (uPA) transgenic mouse and the fumarylacetoacetate hydrolase (FAH)-deficient mouse, in which genetic modifications of the recipient liver provide a tissue environment in which there is extensive liver injury and selection pressure favoring the proliferation and survival of transplanted hepatocytes. Because transplanted hepatocytes do not significantly repopulate the (near-)normal liver, attention has been focused on finding alternative cell types, such as stem or progenitor cells, that have a higher proliferative potential than hepatocytes. Several sources of stem cells or stem-like cells have been identified and their potential to repopulate the recipient liver has been evaluated in certain liver injury models. However, rat fetal liver stem/progenitor cells (FLSPCs) are the only cells identified to date that can effectively repopulate the (near-)normal liver, are morphologically and functionally fully integrated into the recipient liver, and remain viable long-term. Even though primary human fetal liver cells are not likely to be routinely used for clinical liver cell repopulation in the future, using or engineering candidate cells exhibiting the characteristics of FLSPCs suggests a new direction in developing cell transplantation strategies for therapeutic liver replacement. This review will give a brief overview concerning the existing animal models and cell sources that have been used to restore normal liver structure and function, and will focus specifically on the potential of FLSPCs to repopulate the liver.

Activin A, p15INK4b Signaling, and Cell Competition Promote Stem/Progenitor Cell Repopulation of Livers in Aging Rats

BACKGROUND & AIMS Highly proliferative fetal liver stem/progenitor cells (FLSPCs) repopulate livers of normal recipients by cell competition. We investigated the mechanisms by which FLSPCs repopulate livers of older compared with younger rats. METHODS Fetal liver cells were transplanted from DPPIV(+) F344 rats into DPPIV(-) rats of different ages (2, 6, 14, or 18 months); liver tissues were analyzed 6 months later. Cultured cells and liver tissues were analyzed by reverse transcription polymerase chain reaction, immunoblot, histochemistry, laser-capture microscopy, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling analyses. RESULTS We observed 4- to 5-fold increases in liver repopulation when FLSPCs were transplanted into older compared with younger rats. Messenger RNA levels of cyclin-dependent kinase inhibitors increased progressively in livers of older rats; hepatocytes from 20-month-old rats had 6.1-fold higher expression of p15INK4b and were less proliferative in vitro than hepatocytes from 2-month-old rats. Expression of p15INK4b in cultured hepatocytes was up-regulated by activin A, which increased in liver during aging. Activin A inhibited proliferation of adult hepatocytes, whereas FLSPCs were unresponsive because they had reduced expression of activin receptors (eg, ALK-4). In vivo, expanding cell clusters derived from transplanted FLSPCs had lower levels of ALK-4 and p15INK4b and increased levels of Ki-67 compared with the host parenchyma. Liver tissue of older rats had 3-fold more apoptotic cells than that of younger rats. CONCLUSIONS FLSPCs, resistant to activin A signaling, repopulate livers of older rats; hepatocytes in older rats have less proliferation because of increased activin A and p15INK4b levels and increased apoptosis than younger rats. These factors and cell types might be manipulated to improve liver cell transplantation strategies in patients with liver diseases in which activin A levels are increased.

Comparison of hepatic properties and transplantation of Thy‐1+ and Thy‐1− cells isolated from embryonic day 14 rat fetal liver

Thy‐1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy‐1+ cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy‐1+ fetal liver cells. Therefore, we isolated Thy‐1+ and Thy‐1− cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy‐1 expression using immunomagnetic microbeads were enriched from 5.2%‐87.2% Thy‐1+. The vast majority of alpha fetoprotein+, albumin+, cytokine‐19+, and E‐cadherin+ cells were found in cultured Thy‐1− cells, whereas nearly all CD45+ cells were in the Thy‐1+ fraction. In normal rat liver, transplanted Thy‐1+ cells produced only rare, small DPPIV+ cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine‐treated liver, transplanted Thy‐1+ fetal liver cells achieved a 4.6%‐23.5% repopulation. In contrast, Thy‐1− fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine‐treated liver. Regarding the stromal cell–derived factor (SDF)–1/chemokine (C‐X‐C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy‐1+ and Thy‐1− fetal hepatic epithelial cells equally expressed CXCR4. However, SDF‐1α expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy–treated liver, and this correlated with liver repopulation by Thy‐1+ cells. Conclusion: Highly enriched Thy‐1+ ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy‐1− stem/progenitor cells, which repopulate the normal adult liver. (HEPATOLOGY 2007.)