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Progenitor cells isolated from the fetal liver can provide a unique cell source to generate new healthy tissue mass. Almost 20 years ago, it was demonstrated that rat fetal liver cells repopulate the normal host liver environment via a mechanism akin to cell competition. Activin A, which is produced by hepatocytes, was identified as an important player during cell competition. Because of reduced activin receptor expression, highly proliferative fetal liver stem/progenitor cells are resistant to activin A and therefore exhibit a growth advantage compared to hepatocytes. As a result, transplanted fetal liver cells are capable of repopulating normal livers. Important for cell-based therapies, hepatic stem/progenitor cells containing repopulation potential can be separated from fetal hematopoietic cells using the cell surface marker δ-like 1 (Dlk-1). In livers with advanced fibrosis, fetal epithelial stem/progenitor cells differentiate into functional hepatic cells and out-compete injured endogenous hepatocytes, which cause anti-fibrotic effects. Although fetal liver cells efficiently repopulate the liver, they will likely not be used for human cell transplantation. Thus, utilizing the underlying mechanism of repopulation and developed methods to produce similar growth-advantaged cells in vitro, e.g., human induced pluripotent stem cells (iPSCs), this approach has great potential for developing novel cell-based therapies in patients with liver disease. The present review gives a brief overview of the classic cell transplantation models and various cell sources studied as donor cell candidates. The advantages of fetal liver-derived stem/progenitor cells are discussed, as well as the mechanism of liver repopulation. Moreover, this article reviews the potential of in vitro developed synthetic human fetal livers from iPSCs and their therapeutic benefits.

Liver transplantation is the only therapeutic treatment for patients with end-stage liver diseases. However, donor organ scarcity is the major limitation, and therefore, alternative strategies are urgently needed. The ultimate goal for successful cell-based therapies is the ability of transplanted cells to efficiently engraft and reconstitute injured liver mass. To evaluate the repopulation capacity of transplanted cells, it is essential to identify their specific characteristics, as well as to study the mechanism(s) Through which transplanted donor cells replace tissue mass in hepatic microenvironments, using well-established cell transplantation models. To date, rat fetal liver stem/progenitor cells represent the most efficient cell population to reconstitute the near-normal liver and the liver microenvironment with advanced fibrosis/cirrhosis, and therefore, can be used for developing strategies in engineering potential donor cells in the future that will be useful for clinical application in hepatic cell therapy.The present protocol describes the isolation of epithelial stem/progenitor cells derived from ED14/15 fetal livers of DPPIV + F344 or F344-Tg(EGFP) F455/Rrrc rats, the immunohistochemical staining method to detect E-cadherin-positive epithelial cells within unfractionated cell isolates, their transplantation into different DPPIV - liver microenvironments (near-normal, retrorsine-treated, and TAA-induced fibrotic/cirrhotic liver), as well as detection methods to follow the fate of transplanted cells in the recipient liver (see Fig. 1).

Unlabelled: 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., (© 2013 by the American Association for the Study of Liver Diseases.)

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.

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.

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.

Background & Aims: A critical property of stem cells is their ability to repopulate an organ or tissue under nonselective conditions. The aims of this study were to determine whether we could obtain reproducible, high levels of liver repopulation by transplanted fetal liver stem/progenitor cells in normal adult liver and the mechanism by which liver replacement occurred., Methods: Wild-type (dipeptidyl peptidase IV [DPPIV(+)]) embryonic day (ED) 14 fetal liver cells underwent transplantation into DPPIV(-) mutant F344 rats to follow the fate and differentiation of transplanted cells. To determine the mechanism for repopulation, proliferation and apoptosis of transplanted and host liver cells were also followed., Results: Transplanted ED 14 fetal liver cells proliferated continuously for 6 months, differentiated into mature hepatocytes, and replaced 23.5% of total liver mass. The progeny of transplanted cells were morphologically and functionally indistinguishable from host hepatocytes and expressed unique liver-specific genes commensurate with their location in the hepatic lobule. Repopulation was based on greater proliferative activity of transplanted cells and reduced apoptosis of their progeny compared with host hepatocytes, coupled with increased apoptosis of host hepatocytes immediately adjacent to transplanted cells. This process, referred to as cell-cell competition, has been described previously in Drosophila during wing development., Conclusions: We show for the first time that cell-cell competition, a developmental paradigm, can be used to replace functional organ tissue in an adult mammalian species under nonselective conditions and may serve as a strategy for tissue reconstitution in a wide variety of metabolic and other disorders involving the liver, as well as other organs.

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified ( approximately 10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach.

Abstract: 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Total body irradiation is an integral part of many conditioning regimens prior to allogeneic stem cell transplantation. It is a large-field technique affecting all organs at risk, of which the lungs are critical for patient survival. However, the precise rates of long-term pulmonary toxicities are unknown. This analysis provides a large patient cohort with long-term follow-up investigating TBI sequelae. Additionally, we present normal tissue complication probability calculations for acute and chronic lung toxicities to enable comparison between biophysical and real-world data. To our knowledge, this is the first adaption of this model to a total-body irradiation patient cohort, which will help to evaluate the feasibility and appropriateness of this approach. Total body irradiation (TBI) is an essential part of various conditioning regimens prior to allogeneic stem cell transplantation, but is accompanied by relevant (long-term) toxicities. In the lungs, a complex mechanism induces initial inflammation (pneumonitis) followed by chronic fibrosis. The hereby presented analysis investigates the occurrence of pulmonary toxicity in a large patient collective and correlates it with data derived from normal tissue complication probability (NTCP) calculations. The clinical data of 335 hemato-oncological patients undergoing TBI were analyzed with a follow-up of 85 months. Overall, 24.8% of all patients displayed lung toxicities, predominantly pneumonia and pulmonary obstructions (13.4% and 6.0%, respectively). NTCP calculations estimated median risks to be 20.3%, 0.6% and 20.4% for overall pneumonitis (both radiological and clinical), symptomatic pneumonitis and lung fibrosis, respectively. These numbers are consistent with real-world data from the literature and further specify radiological and clinical apparent toxicity rates. Overall, the estimated risk for clinical apparent pneumonitis is very low, corresponding to the probability of non-infectious acute respiratory distress syndrome, although the underlying pathophysiology is not identical. Radiological pneumonitis and lung fibrosis are expected to be more common but require a more precise documentation by the transplantation team, radiologists and radiation oncologists. [ABSTRACT FROM AUTHOR]