Cell-based Therapies For Liver Diseases Research Articles

Optimization of methods for intrasplenic administration of human amniotic epithelial cells in order to perform safe and effective cell-based therapy for liver diseases

In animal experimental models the administration of stem cells into the spleen should ensure high effectiveness of their implantation in the liver due to a direct vascular connection between the two organs. The aim of this study was to update the methods of experimental intrasplenic cell transplantation using human amniotic epithelial cells (hAECs) which are promising cells in the treatment of liver diseases. BALB/c mice were administered intrasplenically with 0.5, 1, and 2 million hAECs by direct bolus injection (400 µl/min) and via a subcutaneous splenic port by fast (20 μl/min) and slow (10 μl/min) infusion. The port was prepared by translocating the spleen to the skin pocket. The spleen, liver, and lungs were collected at 3 h, 6 h, and 24 h after the administration of cells. The distribution of hAECs, histopathological changes in the organs, complete blood count, and biochemical markers of liver damage were assessed. It has been shown that the method of intrasplenic cell administration affects the degree of liver damage. The largest number of mice showing significant liver damage was observed after direct administration and the lowest after slow administration through a port. Liver damage increased with the number of administered cells, which, paradoxically, resulted in increased liver colonization efficiency. It was concluded that the administration of 1 × 106 hAECs by slow infusion via a subcutaneous splenic port reduces the incidence of complications at the expense of a slight decrease in the effectiveness of implantation of the transplanted cells in the liver.

Elimination of Reprogramming Transgenes Facilitates the Differentiation of Induced Pluripotent Stem Cells into Hepatocyte-like Cells and Hepatic Organoids.

Simple SummaryThe elimination of reprogramming transgenes affects the differentiation potential of human induced pluripotent stem cells (hiPSCs) at the early embryonic development stage, but not during the late stage of development into hepatocytes and hepatic organoids. Using an excisable polycistronic lentiviral system (STEMCCA, Cre-loxP system), we generated both transgene-carrying and transgene-free hiPSCs from human fibroblasts and demonstrated that the elimination of transgenes enhances the differentiation potential of iPSCs toward hepatocyte-like cells and the generation of hepatic organoids, exhibiting efficient hepatic differentiation. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids.Hepatocytes and hepatic organoids (HOs) derived from human induced pluripotent stem cells (hiPSCs) are promising cell-based therapies for liver diseases. The removal of reprogramming transgenes can affect hiPSC differentiation potential into the three germ layers but not into hepatocytes and hepatic organoids in the late developmental stage. Herein, we generated hiPSCs from normal human fibroblasts using an excisable polycistronic lentiviral vector based on the Cre recombinase-mediated removal of the loxP-flanked reprogramming cassette. Comparing the properties of transgene-carrying and transgene-free hiPSCs with the same genetic background, the pluripotent states of all hiPSCs were quite similar, as indicated by the expression of pluripotent markers, embryonic body formation, and tri-lineage differentiation in vitro. However, after in vitro differentiation into hepatocytes, transgene-free hiPSCs were superior to the transgene-residual hiPSCs. Interestingly, the generation and hepatic differentiation of human hepatic organoids (hHOs) were significantly enhanced by transgene elimination from hiPSCs, as observed by the upregulated fetal liver (CK19, SOX9, and ITGA6) and functional hepatocyte (albumin, ASGR1, HNF4α, CYP1A2, CYP3A4, and AAT) markers upon culture in differentiation media. Thus, the elimination of reprogramming transgenes facilitates hiPSC differentiation into hepatocyte-like cells and hepatic organoids with properties of liver progenitor cells. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids.

Founder cells for hepatocytes during liver regeneration: from identification to application.

Liver regeneration (LR) capacity in vertebrates developed through natural selection over a hundred million years of evolution. To maintain homeostasis or recover from various injuries, liver cells must regenerate; this process includes the renewal of parenchymal and nonparenchymal cells as well as the formation of liver structures. The cellular origin of newly grown tissue is one of the critical questions in this area and has been a subject of prolonged debate. The regenerative tissue may derive from either hepatocyte self-duplication or liver stem/progenitor cells (LSPCs). Recently, hepatocyte subpopulations and cholangiocytes were also described as important founder cells. The niche that triggers the proliferation of hepatocytes and the differentiation of LSPCs has been extensively studied. Meanwhile, in vitro culture systems for liver founder cells and organoids have been developed rapidly for mechanistic studies and potential therapeutic purposes. This review summarizes the cellular sources and niches that give rise to renewed hepatocytes during LR, and it also describes in vitro culture studies of those founder cells for future applications, as well as current reports for stem cell-based therapies for liver diseases.

Macrophages as a Cell-Based Therapy for Liver Disease

Liver failure arising from acute and chronic liver disease is an unmet clinical need that urgently requires novel therapeutic options in addition to orthotopic liver transplantation. Cell therapies offer new strategies to recover liver function through the reconstitution of healthy parenchyma and resolution of tissue pathology. Macrophages are professional phagocytes that comprise a key part of the innate immune system providing an important defense mechanism against invading pathogens. Macrophages are an inherently diverse cell type with respect to ontogeny, tissue distribution, phenotype, and function. The ability of macrophages to afford innate immunity, efficiently scavenge apoptotic/necrotic cells, and modulate local tissue microenvironment makes them an attractive cell therapy candidate for various diseases. This review aims to outline the rationale and utility of macrophages to serve as a potential cell therapy for liver disease.

Stem Cell-Based Therapies for Liver Diseases: An Overview and Update.

Liver disease is one of the top causes of death globally. Although liver transplantation is a very effective treatment strategy, the shortage of available donor organs, waiting list mortality, and high costs of surgery remain huge problems. Stem cells are undifferentiated cells that can differentiate into a variety of cell types. Scientists are exploring the possibilities of generating hepatocytes from stem cells as an alternative for the treatment of liver diseases. In this review, we summarized the updated researches in the field of stem cell-based therapies for liver diseases as well as the current challenges and future expectations for a successful cell-based liver therapy. Several cell types have been investigated for liver regeneration, such as embryonic stem cells, induced pluripotent stem cells, liver stem cells, mesenchymal stem cells, and hematopoietic stem cells. In vitro and in vivo studies have demonstrated that stem cells are promising cell sources for the liver regeneration. Stem cell-based therapy could be a promising therapeutic method for patients with end-stage liver disease, which may alleviate the need for liver transplantation in the future.

Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors.

The liver provides vital metabolic, exocrine and endocrine functions in the body as such pathological conditions of the liver lead to high morbidity and mortality. The liver is highly regenerative and contains facultative stem cells that become activated during injury to replicate to fully recover mass and function. Canonical Wnt/β-catenin signaling plays an important role in regulating the proliferation and differentiation of liver progenitor cells during liver regeneration. However, possible roles of noncanonical Wnts in liver development and regeneration remain undefined. We previously established a reversibly-immortalized hepatic progenitor cell line (iHPx), which retains hepatic differentiation potential. Here, we analyze the expression pattern of the essential components of both canonical and noncanonical Wnt signaling pathways at different postnatal stages of mouse liver tissues and iHPx cells. We find that noncanonical Wnt4, Wnt5a, Wnt9b, Wnt10a and Wnt10b, are highly expressed concordantly with the high levels of canonical Wnts in late stages of liver tissues. Wnt5a, Wnt9b, Wnt10a and Wnt10b are able to antagonize Wnt3a-induced β-catenin/TCF activity, reduce the stemness of iHPx cells, and promote hepatic differentiation of liver progenitors. Stem cell implantation assay demonstrates that Wnt5a, Wnt9b, Wnt10a and Wnt10b can inhibit cell proliferation and promote hepatic differentiation of the iHPx progenitor cells. Our results strongly suggest that noncanonical Wnts may play an important role in fine-tuning Wnt/β-catenin functions during liver development and liver regeneration. Thus, understanding regulatory mechanisms governing proliferation and differentiation of liver progenitor cells may hold great promise to facilitate liver regeneration and/or progenitor cell-based therapies for liver diseases.

Therapeutic Use of Human Amnion-Derived Products: Cell-Based Therapy for Liver Disease

Since the early 20th century, the placenta has started to regain its status as tissue with great potential in regenerative medicine. In particular, cells isolated from the fetal membrane, amnion epithelial cells (AEC), have been described for their proficient capacity to support regeneration at several organs, primarily the liver. The investigation on regenerative and corrective potential of human AEC has been conducted on relevant preclinical models, showing a satisfactory level of liver regeneration and correction of inborn error of metabolism. When sufficient numbers of proficient cells are engrafted, phenotypic correction of a disease or at least conversion of a life-threatening disease into a milder and more manageable form is attained. Human AEC transplantation offers several advantages in liver cell-based approaches: AEC can be easily isolated by a wide array of donors; isolation and cryopreservation procedures commonly result in a high viability batch of cells, potentially “off the shelf” in every major medical center worldwide; AEC showed satisfactory levels of expression for multiple hepatic features when transplanted, and have been referred to as “immune-privileged” cells since they do not elicit a strong immune response once infused. Finally, unlike other stem cells, human AEC do not have the potential to form any kind of tumor once transplanted. Future and ongoing clinical studies are fundamental to unveil the true power of these immune-privileged cells that might dramatically change regenerative medicine applications.

Progress in stem cell-based therapy for liver disease.

Liver transplantation has been accepted as a useful therapeutic approach for patients with end-stage liver disease. However, the mismatch between the great demand for liver transplants and the number of available donor organs underscores the urgent need for alternative therapeutic strategies for patients with acute and chronic liver failure. The rapidly growing knowledge on stem cell biology has opened new avenues toward stem cell-based therapy for liver disease. As stem cells have capacity for high proliferation and multipotent differentiation, the characteristics of stem cells fit the cell therapy. Several types of cells have been investigated as possible sources of liver regeneration: mesenchymal stem cells, hematopoietic stem cells, liver progenitor cells, induced pluripotent stem cells, and bone marrow mononuclear cells. In vitro and in vivo experiments revealed that these cells have great potential as candidates of stem cell therapy. We reviewed the reports on clinical trials of cell therapy for liver disease that have been recently undertaken using mesenchymal stem cells, hematopoietic stem cells, bone marrow mononuclear cells, and liver progenitor cells. These reports have heterogeneity of description of trial design, types of infused cells, patient population, and efficacy of therapies. We addressed these reports from these viewpoints and clarified their significance. We hope that this review article will provide a perspective on the available approaches based on stem cell-based therapy for liver disease.

Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease. Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

Nuclear receptor gene alteration in human induced pluripotent stem cells with hepatic differentiation propensity

Human induced pluripotent stem (hiPS) cells are an alternative cell source of regenerative medicine for liver disease. Because variations in hepatic differentiation efficacy among hiPS cells exist, it is important to select a hiPS cell line with hepatic differentiation propensity. In addition, nuclear receptors (NR) regulate essential biological processes including differentiation and development. In this study, we identified the hiPS cell line with hepatic differentiation propensity and examined expression levels of 48 NR during this process. We screened 28 hiPS cell lines, which are established from various tissues of healthy persons with various reprogramming methods, using a three-step differentiation method, and examined expression levels of 48 NR by quantitative real-time polymerase chain reaction during the differentiation process in the selected cells. hiPS-RIKEN-2B and hiPS-RIKEN-2F cells have hepatic differentiation propensity. Differentiation propensity towards endoderm was affected by donor origin but not by reprogramming methods or cell type of origins. Expression levels of NR were closely associated with those of hepatic differentiation markers. Furthermore, expression patterns of NR were categorized as five patterns. In particular, seven NR such as chicken ovalbumin upstream promoter transcription factor 1, retinoic acid receptor α, peroxisome proliferator-activated receptor-γ, progesterone receptor, photoreceptor cell-specific nuclear receptor, tailless homolog orphan receptor and glucocorticoid receptor were identified as the genes of which expression gradually goes up with differentiation. These findings will be useful for not only elucidating mechanisms of hepatic differentiation of hiPS cells but also cell-based therapy for liver diseases.

Heps with Pep: Direct Reprogramming into Human Hepatocytes

The limited supply and expansion capacity of primary human hepatocytes presents major challenges for pharmaceutical applications and development of cell-based therapies for liver diseases. Now in Cell Stem Cell, two papers demonstrate efficient direct reprogramming of human fibroblasts into induced hepatocytes, which exhibit metabolic properties similar to primary hepatocytes.

Role of BMSCs in liver regeneration and metastasis after hepatectomy

Hepatocellular carcinoma (HCC), which develops from liver cirrhosis, is highly prevalent worldwide and is a malignancy that leads to liver failure and systemic metastasis. While surgery is the preferred treatment for HCC, intervention and liver transplantation are also treatment options for end-stage liver disease. However, the success of partial hepatectomy and intervention is hindered by the decompensation of liver function. Conversely, liver transplantation is difficult to carry out due to its high cost and the lack of donor organs. Fortunately, research into bone-marrow stromal cells (BMSCs) has opened a new door in this field. BMSCs are a type of stem cell with powerful proliferative and differential potential that represent an attractive tool for the establishment of successful stem cell-based therapy for liver diseases. A number of different stromal cells contribute to the therapeutic effects exerted by BMSCs because BMSCs can differentiate into functional hepatic cells and can produce a series of growth factors and cytokines capable of suppressing inflammatory responses, reducing hepatocyte apoptosis, reversing liver fibrosis and enhancing hepatocyte functionality. Additionally, it has been shown that BMSCs can increase the apoptosis rate of cancer cells and inhibit tumor metastasis in some microenvironments. This review focuses on BMSCs and their possible applications in liver regeneration and metastasis after hepatectomy.

Primary exploration of conditions for hypothermic preservation of rat hepatocyte spheroids

Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium (SFM). Spheroids were then maintained in rocking culture at 37℃ (control condition), or cold stored at 4℃ for 24 or 48 hours in four different cold storage solutions: SFM alone; SFM+1mmol/L deferoxamine (Def ); SFM+1μmol/L cyclosporin A (CsA); and SFM+1mmol/L Def+1μmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def was significantly higher than that stored in SFM or SFM+CsA(P 0.05). Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases. DOI: 10.11855/j.issn.0577-7402.2014.12.02

Hypoxia efficiently induces differentiation of mouse embryonic stem cells into endodermal and hepatic progenitor cells

Although ES cells have potential impact on cell-based therapy for liver diseases, their efficient differentiation into functional hepatocytes remains difficult. One possible approach to this issue is to control the culture environment to recapitulate the in vivo liver development. Given that the embryo is exposed to hypoxic condition in its early stage, hypoxic condition seems suitable for inducing early differentiation of ES cells. However, no studies have evaluated the effect of hypoxia on endodermal or early hepatic differentiation of ES cells. Here we investigated the potential role of hypoxia as a regulatory factor in endodermal and subsequent early hepatic differentiation of mouse ES cells. Under the stimulus of retinoic acid, a typical endodermal morphology emerged more dominantly under hypoxia (5% O2) than normoxia. Accordingly, quantitative RT-PCR revealed significant increase in Foxa2 and GATA4, essential transcription factors for endoderm specification. In addition, hypoxia amplified the effects of hepatic induction factors FGF1, FGF4 and HGF, which was evidenced by dramatically increased ALB-GFP-positive cell fraction, as well as increased gene expression levels of ALB, AFP, and CK18. Furthermore, the hepatic progenitor cells induced under hypoxic condition were efficiently differentiated into mature hepatocytes with ALB secretion ability. We also found that the differentiating cells did not consume oxygen even under normoxia, suggesting that oxygen per se might have a negative effect on the normal endodermal and early hepatic differentiation of ES cells. These results will provide useful insight into efficient production of hepatic progenitor cells, and contribute to stem cell-based liver tissue engineering.

The differentiation of MSCs into functional hepatocyte-like cells in a liver biomatrix scaffold and their transplantation into liver-fibrotic mice

Hepatocytes derived from mesenchymal stem cells (MSCs) hold great potential for cell-based therapies for liver diseases. The cell-based therapies are critically dependent on the hepatic differentiation of the MSCs with a high efficiency and on a considerable scale. Recent results have shown that decellularized organs provide a three-dimensional extracellular matrix for the lineage restriction of stem cell maturation. In this study, we compared the cell proliferation and hepatic differentiation of murine MSCs in a biomatrix scaffold from rat liver and in the presence and absence growth factors (GF) with a two-dimensional substrate. In the absence or presence of GF, the dynamic cultured scaffold (DCS) stimulated the MSCs to express endodermal and hepatocyte-specific genes and proteins associated with improved functions, and the cells exhibited the ultrastructural characteristics of mature hepatocytes. When transplanted into CCl4-injured mice, the cells pretreated with a combination of the DCS and GF exhibited increased survival, liver function, engraftment into the host liver and further hepatic differentiation. The paracrine effect of the transplanted cells on hepatic stellate cells and native hepatocytes played a key role in the treatment of the liver pathology. These studies define an effective method that facilitates the hepatic differentiation of MSCs exhibiting extensive functions and support further research into the use of a decellularized liver matrix as a bioscaffold for liver tissue engineering.

Stem cell-based therapies for liver diseases: state of the art and new perspectives.

Millions of patients worldwide suffer from end-stage liver pathologies, whose only curative therapy is liver transplantation (OLT). Given the donor organ shortage, alternatives to OLT have been evaluated, including cell therapies. Hepatocyte transplantation has been attempted to cure metabolic liver disorders and end-stage liver diseases. The evaluation of its efficacy is complicated by the shortage of human hepatocytes and their difficult expansion and cryopreservation. Recent advances in cell biology have led to the concept of “regenerative medicine”, based on the therapeutic potential of stem cells (SCs). Different types of SCs are theoretically eligible for liver cell replacement. These include embryonic and fetal SCs, induced pluripotent cells, annex SCs, endogenous liver SCs, and extrahepatic adult SCs. Aim of this paper is to critically analyze the possible sources of SCs suitable for liver repopulation and the results of the clinical trials that have been published until now.

Cell Transplantation in the Treatment of Acute Liver Injury: Effect But No Clear Mechanism

Liver transplantation is the current treatment of choice for end-stage liver disease. Nevertheless, cell-based therapy is emerging as an alternative to whole-organ transplantation. It is less invasive and can be performed repeatedly. Hepatocyte transplantation has been used to bridge patients to whole-organ transplantation, which has been done using bioartificial liver techniques such as extracorporeal liver assist device (ELAD), molecular adsorbent recirculating system (MARS), and plasmapheresis. However, these methods are not achieving the desirable goal of effectively reducing mortality in acute liver failure[ 1 O'Grady J. Modern management of acute liver failure. Clin Liver Dis. 2007; 11: 291 Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar ]. An alternative to the primary hepatocyte is stem cell derivative or hepatocyte precursors, which are available within each patient. Research into the ability of these cells to replace hepatocytes and their function is ongoing [ 2 Nussler A. Konig S. Ott M. et al. Present status and perspectives of cell-based therapies for liver diseases. J Hepatol. 2006; 45: 144 Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar ].

Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes

The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

Establishment of Immortalized Human Hepatocytes by Introduction of HPV16 E6/E7 and hTERT as Cell Sources for Liver Cell-Based Therapy.

For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminophen-induced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.

IFATS Collection: In Vivo Therapeutic Potential of Human Adipose Tissue Mesenchymal Stem Cells After Transplantation into Mice with Liver Injury

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl(4)-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor alpha (IL-1Ralpha), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM-MSCs and NHDFs. Thus, our findings suggest that AT-MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment. Disclosure of potential conflicts of interest is found at the end of this article.