Liver cell therapies: cellular sources and grafting strategies

Abstract

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver’s cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Keywords

liver regeneration / hepatocytes / cholangiocytes / stem cells / organoids / regulatory mechanisms / transplantation/grafting strategies

1 Introduction

The liver is the key organ mediating the functions of detoxification, metabolism, glycogen and lipid storage and regulation, and secretion of various factors required systemically. The functions of the liver are closely related to its structural integrity. The liver has strong regenerative and repair abilities to sustain requisite functions under conditions of normal cell turnover, involving 1%–2% of the cells, and is yet able to cope with more extensive regenerative demands when the liver is partially resected or damaged by viruses, drugs, toxins, or radiation [1–3]. The primary cell types of the liver consist of two parenchymal cell populations, hepatocytes and cholangiocytes, complemented by a number of non-parenchymal cells: endothelia, stellate cells, and various hemopoietic cells. The liver’s functions result from the net sum of the interactions among these cellular populations.

Although a maturational lineage of cells within the liver was proposed long ago as defining the liver’s heterogeneity, phenotypic traits, and functions [4,5], that hypothesis has been expanded in recent years with the realization that the parenchymal cell populations can be generated from stem/progenitors within the biliary tree, the ramifying ducts connecting the liver and the pancreas to the duodenum [6–8]. The stem/progenitor cell populations are concentrated in peribiliary glands (PBGs) where the cells appear to migrate along the walls of the PBGs toward the bile duct lumen. These connect to descendants, hepatic stem cells and hepatoblasts, in or near the canals of Hering, and that give rise to hepatocytes and cholangiocytes forming the plates of liver cells [6,7,9]. The cells, the basic functional units of the liver, extend from the portal triads to terminally differentiated cells near the central vein at the center of the lobule. The system constitutes maturational lineages of cells from the most primitive stem cells within the biliary tree to adult parenchymal cells within the lobule to terminally differentiated hepatocytes that undergo apoptosis and are sloughed off into the blood at the central vein.

The ability to analyze the maturational and functional processes has been greatly enhanced in recent years with the improvement of single cell transcription analyses, the establishment of liver injury and other genetic mouse models, and the findings from clinical trials assessing cell transplantation for the treatment of liver injuries. This review summarizes the recent progress on the identity of cells involved in liver repair under various conditions in the hopes of providing effective interventions for end-stage liver diseases.

2 Stem cells in the liver regeneration—progress and challenges

2.1 Stem cells within or near the liver lobule

“The steaming liver” model proposed by Arber and Zajicek in 1985 describes the contribution of liver progenitor cells, also called “oval cells,” to the maintenance of liver homeostasis [10,11]. The model suggests that hepatocytes are derived from hepatic progenitor cells and that slide or “stream” across the liver plates from portal triad to central vein. The model was controversial, since there was no rigorous evidence whether hepatic progenitor cells are true stem cells, and transplants of isolated hepatic progenitor cells into the liver gave variable results depending on the specifics of the experiments. More support for hepatic progenitors in postnatal livers derived from others.

Further clarification of the potential relevance of stem/progenitor populations postnatally was made by a proposal that the liver is a maturational lineage system, in which stem cells in or near the canals of Hering can transit into unipotent committed progenitors (cells with similarities to the oval cells) that mature into the parenchymal cell populations in the liver lobule, and with stepwise progression of the phenotypic traits correlating with ploidy, expansion potential and tissue-specific gene expression [4]. This model offered more realistic clarifications regarding the stem/progenitor populations, the possible mechanisms involved, and the behavior of parenchymal cells under conditions of partial hepatectomy versus effects of periportal versus pericentral injuries [12,13].

Experimental evidence for the maturational lineage process from stem cells to late, mature cells in rat and mouse livers was achieved with evidence that the hepatic parenchymal cells and associated mesenchymal cells showed phenotypic traits that changed progressively within the liver acinus from periportal cells that are diploid to pericentral ones that are polyploid and with evidence for terminal differentiation and apoptosis [4,5,13–24]. Further experimental proof for stem cells in adult mice was achieved by Miyajima and associates, who found stem cells in the canals of Hering in adult murine livers [25–27]. The canals of Hering are the finest branches of the biliary tree, connecting the bile canalicular system to the bile ductules and the interlobular bile duct in the portal tract. The cells found in the canals of Hering have bipotent differentiation potential, giving rise to hepatocytes and cholangiocytes, Therefore, they are candidates for a type of adult stem cells resident in the liver postnatally [21,23].

Katoonizadeh et al. believe that HpSCs are facultative, that is, under healthy conditions, they do not participate in preserving the maintenance of liver homeostasis and tissue renewal. However, during acute and chronic liver injury, when hepatocyte proliferation is inhibited or abundant hepatocytes are lost, HpSCs are activated, differentiate, and mature into hepatocytes and cholangiocytes, and complete the repair of damaged liver parenchyma [28–30]. Whether or not HpSCs are facultative remains unknown given the slow turnover of the quiescent liver, meaning that the slow kinetics may obviate the ability to assess contributions of the stem cells.

Huch and associates [31] found that murine cellular subpopulations expressing leucine-rich repeats containing G protein coupled receptor 5 (LGR5) appear near the bile ducts after adult liver injury. These findings parallel those found by Miyajima and associates [25,26,32]. These cells are smaller (~9–12 µm) than mature hepatocytes (> 17 µm) and have the potential to differentiate into hepatocytes and cholangiocytes, have self-renewal capacity, and so logically can be defined as hepatic stem cells. Lgr5 is a commonly used marker of intestinal stem cells to identify those located in the intestinal crypt [33]. By constructing Lgr5 fluorescent-labeled mice, Huch conducted lineage tracking studies, which proved that these Lgr5⁺ hepatic stem cells produced many hepatocytes and cholangiocytes in the repair stages after liver injury [34]. However, there was no direct evidence to support whether this cell population is the main or sole stem cell subpopulation for liver regeneration (Fig.1).

Analyses of hepatic stem cells and hepatoblasts in human livers indicated that there is an overlap but also with distinctions in the phenotypic traits between two precursor populations, hepatic stem cells and hepatoblasts [22,35–38]. They are both found in or near the canals of Hering; both are bipotent and give rise to hepatocytes and cholangiocytes; both express low to moderate levels of pluripotency genes (e.g., OCT4, SOX2, BMi-1) and express epithelial cell adhesion molecule (EpCAM) and LGR5. Yet they can be distinguished antigenically and behave differently under defined culture conditions. In addition to EpCAM and LGR5, the hepatic stem cells express NCAM, no alpha-fetoprotein (AFP), and have weak, constitutive expression of albumin; by contrast, the hepatoblasts do not express NCAM but instead express ICAM-1; have high levels of AFP; and have much higher, somewhat more regulated expression of albumin. Both express telomerases, but the hepatic stem cells are able to synthesize the mRNA encoding telomerase, whereas the hepatoblasts have high levels of stabilized mRNA and abundant telomerase protein [36]. The hepatic stem cells proved to be precursors of the hepatoblasts, could be sustained for months ex vivo under serum-free, wholly defined conditions, and could be triggered to produce hepatoblasts with supplementation of the culture conditions with particular growth factors and/or matrix components [21,39,40].

A new site for hepatic precursors was found pericentrally in the liver lobule by Wang et al. in which a single layer of Axin2⁺ hepatocytes, located at the central vein, was found to handle some facets of liver turnover [41]. They believed that Axin2⁺ marked a new subgroup of liver stem cells. However, the results of this study were controversial. Investigations by others have resulted in the concept that Axin2⁺ cells are unipotent hepatocytic progenitors handling replacement of terminally differentiated, apoptotic cells near the central vein but that are not true stem cells with self-renewal capacity. True hepatic stem cells are bipotent and found within the acini only periportally, or as discussed below, within the biliary tree, and are the primary sources of precursors for liver turnover [42] (Fig.1).

2.2 Stem cell subpopulations within the biliary tree

Recent collaborative studies by US and Italian investigators have revealed that the stem cells relevant to liver turnover are found also in the larger branches of the biliary tree [7,9,43–45], the ramifying ducts connecting to the liver and to the pancreas [36,37,46]. There are increased numbers of these biliary tree stem cells (BTSCs) in pathological states such as chronic viral hepatitis, primary sclerosing cholangitis [47,48], alcoholic liver disease and nonalcoholic fatty liver disease but are in small numbers in normal liver [22,35–37,43,49–52]. The findings about them are summarized in several reviews [6,7,53].

The BTSC subpopulations are located in extramural peribiliary glands (PBGs), tethered to the outsides of the large bile ducts, and in intramural PBGs (within the bile duct walls) within all branches of the intrahepatic and extrahepatic biliary tree (Fig.2 and 2C). The most primitive of these BTSC subpopulations are in the extramural PBGs and in the Brunner’s glands in the submucosa of the duodenum [6,44] (Fig.3). These connect to stem cells in the intramural PBGs nearest to the centers of the bile duct walls, the sites that are against a rich fibromuscular layer. The stem cells do not express any markers of mature hepatic or pancreatic traits; they co-express hepatic and pancreatic transcription factors, significant levels of pluripotency genes, and have the enzymatic machinery and surface markers associated with hyaluronan synthesis. These regions within the PBGs in the bile duct walls are hypothesized to be presumptive stem cell crypts. With progression of cells toward the bile duct lumens, the cells in the PBGs mature to adult fates, constituting a radial axis of maturation. Within the large intrahepatic bile ducts, the maturing cells resemble bipotent hepato-biliary progenitors and yield hepatic fates; by contrast, in PBGs in the hepato/pancreatic common duct, the stem cells connect into the pancreatic duct glands (PDGs) within the pancreas and mature into pancreatic fates (acinar cells and islets) [52]. Although the connections of the biliary tree into the liver have been characterized rather extensively, further studies are required to define the connections and the network within the pancreas (Fig.3).

The multiple BTSC subpopulations have been shown to be hepato/pancreatic stem cells able to differentiate into hepatic (hepatocytes, cholangiocytes) and pancreatic (acinar cells, islets) cell populations (Fig.4) [43,54,55]. BTSCs express early endodermal transcription factors (SOX9, SOX17, PDX-1), stem cell traits (LGR5, CD44, sodium iodide symporter or NIS) and traits of hyaluronan synthesis (CD44v and CD44s), but do not express mature hepatocyte-specific markers (e.g., ALB, AFP, HNF4A), cholangiocyte specific markers (CFTR), islet endocrine markers (e.g., insulin, glucagon); nor pancreatic acinar cell-specific markers (e.g., digestive enzymes, amylase).

Proof of the multipotential endodermal stemness of the BTSCs has been achieved by transplantation of them into liver or into pancreas using grafting strategies designed both for single cell suspensions and for organoids, aggregates of BTSCs partnered with angioblasts plus precursors to endothelia and stellate cells [55,56]. Using the grafting methods, called “patch grafting,” Zhang et al. were able efficiently to transplant large numbers of BTSCs organoids, > 10⁸, with each organoid being ~100 cells, into the livers of pigs or into the livers or pancreases of mice and found them able rapidly to integrate uniformly amidst the cells within the host organs (Fig.5). The engraftment of the BTSC organoids was found able to generate mature hepatic and pancreatic cells capable of alleviating disease states in those organs such as tyrosinemia (liver) or diabetes (pancreas) [55].

It is worthwhile mentioning that with respect to embryo development, the livers, biliary trees, and pancreases are all derived from the definitive endoderm foregut. It is not surprising that Cardinale et al. identified stem cell populations in the submucosa layer of the duodenum, the Brunner’s glands enriched in stem cells expressing primitive stem cell markers [57]. These duodenal submucosal glands (dSGs) cells were isolated and characterized as stem cells distinct from the known intestinal stem cells located in the crypts of the intestinal mucosa and were able to differentiate into functional hepatocytes to engage in the liver regeneration when transplanted into the livers of hepatic-injury mouse models. The identification of dSGs cells has further extended the network of endodermal stem cells that exist in the biliary tree and related organs, providing more choices of stem/progenitor cells useful for liver or pancreas repair (Fig.2 and 3).

The recent study in pigs has expanded the realization that the network of endodermal stem cells in the biliary tree and duodenum is in all mammalian species, even the pig that is missing the hepato/pancreatic common duct postnatally [54]. In pigs, the stem cells were found located in the peribiliary glands (PBGs) throughout the biliary tree, especially in the extrahepatic bile duct, the cystic duct, and the accessory duct and also in the Brunner’s glands. However, unlike in humans that have only committed progenitors within the pancreas, pigs were found to have stem cells within the pancreas in the pancreatic duct glands (PDGs) that are near to the duodenum but with committed progenitors in PDGs elsewhere [54].

2.3 Challenging of stem cell engagement in liver regeneration with new evidence from lineage tracing

Lineage tracing is a common technique used in developmental biology to study cell proliferation and to trace source cells versus descendants. Genetic engineering technology for gene targeting has provided strategies to do cell lineage tracing. Genetic markers combined with fluorescence probes enable the use of optical microscopy to track and observe cells and their descendants in vivo. A number of groups have made use of such technologies to do lineage tracing of liver cells during liver regeneration. In 2015, two cell populations were identified in mice and relevant to liver homeostasis. Font-Burgada et al. found that a small number of liver cells in the periportal region of the liver lobules can be labeled with Sox9 and were shown to play an important role in repair of liver damage [58]. Wang et al. defined a population of Axin2⁺ diploid cells, linked on their lateral borders to the endothelia of the central vein, which are regulated by Wnt signaling to proliferate, and to expand outwards (from the central vein toward the portal triads) and to differentiate into mature hepatocytes. They were able to replace about 40% of liver cells in the liver acinus within 1 year, suggesting a role in homeostasis [41]. Subsequent studies found that these are not stem cells but unipotent, committed hepatocytic progenitors that replace the terminally differentiated (dying), polyploid hepatocytes that are nearby [59].

These findings were questioned by Jan S. Tchorz’s research team using BAC-transgenic Axin2 CreERT2 mice that had normal Wnt signal strength as well as normal Axin2 expression. When induced with tamoxifen, Axin2⁺ cells were labeled with green fluorescence which could be further tracked. Surprisingly, the results indicated that parenchymal cells throughout the liver were able to proliferate and were not confined to a certain hepatic acinar zone. In the mouse model subjected also to partial hepatectomy, Axin2⁺ cells around the central vein showed no advantage with respect to proliferation compared to hepatocytes in other zones. This widely distributed proliferation pattern throughout the liver lobule was found also by Feng Chen et al., who randomly labeled different cells in the liver with adeno-associated virus (AAV); they found that there was no subpopulation of cells with stronger proliferation in the lobules of the liver; and that liver cells in different regions had different degrees of proliferation [60]. This concept of widespread proliferative ability in hepatocytes was supported also by Tomonori Matsumoto et al.’s study in which even the polyploid hepatocytes demonstrated regenerative capacity in situ and routinely underwent reductive ploidy mitoses during regenerative responses [61].

There was concern that these conflicting conclusions were obtained by tracking mice with different single cell markers, ones that might be leaky and be expressed inappropriately depending on the specifics of the experiments. It is impossible to rule out whether other unlabeled cells also contributed to liver regeneration. In 2021, Yonglong Wei et al. applied the CRISPR-Cas9 technology to integrate IRES-CreERT2 into the 3′-translated region of specific genes in different liver regions, thereby constructing 11 transgenic mice labeled with different regions of liver lobules, together with three Krt19-CreER, to specifically label biliary epithelial cells [62]. They crossed these mice with tdTomato fluorescent transgenic reporter mice, and the resulting offspring mice activated the expression of red fluorescent protein with Tamoxifen, which enabled the hepatocytes and biliary epithelial cells in each region of the liver lobule to be labeled in at least two transgenic mouse lines [62]. By simultaneously tracking the proliferation of these 14 groups of cells at different time points, they showed that under homeostasis conditions, hepatocytes in the zone 2 of the liver lobule continue to divide to replace hepatocytes in the portal triad area and central vein region. Whereas during drug-induced liver injury, hepatocytes in zone 2 were able to protect themselves from damage and replenish other damaged areas of the liver lobule under the regulation of the IGFBP2-mTOR-CCND1 signaling pathway [62].

The above detection methods can detect only cell proliferation at a certain point in time but cannot show the changes of cells during a time course. All proliferating cells are detected at the same time, so that interference by proliferation signals in non-target cells can result in confusion about what is happening. In 2021, to rule out interference from non-hepatocyte proliferative signals, Lingjuan He et al. developed a hepatocyte-specific ProTracer technique based on broad-spectrum ProTracer technology combined with the promoter of the hepatic-specific gene, albumin (Alb). This hepatocyte-specific ProTracer only labels the proliferation of hepatocytes, not only enabling hepatocyte proliferation to be demonstrated directly in the overall level of the liver organ, but also intuitively and accurately displaying the source of newborn hepatocytes. Combined with immunofluorescence co-staining of hepatocyte zonation marker genes, such as E-CAD and GS, they found that most of the newborn hepatocytes were generated from zone 2 instead of zone 1 or zone 3 (E-CAD^–GS^–, zone 2) [63].

The same ProTracer system was used also to detect the liver regeneration in combination with a variety of liver injury models such as hepatic resection, bile duct ligation, and CCl₄, and found that in the hepatectomy model, cells located in zone 1 began the liver regeneration process, and then the liver cells of zone 2 proliferated secondarily and rapidly. While, in the bile duct ligation and CCl₄ acute injury models, hepatocytes located in zone 2 initiate the liver regeneration process. This study is the first to develop a new technology that can trace the proliferation of cells in vivo over a long period of time, and researchers have discovered the source of adult liver cells with this technology, providing new ideas for liver regeneration and clinical treatment of liver diseases.

These lineage tracing investigations have caused excitement but also considerable confusion, especially because the findings from them are so dramatically different from those from past investigations. Moreover, they conflict with the empirical findings of limited hepatocyte proliferation in ex vivo studies and in transplanted hepatocytes in clinical trials of hepatocyte cell therapies [64,65] Miyajima and his associates developed a simple imaging technique to reveal the overall fine structure of the bile duct in the mouse liver [26]. Their results showed that the emergence and expansion of hepatic progenitor cells reflected the structural transformation of the intrahepatic bile duct which corresponds to the pattern of the parenchymal cell injury [26]. Whether it is plausible that this adaptive response is mediated by biliary tree stem/progenitors in the extrahepatic stem cells is a hypothesis yet to be answered. Our recent investigations of grafts of normal biliary tree stem cell organoids, able to yield both hepatic and pancreatic fates, provide proof of determined endodermal stem cells postnatally [55,56], studies discussed further below. However, there is still the need for new animal models to be able to demonstrate the engagement of these native hepato/pancreatic stem cells routinely in liver regeneration.

3 Current status of clinical applications of stem cells (and hepatocytes) in liver diseases

In acute liver injury with loss of a significant portion of the liver mass (such as the classical model of partial hepatectomy), the hepatocytes respond rapidly undergoing nuclear cell division but with most cells not completing cytokinesis and so result in increased polyploidy associated with hypertrophy of the cells. This is followed by increased apoptosis of terminally differentiated cells, located pericentrally, in parallel with continued supply of new parenchymal cells from the periportal zone [12]. Adjustment of liver size to 100% of what is required for homeostasis has been called “hepatostat.” This complex mechanism of liver regeneration promotes the cooperation between hepatocytes and non-parenchymal cells, especially mesenchymal cells, to promote the final restoration of liver homeostasis [66,67]. However, in certain chronic pathological processes (for example, those caused by viruses, metabolic disease, or the hepatotoxic drugs affecting the periportal region), persistent apoptosis and necrosis of hepatocytes exceeds the upper limit of liver regeneration capacity and leads to a wound-healing response involving non-parenchymal cells (NPCs), such as hepatic stellate cells, which deposit extracellular matrix (ECM) that leads to fibrosis and cirrhosis and eventually leads to the cessation of regeneration of the organ to the stage of end-stage live diseases.

The treatment of end-stage liver disease using hepatocyte transplantation has been a targeted treatment strategy in cell transplantation. The world’s first clinical application of hepatocyte transplantation in the treatment of liver disease occurred in 1992 [68]. By 2015, only 143 patients worldwide had received hepatocyte transplantation [69–74]. Most of these patients suffered from liver failure and metabolic liver disease and were waiting for an appropriate liver donor so they could undergo liver transplantation, the only current effective cure to end-stage liver diseases. However, hepatocytes have limited abilities to proliferate in vitro and cannot be expanded on a large scale. The efficiency of hepatocyte engraftment after transplantation is low, typically less than 20%. Although hepatocyte transplantation can offer short-term effects in some patients with genetic metabolic diseases and acute liver failure, there are serious side effects of emboli, ectopic cell delivery and immune rejection. Consequently, hepatocyte cell transplantation is no match for the success of orthotopic liver transplantation when an appropriate donor liver can be identified. It is why so few patients have been subjected to hepatocyte transplantation since its first use in 1992 [68]. For the treatment of liver failure represented by end-stage liver disease, a great deal of interest of clinical trials have resulted in research now devoted to considerations of determined stem cells present postnatally. These are the most likely to be used in clinical programs in the foreseeable future.

3.1 Mesenchymal stem cells

Among the 166 clinical trials of stem cell therapy for liver diseases registered on the website of ClinicalTrials.gov, mesenchymal stem cells (MSCs) are at the forefront of possible treatment options [69,75]. They are relatively easy to isolate, including from donors who can be the recipients and therefore enabling autologous cell therapies without immunological issues; they are easy to cryopreserve and to deliver to relevant target sites; and their effects, primarily via paracrine signaling, are relevant to many tissues [76,77]. Currently, there are more than 500 ongoing or completed MSCs-based cell therapies registered on ClinicalTrials.gov.

Mesenchymal stem cells exist in essentially all human organs, can be characterized by their antigenic traits as precursors to fat, bone and cartilage and are indicative of their capacities of immunomodulatory functions. Transplantation of mesenchymal stem cells has proved to be effective to improve patients’ liver functions [69,78]. Some studies claim that transplanted MSCs can trans-differentiate into functional hepatocytes through the action of transcription factors or small molecular compounds, but these studies remain controversial. The consensus of most investigators is that MSCs alleviate diseases primarily through their paracrine and immune regulation [76,77].

In a clinical trial conducted by Shi et al. in 2012, transfusion of umbilical cord-derived MSCs (UC-MSCs) was proved to be safe and to be efficient in decreasing the serum total bilirubin and alanine aminotransferase levels for the acute-on-chronic liver failure (ACLF) patients associated with hepatitis B virus (HBV) infection [79]. In 2021, the same group had released a long-term follow-up analysis of the open-labeled, randomized controlled study of UC-MSC infusion in 219 patients with HBV-related decompensated liver cirrhosis (DLC). Their results indicated that the UC-MSC treatment markedly improved liver functions during 48 weeks of follow-up, and with no significant side effects [80]. However, according to von Bahr et al.’s study investigating the fate of MSCs after transplantation, MSCs demonstrated limited long-term engraftments, paracrine secretions and immunomodulatory effects, with those remaining as the most commonly accepted functional mechanisms [81]. Co-grafting of MSCs with hepatic parenchymal cells or using microcarriers or biomaterials is becoming a new era of expanding the application potentials for therapies for liver failure.

3.2 Hepatic and biliary tree stem cells

In recent years, increasing numbers of studies have focused on hepatic stem cells (HpSCs), or biliary tree stem cells (BTSCs) resident in livers and in the biliary tree, including in the gallbladder of humans of essentially all donor ages [7,43,51,53]. The earliest explorations of determined stem cell transplantation for the treatment of liver disease derived from the transplantation of fetal liver cells, comprised of hepatic stem cells, hepatoblasts, and diploid hepatocytes [82–85]. As an alternative to transplantation of mature hepatocytes from adult livers, fetal liver cells became the dominant cell type in many basic studies because of their advantages of proliferation in vitro, low immunogenicity and ability to generate mature cells with optimal functions after transplantation [86,87]. However, the availability of fetal liver cells is limited, especially given the prohibitions against the use of fetal or neonatal human tissues in various countries.

Fortunately, the biliary tree is a reservoir of multiple stem cell subpopulations that are precursors for both liver and pancreas, and these populations persist postnatally into adult hosts; indeed, they can be found even in geriatric hosts [6,7,53]. Biliary tree stem cells (BTSCs) exist in the extramural peribiliary glands (PBGs) tethered to the outside of the bile duct walls of the extrahepatic and extra pancreatic biliary tree. They exist also in PBGs that are located intramurally (within the walls of the bile ducts) and with the largest numbers being in the PBGs of the hepato-pancreatic common duct; late stage BTSCs are present in the gallbladder [51]; and a possible start point for the entire network of stem/progenitors is located in Brunner’s glands in the submucosa of the duodenum. Both extramural and intramural subpopulations are found in the biliary tree throughout life, and biliary tree tissue is readily available from organ donation programs. Thus, this source of precursors can provide sufficient tissue for clinical applications. These populations are perhaps the most promising of all for future clinical programs given their expansion potential and their ability to mature into fully functional adult parenchymal cells both for the liver and for the pancreas [6,53].

In early studies, Cardinale et al. demonstrated that BTSCs can give raise to hepatocytes, pancreatic islets and cholangiocytes, findings confirmed by later investigations [43,88]. Wang et al. later conducted a systematic study on hepatic differentiation of BTSCs and revealed the key role of PI3K/Akt signaling pathway in regulating hepatocyte differentiation and maturation [89], which provides an additional strategy for obtaining functional hepatocytes from BTSCs for the treatment of liver failure.

Clinical groups in Hyderabad, India, documented that stem/progenitors more than hepatocytes were able to correct disease conditions long-term both in experimental hosts and in the long term rescue of patients with end-stage liver diseases [82–85,87,90,91]. Hundreds of patients with diverse liver conditions were treated successfully and with long-term effects with hepatic stem cell therapy that consisted of transplanting cells immunoselected for epithelial cell adhesion molecule (EpCAM⁺) from fetal liver cell suspensions. Now the potential for use of biliary tree stem cells (BTSCs) clinically has been indicated also in the first clinical trials using them [92]. Cardinale et al. compared the results of patients with chronic liver disease and treated with transplants of BTSCs versus patients treated by other standard treatments [92]. They showed that within one year after cell transplantation, the patients who received BTSC transplants showed improvement in biochemical indices and clinical symptoms during a 6-month follow-up, whereas the controls with end-stage liver diseases continued to decline. The patients given BTSC transplants continued to improve for more than 12 months and without immune response complications or immunosuppressive agents. Thus, it was confirmed that BTSCs were able to offer alleviation of disease symptoms and could do so with safe treatments in clinical applications. There is now the need for more extensive clinical trials to confirm these preliminary findings with BTSCs.

3.3 Hepatocytes derived from pluripotent stem cells (PSCs)

In recent years, many investigations have explored hepatocyte-like cells derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, hepatocytes obtained by reprogramming methods of somatic cells. Human ESCs were first isolated from the inner cell mass (ICM) of blastocysts in 1998 and demonstrated maintenance of pluripotency accompanied by unlimited proliferation and differentiation potential [93]. The standard establishment process of ESCs has been questioned as ethically controversial since achieved at the expense of destruction of embryos. To overcome this ethical challenge, in 2008, Van de Velde and others successfully extracted ESCs from fertilized eggs at the 4-cell division stage. The success of this technology avoided the ethical obstacles and opened a new prospect application of ESCs in basic research and clinical trials [94].

Human iPSCs were first established from human skin fibroblasts by Yamanaka in 2007 through use of a combination of transcription factors (OCT4, SOX2, Nanog, KLF4) that mediated somatic cell reprogramming [95]. Both iPSCs and ESCs are pluripotent, yet iPSCs can be obtained from a patient’s cells which enables use of cells genetically matched to the patient and so avoids the immune rejection issue that is a serious problem for ESCs [96,97]. Most studies have made use of fibroblasts to generate iPSCS, but these are cells derived from mesoderm; differentiation of them to an endodermal fate, requiring crossing germ layer boundaries, has proved of limited, if any at all, success. Even with efforts for optimizing directed differentiation of iPSCs has yielded results that are distinct with each round of reprogramming [98]. Thus, it is difficult to achieve the desired results reproducibly. Clinical trials using patient-specific induced pluripotency stem cells (PSiPS) derived from cell cultures from skin biopsies of patients for the hepatic disorders and eye disorders from Royan Institute,Tehran, Iran were completed in 2010, yet no results were released to enable further evaluation of PSiPS in liver diseases (Tab.1, NCT00953693).

Through use of combinations of growth factors and specific differentiation methods, it has been possible to generate hepatocytes from PSCs [99,100]; the resulting adult cells have some, but not all traits of hepatocytes and all proved to be immature. Only a subset of the hepatocyte-specific proteins were secreted, and the CYP450 enzyme activity is much weaker in PSCs-derived hepatocytes versus that in normal, freshly isolated hepatocytes. In addition, long-term cultures make hESCs prone to gene mutation or heterologous gene pollution [101] and their potential tumorigenicity remains an unavoidable risk during application. Therefore, to date there are no hESC-derived hepatocytes that have been used in clinical trials. Instead, these models are used for the establishment of disease models and for drug screening [102].

Recent efforts have reworked the strategies to use biopsies of an endodermal tissue, such as intestine, to generate iPSCs more suited to yield endodermal fates [103]. It was found that reprogramming of normal endodermal cells to endodermal iPSCs with small molecules, required fewer factors, and the differentiation of the resultant endodermal iPSCs to an adult fate is easier, more reproducible and results in more functional adult cells with much lower risks of oncogenic events [103]. Thus, the potential for use of PSCs with efficiency and reproducibility has now been achieved. Still, these endodermal iPSCs have yet to be tested for efficacy in candidate clinical programs.

3.4 Hepatocyte-like cells from reprogrammed fibroblasts or reprogrammed hepatocytes

To avoid the problems of low efficiency and incomplete functions of hepatocytes derived from pluripotent stem cells, investigators have reprogrammed murine and human fibroblasts into functional hepatocyte-like cells by transfection of hepatocyte-specific factors (GATA4, HNF1 and FOXA3), which enabled reprogramming of a patient’s fibroblasts into hepatocyte-like cells with much higher efficiency than from PSCs from mesenchymal cells [104,105]. These studies were later confirmed by other teams but with a different combination of two of the four transcription factors (HNF4a, FOXA1, FOXA2 or FOXA3) that reprogram mouse embryonic and adult fibroblasts into functional hepatocyte-like cells (iHep) [106].

To further overcome the expansion limitation of hepatocytes and using a similar reprogramming strategy, Yu et al. obtained hepatic stem cell-like cells that can be differentiated into mature hepatocytes and cholangiocytes. They reprogrammed murine fibroblasts with HNF1β and FOXA3 [107]. The hepatic stem cell-like cells demonstrated considerable expansion potential in vitro. Due to the complexity of the genetic manipulation and the safety of gene transfection, the reprogramming strategy has been replaced by one involving use of small molecular compounds. The small molecules used were a ROCK inhibitor of the ROCK signaling pathway, an inhibitor of the TGFβ/BMP4/SMAD signaling pathway, and inhibitors of GSK3β as activator of the Wnt signaling pathway.

Thereafter, several groups have used different combinations of these small molecules to reprogram mouse and human hepatocytes into hepatic progenitor cells, that can be expanded extensively on a large scale and then can be re-differentiated into mature hepatocytes in vivo and in vitro [108–110]. Others used those small molecules in combination with growth factors such as Wnt3a, Noggin, EGF, etc. to reprogram mouse and human fibroblasts, crossing the germ layer boundaries, to obtain hepatocyte-like cells [111]. The hepatocyte-like cells could be expanded over 10 000 times and once differentiated, expressed functions paralleling those of freshly isolated hepatocytes.

So far, the application of these hepatocyte-like cells derived from reprogrammed and expanded cells has been used for drug screening or for bioartificial livers for functional support of patients who are on the waiting list to be transplanted [112,113]. In Zhang’s study, using a hypoxia culture system, human hepatocyte-like cells were able to proliferate continuously for months. By establishing 3D cultures in a bioartificial liver system, they were able to further prolong and promote the functions of these hepatocytes [114]. The several versions and strategies of reprogrammed and expandable cells used to generate hepatocyte-like cells have demonstrated already their potential for use in drug screening, both in regular culture formats and in bioreactors enabling avoidance of the liver injury murine models.

There are recent studies from Wang et al. that indicated that the long-term, cultured hepatic progenitor cells reprogrammed from primary hepatocytes with a combination of small molecules express dedifferentiation-associated inflammatory factors (DAIF) and long-term in vitro expansion, which caused the loss of hepatocyte differentiation ability [115]. The 3D cultures can reduce DAIF expression enabling hepatic progenitor cells to give rise to functional hepatocytes. To uncover the underlying mechanism of hepatic differentiation from stem cells, we did bioinformatic studies to use the short time-series expression miner (STEM) analysis which indicates signaling pathways that include the PI3K/AKT signal pathway regulation that is important for the maturation of stem cells [89].

3.5 Liver organoids

Organoids are cell aggregates formed by epithelia and mesenchymal cells which have ultimate potential of mimicking structures and functions of organs. Protocols of generating of liver organoids from human pluripotent stem cells (hPSCs), or from Lgr5⁺ hepatic stem cell populations were established based on the mixture of extracellular matrix, growth factors, hormones or small molecule compounds [116,117]. Such systems were originally established for constructing gastric epithelial organoids, and later were adapted to generating organoids from healthy liver tissues, biliary trees [118] or even hepatocellular carcinomas. Compared to monolayer culture-based hepatocytes, liver organoids are more ideal tools for disease modeling and are holding great promise for studies on liver regeneration [31]. However, unlike the intestinal organoids that have spontaneous maturational features during expansion and in vitro maintenance due in part to the conditions used for them, the conditions used for hepatic organoids maintain their stem/progenitor characteristics [56]. These organoids do not mature into functional “mini livers” without adjusting the culture conditions to ones promoting hepatic maturation.

Most studies on hepatic organoids involve use of matrigel or other tumor-derived extracellular matrix-based support systems, conditions that obviate any use of them clinically for treating patients with end-stage liver diseases. In fact, EpCAM⁺ organoids generated from the matrigel system, which presents as a single epithelial holo-structure, are hepatic spheroids that lack mesenchymal cell companions; it is the main reason for their difficulties in maintenance and maturation. In early studies, a “mesenchymal-epithelia” partnership in liver parenchymal cells was established [119]. Angioblasts, progenitors for stellate cells and sinusoidal endothelial cells, play a crucial role in the maintenance of hepatic stem cells under serum free conditions in vitro [55,56]. The maturation of hepatic stem cells to hepatocytes is paralleled by the maturation of angioblasts to mature mesenchymal cells, resulting in maturation-dependent changes in the paracrine signaling, enabling the consistent functionality of hepatocytes.

Organoids can be prepared from biliary tree stem cells (BTSCs) under conditions supportive of stemness traits and that include BTSCs and their natural partners, early lineage stage mesenchymal cells (ELSMCs), precursors to endothelia and to stellate cells. These BTSCs/ELSMCs organoids showed a greater advantage for transplantation and for donor cell maturation due to the recreation of relevant paracrine signaling in vitro [56]. There is greater hope for clinical programs using these biliary tree stem/progenitor organoids and with the only matrix component used being hyaluronan hydrogels. These are able to be transplanted in large numbers ( > 10⁸ organoids per patch).

4 Strategies for delivering cells into solid organs

4.1 Cell transplantation and grafting strategy

Cell transplantation is the crucial step for cell therapies and in the past consisted either of direct injection into the target organ or delivery via the organ’s vascular supply. Whereas hemopoietic cells have evolved to be deliverable via the vasculature and bind into target sites by mechanisms causing activation of relevant adhesion mechanisms, hepatic cells are distinct and, similar to epithelia from solid organs, and must ideally be delivered by grafting mechanisms [120]. Delivery by vascular routes result in poor engraftment efficiency and complications such as emboli formation. Direct injections into the liver or under the Glisson’s capsule have been tried, albeit rarely, since the donor cells remain as a clump; they are slow to integrate amidst the host cells; and are slow to become vascularized, resulting in poor efficiency of engraftment. Also, direct injection transplantation has the limitations of single dose injection volume and injection quantity. Moreover, the mechanical effects caused by injection cause risks to the liver capsule, such as hemorrhaging and for damage to the liver structure. Therefore, this transplantation method is especially not suitable for patients with fibrotic and cirrhotic liver diseases and who are prone to liver bleeding (Fig.5).

Vascular infusion transplantation, with its feasibility and low surgical trauma, has been used for hepatocyte and stem cell transplantation into liver. Most commonly, this is achieved by splenic transplantation resulting in cells delivered into the liver via the portal vasculature. There are problems, such as the retention of cells in the splenic sinuses, and the induction of immune tolerance. The intrahepatic engraftment efficiency of hepatocytes is low (typically ~20% for hepatocytes; ~5% for stem cells), and there are risks of vascular emboli and of ectopic delivery of cells to lymphatic tissues and to other organs. All pose significant safety risks and have resulted in few efforts to use these approaches for clinical programs. Interestingly, Nevi and associates’ method of using stem cells precoated with hyaluronans, glycosaminoglycans with repeating disaccharide chains of N-acetyl-glucosamine and glucuronic acid, the precoating was proved to be not only feasible to increase the engraftment efficiency in vascular delivery, but also during the injection grafting, the number of cells retained at injection sites was significantly improved [121]. The functional parameters of hepatocytes differentiated from these hyaluronan-coated hepatic stem cells also indicated a significantly higher albumin secretion than that of the control group (11% vs. 3%) (Fig.5).

Cell-sheet engineering technology involves use of sheets of cells, bound together by their gap junctions and cell–cell adhesion molecules, prepared on thermolabile dishes, and triggered to detach from the dish so as to be transferrable to the surface of the liver. The bioengineering transplantation technology based on thermal-sensitive matrix material coated with poly (n-isopropylacrylamide) (PIPAAm) was first developed by Takezawa et al. [122,123]. This technology has been applied to the treatment of various damaged tissues such as articular cartilage, bone, periodontal ligament, cornea, blood vessels and myocardium [124]. However, the number of transplanted cells is limited, and the cells do not engraft into the tissue; they only remain at its surface. Therefore, the potential for tissue sheet technology is restricted to situations in which the sheet can provide a requisite function(s) even if the numbers of cells are few (Fig.5).

Patch grafting is the newest of the methods for transplantation into solid organs [55,56]. The cells most easily transplanted by this method are stem/progenitors that can be prepared as organoids, floating aggregates of the epithelial stem cells partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and to stellate cells. The organoids are prepared in a serum-free medium, such as Kubota’s medium, that is devoid of growth factors and hormones other than insulin and transferrin/Fe, and then embedded in a soft layer of a hyaluronan hydrogel, with a rigidity of less than 100 Pa. This hydrogel layer is placed on top of a backing enabling the graft to be tethered to the target organ.

Candidate backings include omentum, amnion-derived matrix, or a generic form of backing is to use more rigid hyaluronan hydrogel (~700 Pa) in a silk mesh and then tethering the silk to the surface of an organ or tissue. The silk is no longer available, but fortunately the amnion-derived matrix is available from multiple commercial sources and has proved the most acceptable for clinical grafting programs. It has not yet been used with the hyaluronan grafts containing organoids but is hypothesized to work readily with them. The conditions enable the organoids to express multiple matrix metalloproteinases (MMPs), especially secreted isoforms, that facilitate the engraftment of the organoids into the organs and are able to integrate uniformly amidst the host cells in a matter of days (a week or less). The patch grafting strategy has proven successful for multiple types of tissues including both liver and pancreas [55,56]. After engraftment, the organs clear the hyaluronans leaving the engrafted cells to be regulated by the synergistic effects of organ-specific soluble signals and extracellular matrix components to mature into adult fates. The engrafted cells transplanted by patch grafting have been found able to alleviate hepatic and pancreatic diseases (Fig.5).

Grafts of mature hepatocytes associated with mature endothelia do not engraft using patch grafting, since the mature cells express plasma membrane-associated, but negligible amounts of secreted MMPs, factors that are required for engraftment. Transplantation of hepatocytes was shown to be achievable by co-transplanting the hepatocytes with fetal tissue-derived mesenchymal stem cells, a source of at least some of the secreted MMPs. Engraftment was achieved but was significantly less than that for organoids of stem/progenitors that produce more of the secreted isoforms of MMPs. Patch grafting strategies are expected in the near future to transition into clinical programs of cell therapies for multiple types of solid organs including liver.

4.2 Biomaterials used for the grafting of stem cells into the liver

Hyaluronan (HA) is a non-sulfated glycosaminoglycan found in the extracellular matrices of stem cell niches in vivo, which has requisite physicochemical properties and cell biological functions [125–127]. The physicochemical property of HA provides a stable yet ideal mechanical force for the maintenance of microenvironment of the stem cells, while its biological effect is related to a group of HA receptors, CD44 and RHAMM, that play an important role in cell proliferation or cell fate determination. HAs have been widely used in transplantation of cells or delivery of molecules in clinical programs in soluble form or as hydrogels [126–129]. In the study conducted by Nevi et al., coating hBTSCs with soluble hyaluronans proved to improve significantly the engraftment of hBTSCs in the liver injury mouse model compared to controls [121]. In patch grafting strategies, we found that the HA hydrogels generated from thiol-modified HA crosslinked by polyethylene glycol diacrylate (PEGDA) proved to be ideal for their precise biochemical and mechanical properties [55,56].

The stability and elasticity of HA hydrogels are critical not only to allow key soluble signals in body fluid to enter the graft, but also to minimize donor cell maturation by providing the stem cells a soft mechanical support and, in parallel, offer conditions supportive of production of both membrane-associated and secreted forms of MMPs [125]. This ability to precisely control the viscoelasticity properties of the HA is central to the success of patch grafting strategies. The soft HA hydrogel was used to embed the stem cell organoids and keep them immature; the stiff HA hydrogels used in the backing enabled one to block cells migrating in inappropriate directions, since the cells confronted with the stiff gels matured sufficiently to mute MMP production; and the HA hydrogel used to coat the outside of the grafts minimized adhesions on the surface of the patch grafts. Thus, the HA prepared in serum-free Kubota’s medium was useful for all three grafting needs simply by tuning the concentration of HA and PEGDA and the mixing ratios to achieve necessary rigidity properties.

The first layer, that of the soft HA hydrogels, the one used for embedding the stem/progenitor cell organoids proved the most important layer for engraftment, due to maintenance of stem cell traits and of expression of multiple MMPs, was dependent on HA hydrogels with a viscoelasticity (G*) less than 100 pascals [55,56]. Of note, hyaluronan receptors were found both on the surface of donor hBTSCs and the hepatic sinusoidal endothelial cells of the host liver. The HA receptor CD44 is one of the hBTSCs markers in early studies, while receptors for HA mediated motility (RHAMM) and LYVE-1 (lymphatic vessel endothelial receptor-1) were demonstrated on the surface of hepatic sinusoidal endothelial cells. Hepatic sinusoidal endothelial cells are known to be largely responsible for the uptake and catabolism of plasma hyaluronan, which allows liver to be one of the primary organs that manages the natural biodegradation of hyaluronans [130]. This biodegradation of HA in the liver might also prompt engraftment of stem cells [121]. Once the HA components in the graft and in the liver are cleared, engrafted hBTSCs receive organ-specific differentiation signals (matrix components and soluble signals) needed to proliferate and to differentiate donor cells into mature hepatocytes and cholangiocytes in the liver (or in the pancreas, to mature pancreatic islet cells or acinar cells) to enable repair of organ dysfunctions.

4.3 Cell engraftment /implantation mechanisms

Several studies are dedicated to the process of hepatocyte engraftment in vivo and the possible signal pathways involved, proving that hepatocytes can home to the damaged areas, by mechanisms paralleling those of the well-known “homing” process of hematopoietic stem cells. For hematopoietic stem cells, the transplantation effect depends on their activity, implantation rate and proliferation ability in vivo. The key factor of homing and colonization after hematopoietic stem cell transplantation and hematopoietic stem cell migration is CXC chemokine ligand 12/stromal cell-derived factor 1α (SDF-1α). The mechanism of hematopoietic stem cell migration activates the expression of CXCL12/CXCR4, then activates p38 MAPK signaling pathway and upregulates the expression levels of MMP-2 and MMP-9 of the matrix metalloproteinase (MMP) family [131,132]. In the liver, transplanted cells, delivered by a vascular route, need to bypass the sinusoidal endothelial cell barrier when entering the liver plate, and the integration of transplanted cells in the liver parenchyma needs to reconstruct the plasma membrane structure and the integrity of liver sinusoidal endothelial cells (LSECs).

Liver sinusoidal endothelial cells (LSECs) are the major liver non-parenchymal cells, accounting for about 70% of the total number of the liver non-parenchymal cells and 15% of the total liver cells [133]. Hepatic sinusoidal endothelial cells are highly specialized endothelial cells that form a continuous layer with the parenchymal cells periportally to pericentrally. However, in humans, whereas the layer of endothelia is continuous periportally in zone 1, it become fenestrated (forms “windows”) constituting gaps in the connections between endothelial cells in zones 2 and 3, such that there is direct contact of blood with the hepatocytes in those gaps. The regions of the sinusoidal plates that are rich in fenestra are an open channel for solute exchange between the blood and the hepatocytes. In the gaps, there is a lack of the extracellular matrix components from the non-parenchymal cells and that contribute to paracrine signaling between hepatocytes and endothelia, and instead there are only the matrix components generated by the hepatocytes. LSECs can regulate material exchange by the parenchyma cells within the hepatic sinusoids by changing the diameter of the gaps and the number of the fenestrae, and so making them an especially permeable endothelial cell layer in mammals [134]. Meanwhile, LSECs act as a physical barrier between hepatocytes and blood in the liver. Amidst those fenestrae are also hepatic stellate cells and Kupffer cells that are essential for the formation and maintenance of the hepatic lobule [135].

In addition, LSECs can release HGF (hepatocyte growth factor) stimulating the proliferation of hepatic parenchymal cells, as well as promoting the repair of the liver after partial hepatectomy [136,137]. Ding et al. confirmed that in the early stage of post 70% hepatectomy, LSECs increased the expression of vascular endothelial growth factor receptor (VEGFr) to inhibit the transcription of the inhibitor of differentiation 1 (ID1) by reducing the expression level of angiotensin II, thus releasing Wnt2 and HGF and promoting the proliferation of hepatocytes [137]. This mechanism enables the liver to complete full regeneration after partial hepatectomy (Fig.6).

The onset of sinusoidal ischemia and reperfusion may activate Kupffer cells and endothelial cells in the liver. Hypoxia combined with monocyte activation during the clearance of non-viable hepatocytes can release adhesion molecules, such as intercellular adhesion molecules (ICAMs) or vascular cell adhesion molecules (VCAMs), promoting the anchoring of transplanted cells and endothelium, and providing the first link to promote the survival of transplanted cells in the liver [138]. In fact, among all the endothelial cells, LSECs uniquely express hyaluronan receptors’ lymphatic vessel hyaluronan receptor-1 (Lyve-1), and Stabilin-2, which not only makes LSECs the main cell types in the liver to clear blood-borne macromolecules and nanoparticles, including hyaluronans, but also promotes the engraftment of hyaluronan-coated cells during transplantation [139,140] (Fig.6).

To allow the transplanted cells to enter the liver plates, endothelial cells need to provide space for cells to reach the perisinusoidal space. Cytokines such as interleukins and tumor necrosis factor-α affect endothelial cells during this procedure. Endothelial cell damage may involve the release of free radicals mediated by oxidase activity and the induction of oxidative stress in Kupffer cells and endothelial cells [141]. In He et al.’s study of the establishment of a humanized liver in chimeric mice, it was found that hepatocytes modified by the FoxM1 gene can significantly enhance their ability to regenerate the liver, and the secretion of IGF2 by aging hepatocytes might promote the transplanted hepatocytes [142,143] (Fig.6). In summary, cytokines produced by various cell types in the liver participate in the engraftment of transplanted cells, which involves a more complicated network of cells and factors influencing integration of cells as compared to the events in “homing” of hematopoietic stem cells. By developing a three-dimensional (3D) live-cell tracing system, studies could reveal responses of the donor cells post-transplantation in the transplanted liver failure mouse model and eventually in human patients in the future [144–146].

4.4 Challenges of cell grafting/transplantation of the diseased liver

In the clinical scenario, candidate stem cells or mature cells often are transplanted to the patients with end-stage diseases, where the microenvironment of the liver is fibrotic and/or with inflammation. What has been found to work in the models of metabolic disorders such as type-I tyrosinemia might not work for the majority of patients. The non-parenchymal cells in the liver, which maintain the microenvironment of the liver and responses to the liver injury constantly, have required more and more attention in liver regeneration. Treating hepatic stellate cells (HSCs) to prevent the transformation of HSCs to myofibroblasts [147], or treating fibrosis patients with macrophages to improve the microenvironment of the liver before transplantation has brought hopes of expanding the stem cell-based treatment for patients with end-stage liver diseases [148].

HSCs, also called Ito cells, are nonparenchymal cells (NPCs) that store vitamin A and retinol esters in lipid droplets throughout the cells [149]. Precursors to hepatic stellate cells are found in or near the canals of Hering [23]. They provide paracrine signaling relevant to the hepatic stem cells, hepatoblasts and hepatic committed progenitors [23]. Their descendants, mature hepatic stellate cells (HSCs), are star-shaped cells located in the perisinusoidal space formed between hepatocytes and sinusoidal endothelial cells in the space of Disse [150] (Fig.1 and Fig.6).

In healthy livers, HSCs maintain a quiescent state, constitute 4%–8% of the liver cells, and express an extracellular matrix protein, reelin, that distinguishes them from myofibroblasts [151]. Upon liver injury, HSCs become activated and highly proliferative, and produce excess extracellular matrix components associated with fibrosis and cirrhosis. There is increased expression of type I collagen and α-smooth muscle actin, as well as the secretion of connective tissue growth factor (CTGF), transforming growth factor-β (TGFβ) and other fibrogenic factors [152].

The activation of HSCs involves signals from parenchymal and nonparenchymal cells. Representative are those from hepatocytes (TGFβ, platelet-derived growth factor β (PDGF-β), vascular endothelial growth factor (VEGF), IGF1, ROS); cholangiocytes (IL-6, TGFβ, endothelin-1); sinusoidal endothelial cells (FGF1, CXCR4); platelets (PDGF-β, CXCL4, etc.); and macrophages (TGFβ, FGF2, IGFBP5) [153]. In the early stages of chronic liver injury, HSCs can inhibit the progress of liver injury and facilitate tissue regeneration by promoting deposition of a form of extracellular matrix (ECM) and secretion of HGF. However, if chronic injuries occur, the fibrogenic process can lead to fibrotic livers that are negatively influenced in their functions (Fig.7 and 7B).

Macrophages are posited to be a key cell type in innate immunity, also playing a major role in maintaining tissue homeostasis, and are involved in the initiation, progression, and termination of liver diseases. Among all solid organs, liver contains the highest percentage of macrophages [154]. In healthy rodent livers, every 100 hepatocytes are accompanied by 20–40 Kupffer cells [155]. There are primarily two sources of hepatic macrophages: (1) liver-resident macrophages, also called Kupffer cells, first discovered and named by pathologist Karl Wilhelm von Kupffer; and (2) monocyte-derived macrophages, originating from the bone-marrow [156]. Known murine Kupffer cell markers include F4/80, CD11b, CD68, CD14, TLR4, CX3CR, etc. [157,158]. By contrast, human Kupffer cells are less well-characterized but can be identified by several markers that include CD14 and CD68 [156,159]. Kupffer cells are difficult to distinguish from other phagocytes due to the overlap in their expression of surface markers. CX3CR1 is a primary tool to differentiate Kupffer cells and monocyte-derived macrophages [160]. Additional studies of surface markers are needed to establish more accurate subtyping of human macrophages.

The core functions of Kupffer cells are continuously to monitor infectious and non-infectious factors in the liver and to modulate corresponding immune responses. Immune responses by Kupffer cells are categorized into two main subtypes: the danger-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). It is generally believed that the responses of Kupffer cells are mainly related to liver injury. Typically, the endogenous danger signals released by host cells primarily activate DAMPs, while exogenous stress mainly activate PAMPs. In both humans and mice, Kupffer cells play major roles in the maintenance of liver homeostasis. However, bone marrow-derived macrophages contribute less. Instead, those are more activated under the injury conditions [161]. During liver injury, Kupffer cells will migrate to inflammatory areas and to sites of dead or dying hepatocytes, such as sites of apoptosis [162]. On the other hand, bone marrow-derived macrophages can selectively aggregate in inflammatory sites mediated by chemokines to supplement the loss of Kupffer cells, and to respond to injuries through a synergistic process with Kupffer cells [163–165] (Fig.7).

5 Discussion

The clarification of the cellular events under different physiological and pathological states provides a favorable scientific basis for the treatment of liver failure. The hope for the future is to establish strategies for cell transplantation to correct various disease states in the liver and to overcome the extreme limitation of the numbers of qualified donor livers for whole liver transplantation. We have summarized the candidate cells that might be used in cell transplantation and have found that (1) those from pluripotent stem cell sources, both ES cells and the several forms of iPS cells, are useful for drug testing and for industry but are not candidates for clinical programs; (2) mesenchymal stem cells can be used clinically for their paracrine signaling effects but are not optimal given their inability to transdifferentiate into parenchymal cells; and (3) determined stem cell subpopulations in the liver, and especially in the biliary tree, are the most promising subpopulations for use in the future for clinical programs.

The methods for delivery of the cells have, in the past, made use of inefficient and ineffective strategies such as direct injection and vascular delivery; both methods are prone to serious and debilitating side effects such as emboli and ectopic delivery of the cells. Grafting methods are now available and, with further study and optimization, should offer routes for efficient and effective transplantation of organoids of stem/progenitors or transplantation of hepatocytes if partnered with a cellular source of MMPs into the liver for alleviation or cure of liver diseases. The powerful promise of the grafting strategies must be tempered by the realizations that advanced liver diseases involve cirrhotic and fibrotic environments that are likely to mute facets of the grafting process. Many additional investigations are required to optimize the grafting process under such conditions.

We did not expand on issues related to cellular immunity. Immunosuppressive treatments are required for orthotopic liver transplantation due to the allogeneic differences between donor and recipient. The hepatic stem cells and biliary tree stem cells have been reported as having low immunogenicity both in experimental systems and in humans [53,69]. However, it is unclear whether the transplanted cells will activate an immune rejection when they differentiate and mature into hepatocytes and cholangiocytes after transplantation. The immune microenvironment modification of transplanted organs has gradually attracted attention in the field. Miyajima et al. found that macrophages reduce liver scar formation by producing matrix metalloproteinases in mice with acute and chronic liver injury fibrosis, and they recruit host immune cells, such as neutrophils and monocytes, to the injury site to strengthen the injury repair mechanisms and reduce liver fibrosis [166], so as to provide a better colonization environment for transplanted cells.

Also, the work of liver zonation summarized in Section “Stem cells in the liver regeneration—progress and challenges” provides tools or methods to predict zonation traits for the study of liver regeneration. Most studies are based on the liver zonation methods as reported by Halpern et al., which simply calculates the correlation between cell populations and representative zone-specific genes to infer the zonation of cell clusters [167]. Spatial transcriptome is not used currently in the field of liver regeneration. Researchers are keen to use single-cell RNA-seq to explore changes in hepatocytes during liver regeneration, and single-cell ATAC-seq can be used to study the transcriptional regulation mechanisms. Unlike lineage tracing, most single-cell studies do not focus on hepatocyte zonation, and only provide a simple description of the zone characteristics of regenerated cells. Moreover, recent studies have not reported specific hepatocyte subpopulations, but have focused on cell plasticity during liver regeneration. For example, Chen et al. used scRNA-Seq and scATAC-seq to study the functional diversification of hepatocytes during liver regeneration. Some cells were found to have acquired chromatin landscapes and transcriptomes of fetal hepatocytes [168]. In a similar study, Chembazhi et al. based on studies of partial hepatectomy (PHx) found that a subset of hepatocytes in liver regeneration instantaneously reactivate similar early postnatal gene expression programs to proliferate, while a different group of metabolically overactive cells appears to compensate for any temporary deficiencies in liver function [169]. In all those gene sequencing studies, the terminologies of hepatocytes, fetal hepatocytes and hepatic stem cells/progenitors were vague, so that verifications of the conclusions based on the bioinformatic analysis still require further validation with standard, carefully designed experiments.

Finally, it is worth mentioning that liver injury repair requires not only replenishment of functional hepatocytes and cholangiocytes but also of non-parenchymal cells. Chen et al. revealed in experimental studies that combined transplantation of human mesenchymal stem cells and rat hepatocytes is superior to single transplantation of parenchymal cells in liver function repair and liver tissue repair. Mesenchymal stem cells can protect hepatocytes from inflammatory damage [170]. Joshi et al. used the co-transplantation of fetal hepatocytes and mesenchymal stem cells to effectively improve the implantation of fetal hepatocytes in the inverted Senecio alkali injury model mice [171]. The strategy of co-transplantation of hepatic parenchymal cells with immunomodulatory mesenchymal stem cells or immune cells has shown a much more promising future in the clinical aspects. It is possible that the use of organoids comprised of both epithelial stem cells and their mesenchymal stem cell partners might prove an optimal route by which to deliver both the cells required for the parenchymal and non-parenchymal cell lineages.

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