Stem cell pools exist in continuously renewing tissues of the body such as bone marrow, skin, and intestinal mucosal epithelium, which generate mature cells through the uninterrupted proliferation and differentiation of stem cells. In the liver, the existence of stem cells in liver tissue has been a long-standing controversial issue because mature hepatocytes still have the ability to divide and proliferate. In recent years, with the rapid development of developmental biology, especially stem cell biology, the existence of hepatic stem cells and their significance in the development of liver diseases have received more and more attention. A variety of pathophysiological processes in the liver may be closely related to the abnormal proliferation and differentiation of stem cells; and the in-depth study of hepatic stem cells has shown attractive prospects for the treatment of various acute and chronic liver diseases as well as hepatocellular carcinoma. The understanding of liver stem cells and related research around this special stem cell has become one of the research hotspots in modern stem cell biology and clinical medicine. Zhang Zhigao, Department of Gastroenterology, General Hospital of Jinan Military Region
1 Concept of hepatic stem cells
1.1 Embryonic liver stem cells Early in human embryonic development, the liver diverticulum at the end of the foregut evolves into two parts, the liver and the tail, of which the liver grows extremely rapidly and fills the large part of the abdominal cavity by the 5th week of embryonic life [1]. These embryonic-like hepatocytes evolving towards the liver are called hepatoblasts, which not only have a vigorous proliferative capacity, but also have the potential to differentiate into hepatocytes and bile duct cells [2], so hepatoblasts are actually embryonic liver stem cells. Only the foregut terminal can be induced into hepatocytes during embryonic development. four families of transcription factors play important roles in the expression of specific differentiation genes in hepatocytes: (i) various hepatocyte nuclear factor 1 (HNF1) with the same structural domain; (ii) a series of hepatocyte nuclear factor 3 proteins (HNF3); (iii) a nuclear receptor superfamily. (iv) leucine zipper family (C/EBP). The target cells are regulated by the above transcription factors to produce phenotypic changes through the expression of specific genes.
The cellular markers of embryonic liver stem cells include differentiation markers such as albumin (ALB), alpha-fetoprotein (AFP) and a series of cytokeratins (CK7, 8, 9, 14, 18, 19, 20), related stem cell factors and receptors such as Thy1, Flt3, SCF/ckit, OV6, and myeloid-derived stem cell markers such as CD34 (Table 1). et al [3] successfully isolated and cultured embryonic liver stem cells based on the above cellular markers by flow cytometric sorting (FACS), and the isolated embryonic liver stem cells could differentiate into hepatocytes and bile duct epithelial cells when transplanted into the liver of homologous animals, suggesting that embryonic liver stem cells do have multidirectional differentiation potential.
1.2 Adult liver stem cells The presence of stem cells in the normal liver is controversial. Some scholars have suggested that the liver itself is a “stem cell pool” based on the fact that mature hepatocytes can regain their proliferative capacity uninterruptedly after liver injury. However, according to the definition of stem cells, in addition to the ability to divide and proliferate, stem cells should also have certain differentiation potential. The progeny of mature hepatocytes that divide are still mature hepatocytes, so in a strict sense, mature hepatocytes should not be considered as stem cells.
It is now believed that epithelial cells with bidirectional differentiation potential exist in the terminal region of the biliary tree, corresponding to the histological canals of Hering, and may be “nondescript” mesenchymal cells of much smaller size originating from the perichondrium of the bile ducts. In the presence of severe liver damage and/or impaired proliferation of mature hepatocytes, they can activate and proliferate abnormally, appearing in large numbers in the peripheral areas of the hepatic lobules and histologically appearing as small, proliferating populations of cells around the lobules. These cells have a large nucleoplasmic ratio and round or ovoid nuclei and are called hepatic oval cells (HOC). The proliferating oval cells migrate along the liver parenchyma to the central region of the liver lobules and differentiate into mature hepatocytes to repair and rebuild the liver. At the same time, oval cells can also differentiate into bile duct epithelial cells and participate in the formation of intrahepatic bile ducts. It is now generally accepted that hepatic oval cells are the stem cells within the adult liver tissue. Another important evidence that oval cells are considered as liver stem cells is that they share similar cellular markers with embryonic liver stem cells, i.e. adult hepatocytes, and share some surface antigens with hematopoietic stem cells (Table 1).
There are two prerequisites for the induction of massive proliferation of adult liver oocytes: first, the presence of factors that stimulate liver tissue regeneration, which is commonly used in animal models where most hepatectomies or toxicants (e.g., carbon tetrachloride) trigger hepatocyte necrosis; and second, inhibition of normal hepatocyte proliferation, and the drugs used in animal experiments to inhibit the division of mature hepatocytes are 2acetylaminofluorene ( 2acetylaminofluorene (2AAF), fumonisin B1 (FB1), retrorsine or cholinedeficient ethionine containing (CDE) diets, etc.
Stem cells do not perform the function of differentiated cells; their function is to produce mature differentiated cells by proliferation. Stem cells divide to produce progeny that can only choose one of two pathways: either they maintain their parental characteristics and remain stem cells, or they follow a specific pathway toward terminal differentiation. Table 1 Cellular markers of adult hepatocytes, oval cells, hepatocytes, bile duct epithelial cells and hepatocellular carcinoma cells Note: +: positive; -: negative; ? : not yet determined 1.3 Multi-origin and plasticity of liver stem cells In recent years, the discovery of multipotent adult progenitor cells (MAPCs) in adult individuals has been particularly exciting in the field of stem cell research. Liver stem cells can be derived from the liver itself, but human and animal studies have demonstrated that non-liver-derived stem cells can differentiate into liver stem cells and even mature hepatocytes. Isolated bone marrow multipotential progenitor cells can be induced to generate phenotypically specific hepatic stem cells and hepatocytes by adding specific growth factors during in vitro culture [4]. The discovery of hepatocytes with donor genetic markers in the liver tissue of patients and animal models undergoing bone marrow transplantation has been confirmed by several studies. Horizontal differentiation of different types of intertissue stem cells has also been reported, for example, Dabeva (1997) found that epithelial cells isolated from rat pancreatic tissue and transplanted into the liver of inbred rats could also differentiate into hepatocytes, which could integrate into hepatic lobular structures and express hepatocyte-specific proteins. It is not clear in what capacity bone marrow-derived progenitor cells travel through the bloodstream to the liver. Research data suggest that the main cellular components of the bone marrow are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial cell progenitors. Current ex vivo experiments have confirmed that hematopoietic stem cells (HSCs) [5] and bone marrow MSCs (MSCs) [6] can differentiate into hepatocytes. Liver oocytes carry bone marrow stem cell markers such as CD34, Thy1, Fly3, and SCF/ckit, which do not yet indicate some necessary link with hematopoietic stem cells. It is not clear whether multipotent progenitor cells in the bone marrow have acquired the ability of transdifferentiation in a different microenvironment or whether they are themselves more primitive multidifferentiated potential progenitors.
Hepatic oocytes are plastic. Oocytes can differentiate into gastrointestinal epithelial cells as well as pancreatic cells with endocrine functions [7]; in brain tissue, oocytes can differentiate into neural cells and glial cells [8]; in the heart, liver stem cells can differentiate into cardiac myocytes [9].Asakura [10] found that stem cells from several organs, including the liver, can differentiate into bone marrow hematopoietic stem cells, and Murase [11] was able to reconstitute bone marrow hematopoiesis after liver transplantation alone in lethally irradiated rats, with exactly the same salvage effect as bone marrow transplantation. In patients treated with liver transplantation, B-cell lymphoma found in the subsequent transplanted liver has been reported to be of donor origin (tumor cells with genetic markers of the donor) [12]; the University of Hong Kong reported that mesenchymal sarcoma found in the transplanted liver of a similar patient was also of donor origin [13].Smith [14] et al. graftversushost disease (GVHD), a large number of immunologically active donor-derived lymphocytes can be found chimerically in the host. The cellular origin of these abnormally proliferating lymphocytes and mesenchymal cells from the donor liver may be twofold: either they are hematopoietic stem cells that originate from the residual blood/hematopoietic stem cells in the donor liver, as some of them remain after the donor liver is isolated despite the implementation of complete liver perfusion, or they may be multipotent progenitor cells that originate from the donor liver, if the liver stem cells have a sufficiently high plasticity.
2 Liver stem cells and liver diseases
2.1 Hepatic stem cells and acute liver injury The regenerative response after liver injury occurs very rapidly and comprehensively (all residual hepatocytes enter a proliferative state), is controlled by precise regulatory mechanisms (synergistic effects of various growth factors, cytokines and hormones), and regenerates while performing their normal physiological functions (e.g. detoxification, bile secretion, glycogen synthesis, etc.). Animal experiments and clinical studies have confirmed that the regeneration of liver tissue induced by acute liver injury is mainly accomplished by the re-entry of residual normal hepatocytes into the cell cycle. Theoretically, for 2/3 hepatectomized rats, the residual hepatocytes can complete the regeneration of liver tissue after an average of 3/2 cell cycles, and the whole regeneration process can be completed in 1~2 weeks. Hepatocyte necrosis caused by partial hepatectomy and acute hepatitis in the clinical setting may undergo a regeneration process similar to that of animal models.
If the division and proliferation of mature hepatocytes are inhibited, or if the extent of hepatocyte necrosis is too large, liver regeneration has to be accomplished by mobilizing stem cell proliferation. In the experimental liver regeneration model, it is clearly observed that oval cells proliferate first in large numbers around the liver lobules and gradually migrate into the lobules over time. Clinically, histological observations of acute massive hepatic necrosis also reveal that oval cells first appear in the region of the border plate around the lobules, and around day 4 of liver injury, oval cells transform into hepatocytes.
2.2 Hepatic stem cells and chronic viral hepatitis In clinical practice, acute liver tissue injury and regeneration are actually not very common; the problem we are often faced with is the recurrent and persistent hepatocellular injury caused by chronic hepatitis and the compensatory proliferation of hepatocytes in this state.
Within the hepatocyte, the HBV virus completes its own replication or even undergoes site-specific integration with the genome of the hepatocyte. At the same time, the viral genes use the protein synthesis apparatus of the hepatocyte to assemble virus-specific proteins. The viral proteins are processed in the hepatocyte as endogenous antigens and then bound to MHCI-like molecules and delivered to the hepatocyte surface, where they are specifically recognized by the CTL or killed by the Fas system. If the virus is cleared by the body through effective immune mechanisms, the hepatocytes are no longer destroyed after compensatory regeneration is completed and the body recovers. However, hepatitis B (or C) is characterized by the difficulty of virus clearance, repeated and persistent hepatocyte damage and regeneration, especially in the late stages of infection, when mature hepatocytes are extensively infected by the virus and virus-infected hepatocytes often lose their ability to proliferate (histochemistry shows very little positive staining for PCNA in cells with positive viral antigens), and proliferation and differentiation of stem cells then becomes the main compensatory proliferative cell population. Experimental studies have also confirmed that in patients with hepatitis cirrhosis, histopathology reveals a proliferative state of stem cells around the proliferative foci [15].
Hepatic stem cells are mainly found in the terminal bile ducts, and one of the histological features of chronic hepatitis (especially active hepatitis) is the presence of massive bile duct proliferation in the confluent area and its surrounding fibrous connective tissue intervals. Although the mechanism of liver fibrosis in chronic hepatitis is not yet understood, there seems to be a definite link between the development of fibrosis and the proliferation of liver stem cells, and high serum AFP levels in patients may also be related to the abnormal proliferation of stem cells.
2.3 Hepatic stem cells and primary hepatocellular carcinoma Oncology is currently debating whether hepatocellular carcinoma arises from differentiation of mature hepatocytes (dedifferentiation) or from maturation arrest of stem cell differentiation or dedifferentiation (dysdifferentiation). The dysdifferentiation view is divided.
The dysdifferentiation view suggests that the target of carcinogenic action is the mature hepatocyte, causing mutations and/or destabilization of the genetic material of the target cell. Experimental evidence in support of this theory is that pre-cancerous lesions can originate in mature hepatocytes in a model of hepatocellular carcinoma induced by 2acetylaminofluorene (2AAF) by labeling mature hepatocytes with retroviral-mediated gene transfection. For human hepatocellular carcinoma, the hypothesis of dedifferentiation of mature hepatocytes seems to be a generally accepted view. Current research in the molecular biology of human hepatocellular carcinoma revolves around the screening of tumor-associated genes and the mutation or instability of the hepatocyte genome that may be caused by the integration of the viral genome.
The blocked differentiation hypothesis suggests that the target of carcinogenic action is the microenvironment in which stem cells differentiate and mature, and that various factors leading to structural abnormalities and/or abnormalities in chemical messenger transmission in this microenvironment are the primary cause of carcinogenic action, and that the essence of the tumor originates from the blocked differentiation of pre-existing stem cells in the tissue [16]. Our study also found that chronic hepatitis tissues, cirrhotic tissues and paraneoplastic liver tissues can express high levels of hepatitis B virus protein products, but in cancerous tissues, the expression of viral antigens mysteriously disappears, and virus-infected hepatocytes often lose their proliferative activity (PCNA negative), and the cells with proliferative activity are often smaller “oval cells” around the liver lobules The proliferating cells are often smaller “ovoid cells” around the liver lobules, i.e. stem cells within the liver tissue.
Other evidence supporting the theory of impaired differentiation include the presence of oval cells within hepatic malignant tissues [17]; hepatocellular carcinoma cells and oval cells express similar cellular markers such as AFP, GGP, cytokeratin series CK (7, 8, 18, 19, 20), transcription factors OC2, OC3, oval cell markers OV1, OV6, stem cell markers SCF/ckit and CD34; many studies found that hepatocellular carcinoma cells also express B-cell differentiation antigens such as CD10 and CD40, but whether this antigen is expressed in oval cells and adult hepatocytes has not been reported; both stem cells and cancer cells possess telomerase activity; low or no replication of viruses and low or no expression of viral antigens in hepatocellular carcinoma cells; the issue of multicentric origin of hepatocellular carcinoma; hepatocellular carcinoma cell lines cultured in vitro with heterogeneous and bidirectional differentiation properties. Although the hypothesis of blocked differentiation of stem cells has been proposed by some authors, there is no visual evidence so far to confirm that hepatocellular carcinoma cells are derived from pre-existing stem cells in vivo.
3 Prospects of liver stem cell applications
The research on liver stem cells is still in its infancy, and the techniques of isolation, culture and identification of liver stem cells are still immature. The relationship between liver stem cells and chronic liver pathological process and liver cancer is yet to be further elucidated, and the regulatory mechanisms of multi-origin and multi-directional differentiation of adult liver stem cells are yet to be clarified, but the clinical application of liver stem cells still shows a very attractive prospect.
3.1 Hepatic Stem Cells and Reconstruction of Liver Function Acute and subacute hepatocellular necrosis caused by various causes can be saved by human liver stem cell transplantation if the basic architecture of the liver survives, taking advantage of the unlimited proliferation of liver stem cells and their potential to differentiate into hepatocytes and bile duct cells.
Human genetically defective liver diseases such as hepatomegaly have the potential to be treated similarly or better than liver transplantation by transplanting liver stem cells. As early as 10 years ago Rhim et al. reported that isolation of genotypically normal adult mouse hepatocytes for transplantation into albumin urokinase (AlbuPA) transgenic mice corrected the defective liver function of the transgenic mice, which showed proliferation of AlbuPA-free toxic hepatocytes that eventually replaced the genetically defective hepatocytes. The following year, this author also reported success in transplanting rat hepatocytes into immune-tolerant AlbuPA transgenic mice, with 100% replacement of genetically defective mouse hepatocytes by rat hepatocytes. In contrast, Overturf used an animal model of fumarylacetoacetate hydrolase (FAH) gene deficient mice, which develop hereditary tyrosinaemia type I (HT1) degenerative liver disease, and he used normal hepatocyte He found that only 1000 hepatocytes were needed to largely replace the original defective hepatocytes, and concluded that hepatocytes have proliferative potential similar to hematopoietic stem cells. However, it is generally believed that differentiated hepatocytes have limited proliferative potential after implantation, have a single direction of differentiation, and mature hepatocytes are much larger than stem cells and are not easily dispersed to the liver parenchyma after portal vein injection, so stem cell transplantation should still be advocated. In clinical practice, Kumar et al [18] reported a case of liver failure due to primary amyloidosis still recurring after liver transplantation, which was cured by performing stem cell transplantation after 10 and 14 months of liver transplantation.
3.2 Treatment of hepatic stem cells and hepatocellular carcinoma As mentioned earlier, hepatocellular carcinoma may occur as a result of blocked differentiation of hepatic stem cells, whereby treatment can be achieved by improving the microenvironment of the liver to induce differentiation of cancer cells to normal cells, or by inducing apoptosis of cancer cells. As early as 10 years ago, Coleman [19] reported that allogeneic hepatocellular carcinoma cell lines GN6TF and GP7TB, malignantly transformed from rat liver stem cell line WBF344, were transplanted into the liver of homozygous rats, resulting in no tumorigenesis of GN6TF and integration of cancer cells into the liver parenchyma to form a normal liver plate; while GP7TB remained tumorigenic in the liver, but the tumor was well differentiated histomorphologically . It is concluded that the microenvironment of liver tissue can influence the differentiation of cancer cells and eliminate or diminish the malignant potential of tumor cells. Several experiments have been reported on the induction of differentiation of hepatocellular carcinoma cell lines to mature hepatocyte phenotypes in vitro, but the molecular mechanisms of induced differentiation and the prospects of clinical application are yet to be further investigated.
3.3 Hepatic stem cells and gene therapy Gene therapy is the most promising treatment for genetic defective diseases and even malignant tumors, without immune rejection and without the need for patients to take long-term immunosuppressive drugs. Given the limitations of in vitro culture of mature hepatocytes, it is more difficult to introduce exogenous genes into hepatocytes, whereas it is easier to use stem cells. Genetically modified liver stem cells or bone marrow stem cells are expected to correct liver metabolic deficiency diseases as long as the microenvironment for host liver cell growth is favorable, and such genetically modified stem cell autotransplantation does show promising applications.