Hepatic Stem/progenitor Cells Research Articles

Functional characterization of CD49f+ hepatic stem/progenitor cells in adult mice liver.

Hepatic Stem/progenitor cells (HSPCs) have gained a large amount of interest for treating acute liver disease. However, the isolation and identification of HSPCs are unclear due to the lack of cell-specific surface markers. To isolate adult HSPCs, we used cell surface-marking antibodies, including CD49f and Sca-1. Two subsets of putative HSPCs, Lin-CD45-Sca-1-CD49f+ (CD49f+) and Lin-CD45-Sca-1+CD49f- (Sca-1+) cells, were isolated from adult mice liver by flow cytometry. Robust proliferative activity and clonogenic activity were found in both CD49f+ and Sca-1+ cells through colony-forming tests and cell cycle analyses. Immunofluorescence staining revealed that CD49f+ cells expressed ALB and CK-19 while Sca-1+ cells expressed only ALB, indicating that CD49f+ cells were bipotential and capable of differentiating into hepatocyte and cholangiocyte. Consequently, PAS stain showed that differentiated CD49f+ and Sca-1+ cells synthesised glycogen, indicating they could differentiate into functional hepatocytes. mRNA expression profile indicated that both CD49f+ and Sca-1+ cells showed differential expression of genes that are associated with liver progenitor function such as Sox9 and EpCam. Moreover, two subsets of putative HSPCs were activated by DDC and we found that their abundance and proliferation increased with age. In summary, wehypothesized that CD49f+ cells were a type of potential HSPCs and may be utilised for clinical stem cell therapy.

Asbestos and Intrahepatic Cholangiocarcinoma.

The link between asbestos exposure and the onset of thoracic malignancies is well established. However epidemiological studies have provided evidences that asbestos may be also involved in the development of gastrointestinal tumors, including intrahepatic cholangiocarcinoma (ICC). In line with this observation, asbestos fibers have been detected in the liver of patients with ICC. Although the exact mechanism still remains unknown, the presence of asbestos fibers in the liver could be explained in the light of their translocation pathway following ingestion/inhalation. In the liver, thin and long asbestos fibers could remain trapped in the smaller bile ducts, particularly in the stem cell niche of the canals of Hering, and exerting their carcinogenic effect for a long time, thus inducing hepatic stem/progenitor cells (HpSCs) malignant transformation. In this scenario, chronic liver damage induced by asbestos fibers over the years could be seen as a classic model of stem cell-derived carcinogenesis, where HpSC malignant transformation represents the first step of this process. This phenomenon could explain the recent epidemiological findings, where asbestos exposure seems mainly involved in ICC, rather than extrahepatic cholangiocarcinoma, development.

Survival of endogenous hepatic stem/progenitor cells in liver tissues during liver cirrhosis

AimsThis study addressed whether endogenous hepatic stem/progenitor (HSP) cells survival were related to the injured signals during liver cirrhosis. Material and methodsLiver cirrhosis was induced in C57BL/6 mice by administering diethylnitrosamine (DEN) in drinking water. Hematoxylin-eosin staining and Masson's trichrome staining were used to identify infiltrative cells and connective tissues, respectively. The inflammatory activity grade and fibrotic stage, represented as G and S respectively, and evaluated by the International Simplified Grading and Staging System (ISGSS). Endogenous HSP cells (Ng2+HSP) were identified by immunofluorescence staining with an anti-neuro/glial antigen 2 (Ng2) antibody. All data were analyzed using SPSS 22.0 and GraphPad Prism 6 and Student's t-test was performed to determine statistical significance. p-values < 0.05 were considered statistically significant. Key findingsAll mice administered oral DEN developed liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). During the fibrosis period, observed a positive correlation of survival of endogenous HSP (Ng2+HSP) cells with inflammatory activity. As the disease developed into the cirrhotic stage, when lost correlation of endogenous HSP cells with inflammatory activity, revealed a dramatically reduced survival rate of endogenous HSP (Ng2+HSP) cells. SignificanceThe DEN-induced liver cirrhosis could develop into three time zone of fibrosis, cirrhosis and cancer, and the histological patterns in the model are similar to those described in humans. Dramatic survival and less apoptosis of endogenous HSP (Ng2+HSP) cells was within fibrotic state, where inflammation signals is strong, indicating such time zone (1–6 weeks) during liver cirrhosis is important for mobilizing endogenous HSP-based regeneration.

Long-Term Culture of Mouse Fetal Hepatic Stem/Progenitor Cells.

Mouse fetal liver includes abundant hepatic stem/progenitor cells (HSPCs). Easy expansion with passage of HSPCs is necessary to obtain steady data. However, it is often difficult to enrich only HSPCs, and HSPCs can die when usual trypsin is used for replating. Here, we introduce serum-free long-term culture with passage of HSPCs using fetal mouse liver without a cell sorter.

Contribution of Resident Stem Cells to Liver and Biliary Tree Regeneration in Human Diseases.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

Hepatic stem/progenitor cell activation differs between primary sclerosing and primary biliary cholangitis

The activation of hepatic stem/progenitor cells (HPCs) is characterized by the appearance of ductular reaction (DR) in the liver parenchyma [1]. The aims of the present study were to evaluate the activation of HPCs in human cholangiopathies. Human liver tissue was obtained from liver donors (N=5), Primary Sclerosing Cholangitis (PSC; N=20), and Primary Biliary Cholangitis (PBC; N=20) patients. Ductular reaction extension was evaluated by Keratin(K) 7 immunoreactivity. HPC phenotype and signalling pathways were investigated by immunohistochemistry and immunofluorescence [2]. Ductular reaction in PBC is more extensive, appears earlier, and has a higher proliferation index compared to PSC. In PBC the extension of DR strongly correlates with clinical prognostic scores. A higher percentage of Sox9+ and K19+ cells characterized DR in PBC versus PSC. In cirrhotic-PSC, the HPC compartment showed signs of hepatocyte commitment. The study of the HPC niche indicated lower levels of laminin and NOTCH1 but higher expression of WNT pathway components in PSC compared to PBC. In conclusion, PSC and PBC are characterized by different patterns of HPC niche activation, reflecting the involvement of different portions of the biliary tree as primary target of damage. These aspects could have implications in the pathogenesis of cholangiopathies and could add prognostic value.

Hepatitis B viral infection of hepatic progenitor cells. Resolving unresolved questions?

Accumulated data to date do not entirely explain the; propensity of the hepatitis B virus (HBV) to cause chronic infections in newborns; failure of antiviral agents to resolve infections or precise mechanism whereby HBV causes hepatocellular carcinoma (HCC). Based on the increased numbers of hepatic stem/progenitor cells (HPCs) present within the neonatal liver, the refractoriness of these cells to the effects of interferons and xenobiotics and their ability to undergo malignant transformation, we hypothesize that HBV infection of HPCs could explain these and perhaps other clinical features of chronic HBV.

Multiple cellular origins and molecular evolution of intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy associated with unfavorable prognosis and for which no effective treatments are available. Its molecular pathogenesis is poorly understood. Genome-wide sequencing and high-throughput technologies have provided critical insights into the molecular basis of ICC while sparking a heated debate on the cellular origin. Cancer exhibits variabilities in origin, progression and cell biology. Recent evidence suggests that ICC has multiple cellular origins, including differentiated hepatocytes; intrahepatic biliary epithelial cells (IBECs)/cholangiocytes; pluripotent stem cells, such as hepatic stem/progenitor cells (HPCs) and biliary tree stem/progenitor cells (BTSCs); and peribiliary gland (PBG). However, both somatic mutagenesis and epigenomic features are highly cell type-specific. Multiple cellular origins may have profoundly different genomic landscapes and key signaling pathways, driving phenotypic variation and thereby posing significant challenges to personalized medicine in terms of achieving the optimal drug response and patient outcome. Considering this information, we have summarized the latest experimental evidence and relevant literature to provide an up-to-date view of the cellular origin of ICC, which will contribute to establishment of a hierarchical model of carcinogenesis and allow for improvement of the anatomical-based classification of ICC. These new insights have important implications for both the diagnosis and treatment of ICC patients.

Members of the Cyr61/CTGF/NOV Protein Family: Emerging Players in Hepatic Progenitor Cell Activation and Intrahepatic Cholangiocarcinoma.

Hepatic stem/progenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactions during severe liver damage when the replicative ability of hepatocytes is inhibited. HPC niches are full of profibrotic stimuli favoring scarring and hepatocarcinogenesis. The Cyr61/CTGF/NOV (CCN) protein family consists of six members, CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3, which function as extracellular signaling modulators to mediate cell-matrix interaction during angiogenesis, wound healing, fibrosis, and tumorigenesis. This study investigated expression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA). Mouse HPC were induced by the biliary toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Differential expression patterns of CCN proteins were found in HPC from DDC damaged mice and in human CCA tumors. In addition, we utilized reporter mice that carried Ccn2/Ctgf promoter driven GFP and detected strong Ccn2/Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and in DDC-induced liver damage. Abundant CCN2/CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that were surrounded by α-smooth muscle actin (α-SMA)+ myofibroblast cells in intrahepatic CCA tumors. These results suggest that CCN proteins, particularly CCN2/CTGF, function in HPC activation and CCA development.

Stem/Progenitor Cell Niches Involved in Hepatic and Biliary Regeneration.

Niches containing stem/progenitor cells are present in different anatomical locations along the human biliary tree and within liver acini. The most primitive stem/progenitors, biliary tree stem/progenitor cells (BTSCs), reside within peribiliary glands located throughout large extrahepatic and intrahepatic bile ducts. BTSCs are multipotent and can differentiate towards hepatic and pancreatic cell fates. These niches' matrix chemistry and other characteristics are undefined. Canals of Hering (bile ductules) are found periportally and contain hepatic stem/progenitor cells (HpSCs), participating in the renewal of small intrahepatic bile ducts and being precursors to hepatocytes and cholangiocytes. The niches also contain precursors to hepatic stellate cells and endothelia, macrophages, and have a matrix chemistry rich in hyaluronans, minimally sulfated proteoglycans, fetal collagens, and laminin. The microenvironment furnishes key signals driving HpSC activation and differentiation. Newly discovered third niches are pericentral within hepatic acini, contain Axin2+ unipotent hepatocytic progenitors linked on their lateral borders to endothelia forming the central vein, and contribute to normal turnover of mature hepatocytes. Their relationship to the other stem/progenitors is undefined. Stem/progenitor niches have important implications in regenerative medicine for the liver and biliary tree and in pathogenic processes leading to diseases of these tissues.

Regulation of the survival and differentiation of hepatic stem/progenitor cells by acyclic retinoid.

During embryonic liver development, hepatic stem/progenitor cells (HpSCs) have a high proliferative ability and bipotency to differentiate into hepatocytes and cholangiocytes. Retinoic acid is a derivative of vitamin A and is involved in the proliferation and differentiation of stem/progenitor cells in several tissues. However, whether retinoic acid regulates the characteristics of HpSCs in the normal liver is still unknown. A recent study has shown that acyclic retinoid regulates the survival and proliferation of HpSCs derived from mouse foetal liver. Acyclic retinoid suppressed the expansion of CD29+CD49f+ HpSCs through the induction of hepatocytic differentiation and progression of apoptosis.

Fibrogenesis and Carcinogenesis in Nonalcoholic Steatohepatitis (NASH): Involvement of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinase (TIMPs)

Nonalcoholic steatohepatitis (NASH) is emerging worldwide because life-styles have changed to include much over-eating and less physical activity. The clinical and pathophysiological features of NASH are very different from those of HBV- and HCV-chronic liver diseases. The prognosis of NASH is worse among those with nonalcoholic fatty liver diseases (NAFLD), and some NASH patients show HCC with or without cirrhosis. In the present review we discuss fibrogenesis and the relationship between fibrosis and HCC occurrence in NASH to clarify the role of MMPs and TIMPs in both mechanisms. Previously we proposed MMP and TIMP expression in the multi-step occurrence of HCC from the literature based on viral-derived HCC. We introduce again these expressions during hepatocarcinogenesis and compare them to those in NASH-derived HCC, although the relationship with hepatic stem/progenitor cells (HPCs) invasion remains unknown. Signal transduction of MMPs and TIMPs is also discussed because it is valuable for the prevention and treatment of NASH and NASH-derived HCC.

Angiogenic factors in chronic liver diseases: the effects on hepatic progenitor cells.

The biliary tree is a complex network of interconnected ducts, lined by epithelial cells called cholangiocytes, that drains the bile produced by the liver into the duodenum (1). At the periphery of the biliary tree, the interlobular bile ducts continue with the bile ductules, which are entirely constituted by cholangiocytes, may traverse the limiting plate, and are connected with the canal of Hering (2). The latter represents the physical link between the bile canaliculus (formed by the apical membranes of hepatocytes) and the biliary tree, and is thought to be an intrahepatic stem cell niche, harboring the multipotent hepatic stem/progenitor cells (HPCs) (3). To this end, the canal of Hering acts as the centre from which the maturation of both hepatocytes and cholangiocytes arises and proceeds in opposite directions. Indeed, HPCs can give rise to hepatoblasts that are also present as single cells or small aggregates in the canal of Hering and are bipotent cells that can further differentiate into committed progenitors of both the hepatocytic and cholangiocytic lineages. While committed hepatocytic progenitors proceed towards to central vein of the lobule to fully differentiate into mature hepatocytes, cholangiocytic progenitors, also known as “small cholangiocytes”, are thought to move towards the periphery of the biliary tree (4). Recent studies suggest that small cholangiocytes, which can be found up to the bile ductules, are the precursors of large cholangiocytes that, on the other hand, line the interlobular (or larger) bile ducts (5). However, peribiliary glands, which are located in the wall of extrahepatic and large intrahepatic bile ducts, also contain progenitor-like cells and might contribute to or drive the maturation of cholangiocytes (6).

Hepatic progenitor cell inhibition during embryonic period with high dose verapamil; liable joint to the cancer therapy

Cancer stem cells (CSCs) have been observed to share certain characteristics with normal stem cells. It was an important argument for cancer therapy and a successful progenitor inhibition could show us targeted cell type for a novel strategy. In this study, we aimed to constitute an inhibition in different stages of hepatic stem/progenitor cells (HPCs) with verapamil. Expression patterns of alpha-fetoprotein (AFP), c-kit (CD117) and p-glycoprotein were investigated in developing mouse on the embryonic day (E) 15, E18 and E21 to characterize early and late stages of HPCs. Proliferation inhibition with 5-Bromo-2-Deoxyuridin (BrdU) incorporation and maturation inhibition with PAS staining results were supported by morphometrical analysis during these periods. AFP, c-kit and p-glycoprotein immunoreactivity increased especially in E15 but decreased in E18 and E21 of the control groups during embryonic development. Verapamil treatment effected particularly E15 cells and immunoexpression of HPCs significantly decreased. Proliferation inhibition was observed in all embryonic days of mouse with verapamil and this drug inhibited not only maturation of HPCs in E18 and E21 embryos, but also decreased HPC number in the same embryonic period. According to our results, we estimated that similar to the early and late progenitor stages of HPCs, CSCc can also be in different stages in a heterogenic tumour bulk and the difficulty of CSC inhibition could be the main mechanism of tumour relapses. In this study, HPCs inhibition by verapamil in E15 was not observed in E18 and E21. As similar, CSCs treatments targeting different stages may be impotent to cells in tumour initiating cell stage. We can speculate that ineffectiveness of CSC-specific therapies may be attributed to the highly selective specificity of the treatment (Fig. 6, Ref. 28).

Liver Regeneration Signature in Hepatitis B Virus (HBV)-Associated Acute Liver Failure Identified by Gene Expression Profiling

IntroductionThe liver has inherent regenerative capacity via mitotic division of mature hepatocytes or, when the hepatic loss is massive or hepatocyte proliferation is impaired, through activation of hepatic stem/progenitor cells (HSPC). The dramatic clinical course of acute liver failure (ALF) has posed major limitations to investigating the molecular mechanisms of liver regeneration and the role of HSPC in this setting. We investigated the molecular mechanisms of liver regeneration in 4 patients who underwent liver transplantation for hepatitis B virus (HBV)-associated ALF.Methods and FindingsGene expression profiling of 17 liver specimens from the 4 ALF cases and individual specimens from 10 liver donors documented a distinct gene signature for ALF. However, unsupervised multidimensional scaling and hierarchical clustering identified two clusters of ALF that segregated according to histopathological severity massive hepatic necrosis (MHN; 2 patients) and submassive hepatic necrosis (SHN; 2 patients). We found that ALF is characterized by a strong HSPC gene signature, along with ductular reaction, both of which are more prominent in MHN. Interestingly, no evidence of further lineage differentiation was seen in MHN, whereas in SHN we detected cells with hepatocyte-like morphology. Strikingly, ALF was associated with a strong tumorigenesis gene signature. MHN had the greatest upregulation of stem cell genes (EpCAM, CK19, CK7), whereas the most up-regulated genes in SHN were related to cellular growth and proliferation. The extent of liver necrosis correlated with an overriding fibrogenesis gene signature, reflecting the wound-healing process.ConclusionOur data provide evidence for a distinct gene signature in HBV-associated ALF whose intensity is directly correlated with the histopathological severity. HSPC activation and fibrogenesis positively correlated with the extent of liver necrosis. Moreover, we detected a tumorigenesis gene signature in ALF, emphasizing the close relationship between liver regeneration and liver cancer.

Expression of vascular endothelial growth factors and their receptors by hepatic progenitor cells in human liver diseases.

Hepatic stem/progenitor cells (HPCs) are stem cells residing in the most peripheral branches of the biliary tree; these cells are able to differentiate towards mature hepatocyte or mature cholangiocyte; moreover in normal conditions, they are mostly quiescent cells. HPC activation has been involved in the progression of chronic parenchymal diseases (chronic viral hepatitis) and chronic biliary diseases (such as Primary Biliary Cirrhosis: PBC) and in the occurrence of intrahepatic cholangiocarcinoma. The HPCs participate in the repair of liver damage either through the replacement of dead cells or by driving fundamental repair processes, including fibrosis and angiogenesis. Little information exists regarding the expression of VEGF by HPC in the course of liver non-malignant pathologies. In this study, we evaluated: (I) the presence of HPCs in PBC and HCV-related Cirrhosis (HCV-C) samples, and (II) the expression of VEGFs and VEGF-Rs in PBC and HCV-C samples. Our results showed (I) PBC samples presented a more extensive expansion of HPC population in comparison with those of HCV-C samples; (II) PBC samples showed a more extensive angiogenesis if compared to HCV-C; and (III) PBC samples were characterized by an increased expression of VEGF-A and VEGF-C if compared to HCV-C and the number of HPCs expressing VEGFs was correlated with the extension of ductular reaction and angiogenesis. The role of VEGFs in the expansion of HPC niche could have important implication in the management of fibrogenic processes and carcinogenesis.

Hepatic stellate cells are involved in the pathogenesis of acute-on-chronic liver failure (ACLF)

ACLF is a condition with varied acute and chronic underlying etiologies and high mortality. Hepatic stellate cells (HSCs) are known to play an important role in hepatic fibrogenesis. Experimental data suggest that HSCs also display characteristics of hepatic stem/progenitor cells (HPCs). We investigated the number of activated HSC and their relationship to HPCs in ACLF in comparison to acute and chronic stages of other liver diseases. Immunohistochemical analysis of the number of activated HSCs and HPCs was performed in liver biopsies of 70 cases of ACLF and in the biopsies of patients with cirrhosis (45), acute hepatitis (25), chronic hepatitis (25), and normal live-related liver transplantation (LDLT) donor biopsies (15). The number of α-SMA-positive HSCs was assessed both quantitatively and semi-quantitatively. Keratin-19 (K19)-positive HPCs were estimated semi-quantitatively, and we looked for correlations between numbers of HSCs and HPCs. We found significantly more α-SMA activated HSCs/1,000 hepatocytes in ACLF biopsies (231 ± 91) than in biopsies of patients with acute hepatitis (147 ± 77), cirrhosis (73 ± 35), chronic hepatitis (66 ± 30), and normal LDLT donor biopsies (25 ± 10), respectively (p < 0.0001). A significant correlation between the presence of HPCs and ACLF versus other etiologies of liver disease was found (p < 0.001). There was a significant correlation between the numbers of HPCs and of activated HSCs (p < 0.001). ACLF is associated with more significant HSC activation than in acute hepatitis and other chronic liver diseases. There is a significant relationship between the presence of HSCs and that of HPCs, indicating a possible dynamic role of HSCs in liver regeneration and pathobiology of ACLF.

Prospective Isolation and Characterization of Bipotent Progenitor Cells in Early Mouse Liver Development

Outgrowth of the foregut endoderm to form the liver bud is considered the initial event of liver development. Hepatic stem/progenitor cells (HSPCs) in the liver bud are postulated to migrate into septum transversum mesenchyme at around embryonic day (E) 9 in mice. The studies of liver development focused on the mid-fetal stage (E11.5-14.5) have identified HSPCs at this stage. However, the in vitro characteristics of HSPCs before E11.5 have not been elucidated. This is probably partly because purification and characterization of HSPCs in early fetal livers have not been fully established. To permit detailed phenotypic analyses of early fetal HSPC candidates, we developed a new coculture system, using mouse embryonic fibroblast cells. In this coculture system, CD13(+)Dlk(+) cells purified from mouse early fetal livers (E9.5 and E10.5) formed colonies composed of both albumin-positive hepatocytic cells and cytokeratin (CK) 19-positive cholangiocytic cells, indicating that early fetal CD13(+)Dlk(+) cells have properties of bipotent progenitor cells. Inhibition of signaling by Rho-associated coiled-coil containing protein kinase (Rock) or by nonmuscle myosin II (downstream from Rock) was necessary for effective expansion of early fetal CD13(+)Dlk(+) cells in vitro. In sorted CD13(+)Dlk(+) cells, expression of the hepatocyte marker genes albumin and α-fetoprotein increased with fetal liver age, whereas expression of CK19 and Sox17, endodermal progenitor cell markers, was highest at E9.5 but decreased dramatically thereafter. These first prospective studies of early fetal HSPC candidates demonstrate that bipotent stem/progenitor cells exist before E11.5 and implicate Rock-myosin II signaling in their development.

Identification and location of label retaining cells in mouse liver

In most somatic tissues, adult stem cells are crucial for the maintenance of tissue homeostasis under normal physiological states and during recovery from injuries. Label retaining cell (LRC) assay remains the well-known method to identify possible somatic stem/progenitor cells and their location both in situ and in vivo. Here, BrdU was used to tag the possible hepatic stem/progenitor cells (HSPCs) in newborn pups, followed by a trace period of up to 23 months. Additionally, we report a method to rapidly kill proliferating cells in adult liver tissue, and activate and label (KAL) surviving possible HSPCs. We found that LRCs definitively exist in the liver tissues of adult mice, that LRCs express cell cycle proteins cyclind3 and cdk6, but do not express sca-1 or c-kit, and that LRCs locate primarily in the periportal and pericentral regions. Moreover, the number of these LRCs remains nearly constant during the lifespan of the mice. After injury induced by 5-fluorouracil, we observed that the activation of possible HSPCs tagged by the BrdU label was almost completely inhibited at day 4. The cellular kinetics of repair of BrdU-tagged HSPCs were different every 12 h between day 3 and day 4. Moreover, HSPCs still retained labels and located definitively in the periportal region after a prolonged chase. The LRC method together with our novel KAL method reported here may be used to identify and locate possible HSPCs.