Passage of bone-marrow-derived liver stem cells in a proliferating culture system

AIM： To explore the feasibility of passage of bone- marrow-derived liver stem cells （BDLSCs） in culture systems that contain cholestatic serum.METHODS： Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction （RT-PCR） and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.RESULTS： The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units （H-CFUs） were formed. H-CFUs expressed markers of embryonic hepatocytes （alpha-fetoprotein, albumin and cytokeratin 8/18）, biliary cells （cytokeratin 19）, hepatocyte functional proteins （transthyretin and cytochrome P450-261）, and hepatocyte nuclear factors 1α and -3β）. They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.CONCLUSION： BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Role of liver stem cells in hepatocarcinogenesis

Liver cancer is an aggressive disease with a high mortality rate. Management of liver cancer is strongly dependent on the tumor stage and underlying liver disease. Unfortunately, most cases are discovered when the cancer is already advanced, missing the opportunity for surgical resection. Thus, an improved understanding of the mechanisms responsible for liver cancer initiation and progression will facilitate the detection of more reliable tumor markers and the development of new small molecules for targeted therapy of liver cancer. Recently, there is increasing evidence for the "cancer stem cell hypothesis", which postulates that liver cancer originates from the malignant transformation of liver stem/progenitor cells(liver cancer stem cells). This cancer stem cell model has important significance for understanding the basic biology of liver cancer and has profound importance for the development of new strategies for cancer prevention and treatment. In this review, we highlight recent advances in the role of liver stem cells in hepatocarcinogenesis. Our review of the literature shows that identification of the cellular origin and the signaling pathways involved is challenging issues in liver cancer with pivotal implications in therapeutic perspectives. Although the dedifferentiation of mature hepatocytes/cholangiocytes in hepatocarcinogenesis cannot be excluded, neoplastic transformation of a stem cell subpopulation more easily explains hepatocarcinogenesis. Elimination of liver cancer stem cells in liver cancer could result in the degeneration of downstream cells, which makes them potential targets for liver cancer therapies. Therefore, liver stem cells could represent a new target for therapeutic approaches to liver cancer in the near future.

An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

AIM： To develop a method to isolate liver stem cells fast and efficiently. METHODS： Fetal mouse liver cells were characterized by cell surface antigens （c-Kit and CD45/TER119） using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by -lone-forming culture, RT-PCR and immunofluorescence says. RESULTS： The c-Kit-（CD45/TER119）- cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells. Functionally mature hepatocytes were observed after 21 d of culture. CONCLUSION： This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.

Chimera formation of platelet GPⅡb Bak a/b by intrauterine transplantation of fetal liver stem cells

Abstract:Objective To investigate whether artificial heterozygous chimeras of platelets can be established by intrauterine transplantation of fetal liver stem cells and evaluate its potential use for the treatment of Glanzmann thrombasthenia.Methods Platelet glycoprotein (GP) Ⅱb Bak a/b (or GPⅡb Ⅰle843Ser) was used as a genetic marker. A homozygous 16-week-old Bak a/a fetus (as donor) and a homozygous 16.5-week-old Bak b/b fetus (as recipient) were screened from 42 pregnant women hospitalized for abortion. PCR with allele specific primers and FOK Ⅰ digestion based on PCR products were used. Aborted donor fetal liver cell suspensions were prepared and intrauterine transplantation was carried out by infusion of 4?ml fetal liver cells (22×105) into the recipient umbilical vein under ultrasonic visualization.Results At gestation termination (abortion), 21 days after transplantation, chimera GPⅡb Bak a/b of the recipient were detected by FOK 1 digestion based on PCR from DNA and RT-PCR from platelet RNA. Conclusion Intrauterine transplantation of fetal liver cell may provide an effective way for curing GT or other inherited diseases.

Influence of adriamycin on changes in Nanog, Oct-4, Sox2, ARID1 and Wnt5b expression in liver cancer stem cells

AIM: To determine the influence of Adriamycin (ADM) on the changes in Nanog, Oct4, Sox2, as well as, in ARID1 and Wnt5b expression in liver cancer stem cells.

Epithelial cells with hepatobiliary phenotype:Is it another stem cell candidate for healthy adult human liver?

AIM： To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS： Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and sub- cultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CDl17. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature （α-fetoprotein） but also differentiated hepatocyte （albumin and CK-18） and biliary markers （CK-7 and 19）, whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma. CONCLUSION： According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.

Regulators of liver cancer stem cells

Hepatocellular carcinoma(HCC)is a leading cause of cancer deaths.It is often detected at a stage when there are few therapeutic options.Liver cancer stem cells(LCSCs)are highly tumorigenic and resistant to chemotherapy and radiation therapy.Their presence in HCC is a major reason why HCC is difficult to treat.The development of LCSCs is regulated by a variety of factors.This review summarizes recent advances on the factors that regulate the development of LCSCs.Due to the importance of LCSCs in the development of HCC,a better understanding of how LCSCs are regulated will help to improve the treatments for HCC patients.

Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure

AIM:To study the clinical efficacy of traditional Chinese medicine(TCM)intervention"tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment"("TTK")for treating liver failure due to chronic hepatitis B.METHODS:We designed the study as a randomized controlled clinical trial.Registration number of Chinese Clinical Trial Registry is Chi CTR-TRC-12002961.A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study.Participants were randomly assigned to the following three groups:(1)a modern medicine control group(MMC group,36patients);(2)a"tonifying qi and detoxification"("TQD")group(72 patients);and(3)a"tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment"("TTK")group(36patients).Patients in the MMC group received general internal medicine treatment;patients in the"TQD"group were given a TCM formula"tonifying qi and detoxification"and general internal medicine treatment;patients in the"TTK"group were given a TCM formula of"TTK"and general internal medicine treatment.All participants were treated for 8 wk and then followed at 48 wk following their final treatment.The primaryefficacy end point was the patient fatality rate in each group.Measurements of various virological and biochemical indicators served as secondary endpoints.The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.RESULTS:At the 48-wk post-treatment time point,the patient fatality rates in the MMC,"TQD",and"TTK"groups were 51.61%,35.38%,and 16.67%,respectively,and the differences between groups were statistically significant(P<0.05).However,there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups(P>0.05).Patients in the"TTK"group had significantly higher levels of serum total bilirubin compared to MMC subjects(339.40μmol/L±270.09μmol/L vs 176.13μmol/L±185.70μmol/L,P=0.014).Serum albumin levels were significantly increased in both the"TQD"group and"TTK"group as compared with the MMC group(31.30 g/L±4.77g/L,30.72 g/L±2.89 g/L vs 28.57 g/L±4.56 g/L,P<0.05).There were no significant differences in levels of alanine transaminase among the three groups(P>0.05).Safety data showed that there was one case of stomachache in the"TQD"group and one case of gastrointestinal side effect in the"TTK"group.CONCLUSION:Treatment with"TTK"improved the survival rates of patients with liver failure due to chronic hepatitis B.Additionally,liver tissue was regenerated and liver function was restored.

Human liver stem/progenitor cells decrease serum bilirubin in hyperbilirubinemic Gunn rat

AIM: To test the ability of adult-derived human liver stem/progenitor cells (ADHLSC) from large scale cultures to conjugate bilirubin in vitro and in bilirubin conjugation deficient rat.

Stem cell differentiation and human liver disease

Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,eff icient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the f ield and discuss the future potential and limitations of stem cell technology.

Therapeutic application of stem cells in gastroenterology:An up-date

Adult stem cells represent the self-renewing progenitors of numerous body tissues, and they are currently classified according to their origin and differentiation ability. In recent years, the research on stem cells has expanded enormously and holds therapeutic promises for many patients suffering from currently disabling diseases. This paper focuses on the possible use of stem cells in the two main clinical settings in gastro-enterology, i.e., hepatic and intestinal diseases, which have a strong impact on public health worldwide. Despite encouraging results obtained in both regenerative medicine and immunemediated conditions,further studies are needed to fully understand the biology of stem cellsand carefully assess their put ativeonco- genicproperties.Moreover,there searchonstemcellsarousesferventethical,socialandpoliticaldebate.TheItalianSocietyofGastroenterologysponsoredaworkshoponstemcellsheldinVeronaduringtheⅩⅥCongressoftheFederationofItalianSocietiesofDigestiveDiseases(March 6-9,2010).Here,we report on the issues discussed,including liver and intestinal diseases that may benefit from stemcell therapy,the biology of hepatic and intestinal tissue repair,and stem cell usage inclinical trials.

road to stemness in hepatocellular carcinoma

Carcinogenic process has been proposed to relay on the capacity to induce local tissue damage and proliferative repair. Liver has a great regeneration capacity and currently, most studies point towards the dominant role of hepatocytes in regeneration at all levels of liver damage. The most frequent liver cancer is hepatocellular carcinoma(HCC). Historical findings originally led to the idea that the cell of origin of HCC might be a progenitor cell. However, current linage tracing studies put the progenitor hypothesis of HCC origin into question. In agreement with their dominant role in liver regeneration, mature hepatocytes are emerging as the cell of origin of HCC, although, the specific hepatocyte subpopulation of origin is yet to be determined. The relationship between the cancer cell of origin(CCO) and cancer-propagating cells, known as hepatic cancer stem cell(HCSC) is unknown. It has been challenging to identify the definitive phenotypic marker of HCSC, probably due to the existence of different cancer stem cells(CSC) subpopulations with different functions within HCC. There is a dynamic interconversion among different CSCs, and between CSC and non-CSCs. Because of that, CSC-state is currently defined as a description of a highly adaptable and dynamic intrinsic property of tumor cells, instead of a static subpopulation of a tumor. Altered conditions could trigger the gain of stemness, some of them include: EMT-MET, epigenetics, microenvironment and selective stimulus such as chemotherapy. This CSC heterogeneity and dynamism makes them out reach from therapeutic protocols directed to a single target. A further avenue of research in this line will be to uncover mechanisms that trigger this interconversion of cell populations within tumors and target it.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

COL14A1 promotes self-renewal of human liver cancer stem cells through activation of ERK signaling

Objective:Liver cancer stem cells(CSCs)are the culprits of hepatocellular carcinoma metastasis and recurrence.Only by eliminating tumor stem cells can malignant tumors be fundamentally cured.This study aimed to identify the role and underlying mechanism of aberrant Collagen Type XIV Alpha 1 Chain(COL14A1)overexpression in liver CSCs,and improve understanding of the molecular basis of hepatocellular carcinoma metastasis and recurrence.Methods:First,quantitative real-time polymerase chain reaction was used to confirm aberrant high-expression of COL14A1 in liver CSCs.Next,interference experiments were performed to determine the key role of COL14A1.To explore the mechanism of COL14A1 overexpression in liver CSCs,putative microRNA(miRNAs)targeting COL14A1 were analyzed using the miRTarBase database.Next,quantitative real-time polymerase chain reaction,western blotting,and luciferase reporter assays were performed to verify the interaction between miR-7108-3p and COL14A1.Lastly,key target proteins of the COL14A1-extracellular-regulated signal kinase(ERK)signaling pathway were identified through western blotting analysis.This study was approved by the Ethics Committee of Shanghai Fourth People’s Hospital,Tongji University School of Medicine,China(approval No.2019tjdx17)on February 21,2019.Results:COL14A1 is abnormally highly expressed in liver CSCs,which is necessary for liver CSCs to maintain their self-renewal capability.Mechanistically,COL14A1 is post-transcriptionally regulated by miR-7108-3p in a negative manner.Low expression of miR-7108-3p increased translation of COL14A1,which subsequently activated ERK signaling,ultimately maintaining the self-renewal and stem cell-like properties of liver CSCs.Conclusion:COL14A1,which is negatively regulated by miR-7108-3p,was found to play a crucial role in maintaining the selfrenewal and stem cell-like properties of liver CSCs through activation of ERK signaling.

Microenvironment of Liver Regeneration in Liver Cancer

The occurrence and development of liver cancer are essentially the most serious outcomes of uncontrolled liver regeneration. The progression of liver cancer is inevitably related to the abnormal microenvironment of liver regeneration. The deterioration observed in the microenvironment of liver regeneration is a necessary condition for the occurrence, development and metastasis of cancer. Therefore, the use of a technique to prevent and treat liver cancer via changes in the microenvironment of liver regeneration is a novel strategy. This strategy would be an effective way to delay, prevent or even reverse cancer occurrence, development and metastasis through an improvement in the liver regeneration microenvironment along with the integrated regulation of multiple components, targets, levels, channels and time sequences. In addition, the treatment of ＂tonifying Shen（Kidney） to regulate liver regeneration and repair by affecting stem cells and their microenvironment＂ can regulate ＂the dynamic imbalance between the normal liver regeneration and the abnormal liver regeneration＂; this would improve the microenvironment of liver regeneration, which is also a mechanism by which liver cancer may be prevented or treated.

Role of Liver Progenitor Cell in Liver Regeneration Cellular Cross-Talks and Signals

The liver is well known for its ability to regenerate in response to injury. After partial hepatectomy and some chemicals induced acute liver injury, existing hepatocytes can expand to repair the liver function. While adult liver stem/progenitor cells(LPCs) are evoked and differentiate into functional hepatocytes and cholangiocytes to compensate the damaged liver once hepatocyte proliferation is severely impaired. A number of evidences suggest that adjacent hepatic stellate cells(HSCs) or invading leukocytes may be involved in LPCs directed regeneration through governning two major events including fibrogenic and inflammatory responses respectively or simultaneously. As such, a microenvironment(or "niche") composed of different cell sources or factors presents diversity, which eventually mediates LPCs response to biliary or hepatocellular regeneration. This mini review aims at summarizing the latest development on the roles of HSCs, macrophages and lymphocytes as well as corresponding signaling pathways in liver progenitor cells mediated biliary and hepatocellular regeneration, and discussing therapeutic potential of liver progenitor cells in hepatic diseases.