Construction Of A Defined Glycogel Culture System For The Expansion Of Biliary Tree Stem Cells

ObjectiveEnd-stage liver disease(ESLDs)are a clinical syndrome characterized by abnormal liver function and systemic manifestations.Both acute liver failure and chronic liver disease can lead to end-stage liver disease.According to the statistics of the World Health Organization(WHO),about 3 million people die of ESLDs each year.In China,as a large country with hepatitis B,20% to 30% of the patients will develop ESLDs.Therefore,ESLDs has become a global public health problem with high morbidity and mortality,and it is a major disease that endangers national health and economic burden.Orthotopic liver transplantation(OLT)is the most effective method for clinical treatment of liver failure.However,due to the lack of donor liver,only a few patients are likely to receive liver transplantation,and many patients die while waiting for the liver source.Other problems include rejection after transplantation,high risk and high cost of operation,which limit its application and promotion in clinical treatment of endstage liver disease to a great extent.In recent years,regenerative medicine and stem cell research have been carried out rapidly.Domestic and foreign researchers have carried out a large number of preclinical and clinical studies using a variety of cells and stem cells,and achieved certain results.At present,gratifying progress has been made in the basic research of stem cell transplantation in the treatment of liver failure.The source of therapeutic cells includes hepatocytes,human embryonic stem cells(h ESCs),human induced pluripotent stem cells(hi PSCs),mesenchymal stem cells(MSCs),biliary tree stem cells(BTSCs),hematopoietic stem cells(HSCs)and so on.Screening and determining the types of stem cells that can be used for disease treatment is the basis for the development of stem cell-based ESLDs.BTSCs are ideal candidates for the treatment of ESLDs due to their potentials to differentiate into mature hepatocytes,bile duct epithelial cells(cholangiocytes)or functional islets.They are a kind of stem cells in the peribiliary glands of bile duct system.Compared to mature hepatocytes,BTSCs has the advantages of a wide range of tissue acquisition sources and the ability of cell proliferation in vitro.The study found that by transplanting fetal BTSCs into the patient,the patient’s MELD score decreased,liver function improved after 3 months,and did not need continuous immunosuppressive therapy.Compared to h ESCs and other pluripotent stem cells,BTSCs is safe in application and can effectively induce and mature into functional cells.Compared with umbilical cord MSCs,BTSCs can be engrafted into the patient`s liver to differentiate and mature into hepatocytes or pancreatic islet cells in vivo,and play the function of tissue functional repair and substitution.These basic and applied studies have proved that as a type of stem cells with low immunogenicity and can differentiate into functional hepatocytes in vivo and in vitro,BTSCs has advantages in stem cellbased ESLDs and even in the treatment of diabetes,and has a wide application prospect.However,BTSCs are adult stem cells with a limited number.In the previous study,the purified primary BTSCs could be obtained in a defined serum-free Kubota`s Medium(KM),but the passage and expansion of BTSCs could not be realized in this system.Therefore,the limited number of cells in the clinical application of BTSCs needs to be solved.The establishment of an ideal in vitro expansion system is the key to maintain cell stemness and normal physiological function.At present,the culture and amplification of h BTSCs is mainly based on the in vitro expansion system of other endodermal organ stem cells(such as intestinal stem cells,hepatic stem cells,etc.),which mainly includes two systems: co-culture and Matrigel-based culture.In view of the complicated operation of the co-culture system and the possible contamination in the preparation process of the feeder layer,the threedimensional culture method based on hydrogel has gradually become the preferred method to realize the in vitro expansion of endoderm organ-derived epithelial stem cells.At present,the three-dimensional system of hydrogel is mainly based on Matrigel base hydrogel.Matrigel is a matrix component extracted from mouse chondrosarcoma.Its main components include laminin,type IV collagen,heparan sulfate glycoprotein,matrix metalloproteinases(MMPs),and so on.It has been proved to be used for long-term culture and expansion of a variety of endodermal stem cells by adding a combination of growth factors to the Matrigel.Huch M et al.expanded the hepatic stem cells into organoids in vitro through Matrigel-based system for several months.Small molecular compounds are the key components in the expansion of endodermal stem cells in vitro.Small molecular compounds are monomers with molecular weights less than 500 Daltons,which have been proved to be effective in improving the efficiency of somatic reprogramming in i PSCs studies.Wang et al.used small molecular compounds(transforming growth factor-superfamily type I activator receptor-like kinase receptor ALK4,SB431542,inhibitor of ALK5 and ALK7,non-ATP competitive MEK inhibitor PD0325901,G9a-like protein and G9 a histone lysine methyltransferase inhibitor Bix01294,non-nucleoside inhibitor RG108 of DNA methyltransferase,calcium channel inhibitor Bay K8644,etc.)to successfully induced gastric epithelial cells into an endodermal stem cells-liked human induced endodermal progenitor cells(hi Endo PCs),and continue to achieve the long-term passage and expansion of hi Endo PCs in this system.This method based on small molecular compounds instead of growth factors greatly increases the safety in the process of stem cell expansion and effectively reduces the cost of stem cell expansion.Therefore,the combination of Matrigel and small molecular compounds can provide a feasible scheme for the amplification of BTSCs in vitro.In this study,we optimized the original KM medium and constructing Glycogel system.The improved amplification medium p KM is formed by screening the cytokines and small molecular compounds that promote the expansion of BTSCs,and additional biomatrix is added to realize the passage and expansion of biliary tree stem cells.The biomatrix in the Glycogel system may be chemically defined,such as collagen or collagen hydrogel,or nonchemically defined,such as decellularized bio-scaffold.Suitable composite protein hydrogels may contain extracellular matrix components,such as laminin,collagen IV,hyaluronic acid and heparan sulfate proteoglycans(HSPGs).And,here we use two kinds: The first one is the biomatrix which can be used for embedding BTSCs which based on the hydrogel of extracellular matrix proteins from mouse sarcoma cells.It is available from commercial sources and includes Matrigel(Corning).The other one is based on the biomatrix which can be added into the culture medium,a HAHS hydrogel which generated by mixing hyaluronic acid(HA)with heparan sulfate(HS).Based on our previous research,a Glycogel culture system composed of glycosaminoglycan(GAGs)was established.The BTSCs expansion method provided by this system can not only provide a stable 3D microenvironment for BTSCs,but also increase the expansion dimension while reduce the adverse effects of external stimulation on stem cells,ensure the proliferation while maintaining the stemness of BTSCs,and provide sufficient and functionally stable seed cells for the treatment of ESLDs using BTSCs.At the same time,the establishment of this system can also provide a reference experimental scheme for the clinical application of other stem cell-derived hepatocytes,and finally achieve a breakthrough in the treatment of liver diseases by cell transplantation.Methods1.According to the experimental methods of human BTSCs,the murine extrahepatic BTSCs were isolated and cultured in KM medium.According to the biological characteristics of BTSCs obtained by other species,the evaluation scheme was established to identify the target cells and identify the developmental lineage stages.The similarity between murine BTSCs and human BTSCs was analyzed by transcriptome sequencing.2.Referring to the related studies,the Glycogel components of cytokines and small molecular compounds that promote the expansion of BTSCs were screened to construct the expansion medium.The growth state of cells was observed after changing culture medium.Through the comparison of q PCR,immunohistochemistry,flow analysis and other experimental techniques to verify the effect of the amplification medium,and to identify whether to maintain the stability of BTSCs dryness during the rapid expansion of the system.3.To explore the expansion conditions of glycosaminoglycan(GAGs)combination(Matrigel and HAHS)to realize the 2D and 3D passage of murine BTSCs.4.To judge the viability of BTSC organoids with the live/dead assay staining kit.5.The p KM components for the expansion of primary BTSCs include 4 growth factors(EGF,R-Spindin1,Noggin,Wnt3a),and 6 small molecular compounds(Bix01294,Bay K8644,RG108,SB431542,A83-01,Forskolin),which was named as 10p(p KM).10 p was divided into groups according to signal pathway,and 10 p was evaluated by the formation efficiency and number of BTSC organoids.6.The reason why 3D Glycogel promotes BTSCs amplification compared with 2D Glycogel was analyzed by transcriptome sequencing.7.3-O-HS sulfotransferase(HS3ST1)were used to study the mechanism of 3-O-heparan sulfate on maintenance and in vitro expansion of murine BTSCs.Results1.The primary murine BTSCs isolated from mice extrahepatic bile duct formed colonies and grew slowly in the Kubota`s medium.They express the typical BTSCs marker genes Ep CAM,SOX17 and PDX1 both with the detection of q PCR and by immunohistochemistry(IHC)staining.Flow cytometry analysis of the colonies cultured for 21 days showed that the percentage of Ep CAM+ cells in cultured cells was about 84%.The cells were counted on the 14 th day,and about 2 × 104 target cells were obtained per mouse.2.The isolated primary murine BTSCs were rapidly expanded in 10 p KM expansion medium,and 4.2x105 target cells were obtained by cell counting on the 7th day,and the number of cells increased greatly.The morphology of the cells showed epithelioid cells adhered to the plate,with high nucleus/ cytoplasm ratio.They also expressed BTSCs specific marker genes Ep CAM,SOX17 and PDX1 both by q PCR and IHC staining.Flow cytometry analysis on the 7th day showed that the proportion of Ep CAM+ in cultured cells was about 82%.3.The 10 p in the expansion medium was evaluated,and it was found that RG108,A83-01,Forskolin,Bay K8644 and RSPO1 were essential for the expansion and maintenance of BTSCs,which lead to 5p KM as the final selected BTSCs expansion medium.4.Rapid expansion and subculture can be achieved by using Matrigel embedding method to form organoids during different passages.This 3D organoid expansion method was sufficient for expanding BTSCs for least 6 passages with stable expression of BTSCs specific genes In total of 3x107 cells can be obtained from 2x104 P0 BTSCs.5.In addition,in the absence of Matrigel,hyaluronic acid hydrogels alone are not enough to support the in vitro expansion and maintenance of BTSCs,but the addition of heparan sulfate and hyaluronic acid hydrogels can promote the attachment of BTSCs and achieve the passage expansion of BTSCs.6.Bioinformatics data indicated that 3-O modified heparan sulfate enzymes(HS3ST1 and HS3 ST 3A)were expressed in BTSCs.The results of HS3ST1 expression were consistent with q PCR.Conclusions1.Murine BTSCs colonies could be obtained by culture in KM medium.Flow cytometry analysis showed that the proportion of Ep CAM+ cells was 84 ±10.58%.2.The BTSCs expansion medium composed of growth factors and small molecular compounds(10p)was 10 p KM,which could effectively expand the primary BTSCs,obtain a large number of cells and the stemness is stable.3.The 5pKM that play a key role in dry maintenance and proliferation of BTSCs in vitro were identified through stepwise screening.4.The Glycogel components suitable for in vitro amplification of BTSCs were screened.Two-dimensional and three-dimensional pass amplification methods based on Glycogel system BTSCs are established.BTSC organoids can be obtained by Matrigel based Glycogel system,which can pass through at least 6 generations.5.In the Glycogel of hyaluronic acid plus heparan sulfate(HAHS),heparan sulfate is the key to support the proliferation of BTSCs in vitro in HAHS.6.HS3ST1 was highly expressed in BTSCs.It was confirmed that 3-Oheparan sulfate was the key subtype of heparan sulfate supporting the proliferation of BTSCs in vitro.In summary,we established a defined Glycogel system through murine BTSCs,and provided two culture methods of 2D and 3D of BTSCs,and can achieve stable passage and expansion through 3D organoids culture,which provides a reference for the expansion of human BTSCs in the future.This work will solve the problem of insufficient clinical application of BTSCs and create conditions for the clinical application of BTSCs.