Expansion Of Human Pluripotent Stem Cells In 3D Suspension Culture And Their Differentiation Toward Hepatocytes

Human pluripotent stem cells(hPSCs)and their derivatives widely used in drug screening,disease models,cell transplantation and clinical treatment,whichrequires to obtain a large quantity and highquality of human pluripotent stem cells as a basis at first.Combined stirred bioreactors withthe three-dimensional(3D)systems in the form of cell aggregates or microcarriers has emerged as a promising strategy for large-scale production of cells.Liver transplantation is the first choice for patients withacute liver failure.However,due to the lack of donor livers,liver cell therapy and bioartificial liver therapy are currently important treatments for prolonging the life of patients withacute liver failure.Eachtreatment requires at least 10¹⁰functional liver cells,the need is more urgent to develop a protocol to scalability produce clinically relevant numbers of functional liver cells.The differentiation of human pluripotent stem cells into functional liver cells brings hope to these patients.Objective:To overcome the significant challenge withregards to culture scalability to produce clinically relevant numbers of human pluripotent stem cells and their derivatives,we tried to develop a protocol for efficient expansion of hPSCs and establisha protocol for directional differentiation of liver cells from hPSCs in the 3D suspension culture system.These routes are promising strategies for obtaining large-scale production and high-quality hPSCs as important seed cells for functional liver cells differentiation.Methods:1.3D suspension culture of human pluripotent stem cells in the form of cell aggregates under static or dynamic conditions;2.Photographing of cell aggregates,cell counting,size analysis of cell aggregates,and cell viability was assessed using the Cell Counting Kit(CCK-8)assay;3.Real-time quantitative polymerase chain reaction(RT-q PCR),western blot(WB),flow cytometry(FACS)analysis and immunofluorescence staining of the aggregates verified that the cells harvested from the 3D suspension culture system can maintain self-renewal ability;4.Embryoid body(EB)formation experiment,immunofluorescence staining analysis of EB and teratoma formation experiments verified that the cells harvested from 3D suspension culture system can maintain the potential for multidirectional differentiation;5.Assessed the possible mechanisms of controlled the size of aggregation of dextran sulfate(DS)and promoted cell proliferation of polyvinyl alcohol(PVA)by transcriptome sequencing(m RNA-seq)and data analysis;6.Using water/oil/water(W/O/W)double emulsification technology to develop porous and solid PLGA-microspheres;7.Using tungsten filament scanning electron microscope(SEM)to perform gold-sprayed scanning imaging on the developed porous and solid microcarriers;8.Using RT-q PCR and FACS to analyze differentiation efficiency of definitive endoderm,hepatoblasts and hepatocytes from human pluripotent stem cells;9.Using immunofluorescence staining,agarose-embedded sections and H&E staining to analyze the structure and function of liver progenitor cell spheroids;10.Using ELISA to detect the secretion of ALB in the supernatant of the culture medium,and performing drug induction by omeprazole and rifampin to detect the expression level of drug-metabolizing enzymes,whichcan assess the function of the hepatocytes spheroids and the ability of drug metabolism.Results:In the present study of this subject,we developed a chemical-based approachfor expansion of ex vivo hPSCs suspension culture supplemented withpoly(vinyl alcohol)(PVA)and dextran sulfate(DS).Our results demonstrated that the combination of DS and PVA not only promoted cell proliferation of hPSCs but also produced uniform and size-controlled cell aggregates.Moreover,hPSCs treated withPVA,or DS or a combination,maintained the pluripotency and were capable of differentiating into all three germ layers.m RNA-sequencing analyses demonstrated that the combination of PVA and DS significantly promoted hPSCs proliferation and prevented cell aggregation throughimproving energy metabolism-related processes,regulating cell growth,cell proliferation and cell division,as well as reducing the adhesion among hPSCs aggregates by affecting expression of genes related to cell adhesion.Our results represent a significant step towards developing a simple and robust approachfor the expansion of hPSCs in large-scale.In addition,highquality,solid and porous PLGA-microcarriers were developed,whichwere subsequently applied on expansion and differentiation of human pluripotent stem cells.Finally,a high-efficiency and low-cost protocol of directional differentiation of liver cells from hPSCs in the 3D suspension culture system was established.Our hepatocytes spheroids exhibit well function of secreting albumin and ability of drug metabolism.All in all,we have developed a simple and reliable method for 3D suspension large-scale culture of hPSCs based on stirred bioreactor,to solve the problem of the urgent clinical demand for human pluripotent stem cells and their derivatives.