Modulation Of Wnt And Activin/Nodal Supports Efficient Derivation,Cloning And Suspension Expansion Of Human Pluripotent Stem Cells

Pluripotent stem cells(PSCs)include embryonic stem cells and induced pluripotent stem cells.Because PSCs have the ability of unlimited proliferation and differentiation into different three germ cells,they have important application value in the study of embryo or organ development,disease model construction,cell therapy,drug development and toxicity testing.However,there are some problems in the quantity and quality control of pluripotent stem cells.To solve the obstacles in the application of pluripotent stem cells,we need a stable culture system as a support,so that we can produce a large number of high-quality human PSCs in vitro.At present,there are many systems for the culture of human PSCs,such as MEF-CM?KSR/bFGF?m Te SR?E8?NHSM?5i LAF?t2i LG??LCDM.However,there are some problems in these different culture systems and cells cultured in different systems.Firstly,the complex components and heterologous proteins of the system restrict the clinical application;Secondly,it is difficult to carry out single cell passage or low cell survival rate in single cell passage,which leads to unstable and inefficient culture,which is not conducive to gene editing and single cell cloning screening,and limits the application of pluripotent stem cells;Thirdly,stem cells differentiate spontaneously in the process of culture,which affects the quality of cells;Fourthly,some systems will lead to the instability of the genome and the loss of genetic imprint of pluripotent stem cells;Fifthly,it is difficult to carry out large-scale amplification or low yield in large-scale amplification,which is difficult to meet the number requirements of cell application.Sixthly,poor compatibility with pluripotent stem cells.These problems greatly limit the application of PSCs.The establishment of a new culture system is of great significance for the high-quality production,efficient establishment and large-scale expansion of pluripotent stem cells,which can better promote the application of pluripotent stem cells.Recent studies have shown that the self-renewal and differentiation potential of pluripotent stem cells are regulated by different signaling pathways.For example,Wnt signaling pathway controls cell fate and Histogenesis,and regulates the proliferation or differentiation of pluripotent stem cells during embryonic development in rodents and primates.Activin/Nodal signaling pathway can directly regulate the expression of Nanog and other pluripotent transcription factors during the embryonic development of primates,and can also maintain the self-renewal and promote the proliferation of stem cells in human pluripotent stem cells.Therefore,this signaling pathway plays an important role in the maintenance of human pluripotency both in vivo and in vitro.In general,Wnt and Activin/Nodal signaling pathways play an important role in regulating the pluripotency of human stem cells.However,whether Wnt and Activin/Nodal signaling can be precisely regulated to maintain the self-renewal of pluripotent stem cells remains unclear.In this study,we used different concentrations of small molecules and growth factors that can regulate these two signaling pathways to screen a new culture system of pluripotent stem cells.Finally,a new pluripotent stem cell culture system named AIC system(10 ng/ml Activin-A,2?M IWP2 and 0.6?M CHIR).The composition of the system is clear and there is no heterologous component,which is conducive to clinical application.This system can co regulate Wnt and Activin/Nodal signaling pathways to maintain the self-renewal of pluripotent stem cells.AIC system supports the establishment of new stem cell lines directly from blastocysts or by somatic cell reprogramming,which is more efficient than the current commonly used system.It also supports the transformation of existing stem cell lines to AIC culture system.Moreover,AIC system can support feeder layer,Feeder-free(matrix: Matrigel,fibronectin,etc.)and suspension culture mode to meet different experimental needs.At the same time,AIC system also supports efficient suspension expansion,and can proliferate about 25 times in 4 days,which lays a foundation for large-scale culture of stem cells;AIC system has better viability and higher efficiency of single cell cloning(> 90%),which is conducive to gene editing and other operations;AIC system had high homogeneity,reduced spontaneous differentiation and improved cell quality.The long-term cultured cells in AIC system maintained the stability of genome and improved the safety;At the same time,AIC system has good compatibility with different stem cell lines and can maintain the pluripotency of different stem cell lines.We also explored the maintenance mechanism of Wnt and Activin/Nodal signaling pathway on stem cell pluripotency.In a word,AIC system established by regulating Wnt and Activin/Nodal signal can solve the problems existing in the existing culture system of pluripotent stem cells,and then solve the requirements of cell quality and quantity in the application process of pluripotent stem cells,which is conducive to the establishment of GMP grade stem cells,and lay the foundation for the clinical transformation and application of pluripotent stem cells.