Optimization Of A Hepatic Differentiation Protocol For Human Umbilical Cord Mesenchymal Stem Cells And A Culture System For Liver Organoid Of Their Origin

Objective: To design and optimise a cytokine-drived differentiation protocol for stem cells(SCs)into hepatocytes(HLCs).The goal is to produce large numbers of homogeneous and functional HLCs in vitro in a stable and efficient manner.At the same time,a culture system for complex liver organoids(LOs)of HLCs origin is established to promote HLCs differentiation and maturation and overcome the disadvantages of conventional cell transplantation,such as a low survival rate,poor efficacy,and easy embolisation.Methods: Human umbilical cord mesenchymal stem cells(h UCMSCs)were isolated and extracted from human umbilical cord tissue.b FGF,EGF,HGF,OSM and other cytokines were added to the culture medium to prepare different stages of the differentiation medium.h UCMSCs were induced into HLCs in vitro after 23 d of continuous irrigation.Morphological cell changes were observed under a microscope.The expression of characteristic genes and proteins of hepatocytes within the HLCs were further detected by RT-PCR and cellular immunofluorescence.A 10:7:2 mixture of HLCs,human umbilical vein endothelial cells(HUVECs),and h UCMSCs cell suspension was added to Matrigel.After 6-7 d of culture,the three cells spontaneously assembled on Matrigel to form complex LOs,and the spatial distribution of cells within the LOs was observed by 3D live cell imaging.The morphology of the LOs or the expression and distribution of the three cell surface markers ALB,CD31,and Vimentin were determined by HE staining,immunohistochemical staining and tissue immunofluorescence.The expression of hepatocyte characteristic genes within the LOs were detected by RT-PCR.Results: Morphology showed that the cells changed from a long spindle-shaped,swirling arrangement to a round-like,partially binucleated,pavement-like arrangement under this differentiation protocol.RT-PCR results showed that,compared to undifferentiated h UCMSCs,the levels of ALB(P<0.001),A1AT(P<0.001),HNF4α(P<0.01),CYP1A1(P<0.05),and CYP1B1(P<0.001)m RNA increased with statistically significant differences.At the same time,AFP(P=0.204)m RNA changed but without statistically significant differences,whilst CK18(P<0.001)and CK19(P<0.001)m RNA decreased with statistically significant differences.Cellular immunofluorescence showed that,compared to undifferentiated h UCMSCs,differentiated 23 d HLCs were able to synthesise ALB,A1 AT,and AFP with green fluorescence in the cytoplasm of HLCs under fluorescence microscopy.Furthermore,HLCs,HUVECs,and h UCMSCs were mixed in proportion to suspensions and aggregated to form visible complex LOs at 24 h.With increasing time,complex LOs aggregated more and more tightly.After 6 d of complex LOs aggregation,3D live cell imaging showed that GFP-HLCs were distributed at the edges of the cell clusters,whilst more m Cherry-HUVECs were distributed in the centre of the clusters.HE staining showed that the LOs were single,oval-shaped clusters with large diameters,deep purple stained nuclei and light pink cytoplasm.Immunohistochemistry and tissue immunofluorescence showed that the three cells were not randomly distributed within the LOs.Instead,they showed an arrangement similar to the cells in liver tissue,with the HLCs arranged in tightly adherent clusters and those located in the central region of the LOs being more tightly arranged and highly differentiated.The HUVECs spontaneously aggregated to form tubular structures and were scattered within the LOs.The h UCMSCs were diffused between HLCs and HUVECs and served as mesenchymal cells supporting LOs growth.RT-PCR results showed that,compared to HLCs in 2D culture conditions,the m RNA levels of ALB(P<0.01),A1AT(P<0.05),and G6P(P<0.001)were significantly increased,whilst AFP(P<0.001)were significantly decreased in 3D complex LOs with statistically significant differences.Conclusions: This study designed and optimized a cytokine-derived differentiation protocol for SCs into HLCs.This protocol can produce a large number of homogeneous and functional h UCMSCs-derived HLCs in vitro in a stable and efficient manner.A culture system was established for HLCs-derived complex LOs,complex LOs possess more mature liver functions and phenotypes.Moreover,the complex LOs are about 0.1 cm in diameter and can be transplanted with cells in situ,overcoming the the shortcomings of low survival rate,poor efficacy and easy embolisation of cell infusion.