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CN-121975724-A - Construction method and application of full-lineage liver organoid

CN121975724ACN 121975724 ACN121975724 ACN 121975724ACN-121975724-A

Abstract

The invention relates to the field of biomedical engineering and disease model construction, in particular to a construction method and application of full-lineage liver organoids. The construction method comprises the steps of respectively differentiating induced pluripotent stem cells from human umbilical cord sources to obtain transitional hepatic endoderm cells, endothelial progenitor cells, erythroid progenitor cells, hepatic stellate cells and diaphragm mesenchymal stem cells, respectively inoculating the transitional hepatic endoderm cells, the endothelial progenitor cells, the erythroid progenitor cells, the hepatic stellate cells and the diaphragm mesenchymal stem cells into a 3D culture system according to the cell number ratio of 9-11:6-8:1-2:1-2:1-2, and performing self-assembly co-culture on the various seed cells in the 3D culture system until liver functions are mature to obtain the full-lineage liver organoid. The invention solves the technical problems that the physiological reality, the functional integrity and the application universality of the existing liver model are all to be improved.

Inventors

  • WU YIXING
  • WANG YANG
  • ZHANG WENJIE
  • YUAN LILI

Assignees

  • 中国计量大学

Dates

Publication Date
20260505
Application Date
20260408

Claims (10)

  1. 1. A method for constructing a full-lineage liver organoid, comprising the steps of: s100, respectively differentiating induced pluripotent stem cells from human umbilical cord sources to obtain seed cells of transitional hepatic endoderm cells, seed cells of endothelial progenitor cells, seed cells of erythroid progenitor cells, seed cells of hepatic stellate cells and seed cells of diaphragm mesenchymal stem cells; S200, respectively inoculating seed cells of transitional liver endoderm cells, seed cells of endothelial progenitor cells, seed cells of erythrocyte progenitor cells, seed cells of hepatic stellate cells and seed cells of diaphragm mesenchymal stem cells into a 3D culture system according to the cell number ratio of 9-11:6-8:1-2:1-2:1-2; s300, performing self-assembly co-culture on various sub-cells in a 3D culture system until liver functions are mature, and obtaining the full-lineage liver organoid.
  2. 2. The method for constructing a full-lineage liver organoid according to claim 1, wherein the step of obtaining transitional liver endoderm cell seed cells from induced pluripotent stem cells derived from human umbilical cord comprises: s111, passaging induced pluripotent stem cells from the recovered human umbilical cord to cell stability to obtain hiPSC cells, and amplifying the hiPSC cells in a pore plate paved with matrigel, wherein the hiPSC cells are used for differentiation when growing to 60% -70% of cell confluence; s112, culturing hiPSC cells by using a DE-1 differentiation medium to start and induce DE differentiation of the hiPSC cells; S113, replacing the culture medium with a DE-2 differentiation medium to continuously culture hiPSC cells so as to strengthen and induce the formation of DE cells; S114, continuously culturing the hiPSC cells by replacing the culture medium with the HE differentiation medium to perform differentiation culture at the HE stage, so as to obtain the hiPSC-HE cells, and ending the differentiation culture until the expression of the hiPSC-HE cells is down-regulated relative to the expression of genes NANOG and OCT4 in the hiPSC cells, and meanwhile, the expression of endodermal marker genes FOXA2, SOX17, EOMES, GATA4, GATA6, HNF4a and HHEX are up-regulated, so that the obtained hiPSC-HE cells are seed cells of transitional intrahepatic endodermal cells.
  3. 3. The method for constructing a full-lineage liver organoid according to claim 1, wherein the step of obtaining seed cells of endothelial progenitor cells from human umbilical cord-derived induced pluripotent stem cells comprises: S121, passaging induced pluripotent stem cells from the recovered human umbilical cord to cell stability to obtain hiPSC cells, and amplifying the hiPSC cells in a pore plate paved with matrigel, wherein the hiPSC cells are used for differentiation when growing to 60% -70% of cell confluence; s122, culturing hiPSC cells by adopting an EPC mesoderm differentiation medium to perform EPC differentiation so as to induce the differentiation of the hiPSC cells to form mesoderm; s123, replacing the culture medium with an EPC induction culture medium, continuously culturing hiPSC cells to perform EPC differentiation, differentiating the hiPSC cells into EPC on the basis of mesoderm to obtain hiPSC-EPCs cells, and ending differentiation culture until the expression of CD31 and CD144 proteins in the hiPSC-EPCs cells is positive, wherein the obtained hiPSC-EPCs cells are seed cells of endothelial progenitor cells.
  4. 4. The method for constructing a full-lineage liver organoid according to claim 1, wherein the step of obtaining seed cells of erythroid progenitor cells from human umbilical cord-derived induced pluripotent stem cells by differentiation comprises: S131, passaging the induced pluripotent stem cells of the human umbilical cord source recovered by S131 to cell stability to obtain hiPSC cells, and amplifying the hiPSC cells in a pore plate paved with matrigel, wherein the hiPSC cells are used for differentiation when growing to 60% -70% of cell confluence; s132, culturing hiPSC cells by using an EMP mesoderm induction medium to induce the generation of premature germ cells; S133, replacing the culture medium with the EMP hematopoietic induction medium to continuously culture the hiPSC cells so as to specialize and generate hematopoietic progenitor cells, thereby obtaining hiPSC-EMP cells, and ending the differentiation culture until the expression of the hiPSC-EMP cells is up-regulated relative to genes CD34, CD43, CSF1R, CD14, CD68, CD163 and ID3 in the hiPSC cells, thereby obtaining the hiPSC-EMP cells, namely seed cells of the erythroid progenitor cells.
  5. 5. The method for constructing a full-lineage liver organoid according to claim 1, wherein the step of obtaining seed cells of hepatic stellate cells from human umbilical cord-derived induced pluripotent stem cells comprises: s141, passaging induced pluripotent stem cells from human umbilical cord recovered by S141 to cell stability to obtain hiPSC cells, amplifying the hiPSC cells in a pore plate paved with matrigel, and differentiating when the hiPSC cells grow to 60% -70% of cell confluency; S142, culturing hiPSC cells by sequentially adopting a first LDM culture medium, a second LDM culture medium, a third LDM culture medium and a fourth LDM culture medium to obtain hiPSC-HSC cells, and ending differentiation culture until the expression levels of the gene Desmin, GFAP, PCDH and the PDGFRB are in an ascending trend along with the time, wherein the obtained hiPSC-HSC cells are seed cells of hepatic stellate cells.
  6. 6. The method for constructing a full-lineage liver organoid according to claim 1, wherein the step of obtaining seed cells of diaphragm mesenchymal stem cells from induced pluripotent stem cells derived from human umbilical cord is: S151, passaging induced pluripotent stem cells from the human umbilical cord recovered by S151 to cell stability to obtain hiPSC cells, and amplifying the hiPSC cells in a pore plate paved with matrigel, wherein the hiPSC cells are used for differentiation when growing to 60% -70% of cell confluence; S152, culturing hiPSC cells by adopting STM mesoderm induction culture medium to induce differentiation of mesoderm cells; S153, replacing the culture medium with STM differentiation medium to continuously culture the hiPSC cells, so as to specialize and expand the mesenchymal stem cells of the diaphragm, obtain hiPSC-STM cells, and ending the differentiation culture until the expression of the hiPSC-STM cells relative to genes GATA4, FOXF1, COL4A1 and ALCAM in the hiPSC cells is up-regulated, wherein the obtained hiPSC-STM cells are seed cells of the mesenchymal stem cells of the diaphragm.
  7. 7. The method of claim 1, wherein the 3D culture system is one of Matrigel-bed, ELPLASIA PLATES and ultra-low adsorption plate, and 5% GFR-Matrigel is additionally added to LOM of the ultra-low adsorption plate.
  8. 8. The method according to claim 1, wherein the step S300 is performed for 13-15 days by self-assembled co-culture, and wherein the liver function is mature to the full-lineage liver organoid expressing ALB, CYP3A4, A1AT, FVIII and NTCP and urea is produced.
  9. 9. Use of a method of constructing a full-lineage liver organoid according to claim 1 in constructing a liver disease model, a drug hepatotoxicity evaluation model, or a hepatocyte-to-hepatocyte interaction study model.
  10. 10. Use of a method of constructing a full lineage liver organoid according to claim 1, in constructing a liver inflammation model.

Description

Construction method and application of full-lineage liver organoid Technical Field The invention relates to the field of biomedical engineering and disease model construction, in particular to a construction method and application of full-lineage liver organoids. Background The liver is the most important metabolic organ of human body and plays the role of detoxification, protein synthesis, drug metabolism and other complex physiological functions. Worldwide, the incidence and mortality of liver diseases such as fatty liver disease, viral hepatitis, liver cirrhosis and liver cancer continue to rise, and thus, the disease has become a significant public health burden. This severe disease situation has created an urgent need for the deep revealing of liver disease mechanisms, speeding up the safe and effective drug development, all of which are highly dependent on reliable models that can highly mimic the complex physiological and pathological states of the human liver in vitro. Currently, the mainstream liver model is mainly divided into three types, namely liver cancer cell lines (such as HepG2 and Huh 7), primary human liver cells (PHH) and liver organoids based on different cell sources, wherein the liver organoids of different cell sources mainly comprise two types of multipotent stem cell differentiation and adult stem cell sources. Among them, pluripotent stem cells include Human induced pluripotent stem cells (Human induced pluripotent STEM CELLS, hiPSC) and Human embryonic stem cells (hESC) STEM CELLS. Although the liver cancer cell line model has the operational advantages of easy acquisition, high standardization degree, convenient large-scale culture and the like, the liver cancer cell line model has essential differences with normal liver tissues in the aspects of metabolic functions, gene expression profiles and the like. Primary human hepatocytes, although acting as "gold standard" for traditional liver studies, face three major core challenges, namely scarcity of donor sources, significant batch-to-batch differences, and rapid dedifferentiation in vitro culture. Although the liver organoids derived from adult stem cells solve the problems of long-term culture and expansion, key defects of single cell component (usually only containing liver parenchymal cells or cholangiocytes) are common, and the complex structure and micro-environmental system of the liver are difficult to simulate. Wherein, the differentiation system of the pluripotent stem cells (human induced pluripotent stem cells/human embryonic stem cells) can realize the self-assembly of various types of liver cells through three-dimensional culture, and the method has unique advantages in the aspect of reproducing the liver tissue structure and cell interaction. However, to date, a full-lineage organoid model integrating five key hepatocyte types (liver parenchymal cells, cholangiocytes, kupffer cells, liver sinus endothelial cells and hepatic stellate cells) at the same time has not been established, and this blank seriously affects the physiological relevance of the liver model, and is a bottleneck problem restricting liver disease research and related drug development. In summary, the existing liver model has significant shortcomings in physiological reality, functional integrity and application universality. Thus, there is a need in the art to develop a novel full lineage liver organoid model. The establishment of the model can fundamentally break through the bottleneck of the current liver disease mechanism research and drug screening, and provides an irreplaceable powerful tool for liver physiology research, disease modeling, personalized medicine and drug hepatotoxicity evaluation. Disclosure of Invention The invention provides a construction method and application of a full-lineage liver organoid, and aims to solve the technical problems that the physiological reality, the functional integrity and the application universality of the existing liver model are to be improved. The technical scheme is as follows: the first aspect of the invention provides a method for constructing liver organoids of full lineages, which comprises the following steps: s100, respectively differentiating induced pluripotent stem cells from human umbilical cord sources to obtain seed cells of transitional hepatic endoderm cells, seed cells of endothelial progenitor cells, seed cells of erythroid progenitor cells, seed cells of hepatic stellate cells and seed cells of diaphragm mesenchymal stem cells; S200, respectively inoculating seed cells of transitional liver endoderm cells, seed cells of endothelial progenitor cells, seed cells of erythrocyte progenitor cells, seed cells of hepatic stellate cells and seed cells of diaphragm mesenchymal stem cells into a 3D culture system according to the cell number ratio of 9-11:6-8:1-2:1-2:1-2; s300, performing self-assembly co-culture on various sub-cells in a 3D culture system until liver functions are mature,