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Culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets

A medium and cell technology, applied in the field of stem cells, can solve the problems affecting the clinical application of induced pancreatic islets, low repeatability, and long differentiation time.

Active Publication Date: 2021-01-22
ALLIFE MEDICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the induction process is often divided into dry and dry stages, and the differentiation time is long, in the actual operation process, the repeatability is often low, the induction efficiency is poor, and the by-products seriously affect the clinical application of induced islets.

Method used

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  • Culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets
  • Culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets
  • Culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0471] Stage1: Differentiation of iPSCs to Committed Endoderm

[0472] First, the Matrigel matrix was plated on a cell culture plate, and E8 complete medium was added, and placed in a cell culture incubator for one day to obtain a plated cell culture plate, and then the E8 medium was discarded.

[0473] Use E8 medium to inoculate iPS cells to a cell density of 150,000-200,000 / cm 2 , when the cell confluency reached over 90%, the medium was replaced with stage1 medium A (containing 100ng / ml GDF8, 3μmol / L CHIR-99021, 3g / L sodium bicarbonate, 2mmol / L (100×) glutamine , 20mmol / L glucose, 2% BSA (fetal bovine albumin) in MCDB131 medium), induced 1d.

[0474] On the 2nd day, replace the culture with stage1 medium B (containing 100ng / ml GDF8, 3g / L sodium bicarbonate, 2mmol / L (100×) glutamine, 20mmol / L glucose, 2% BSA (fetal bovine albumin) MCDB131 medium) induced 1d;

[0475] On the 3rd day, replace with fresh stage1 medium B (containing 100ng / ml GDF8, 3g / L sodium bicarbonate, 2mm...

Embodiment 2

[0481] Stage 2: Differentiation of directed endoderm to gastrula tube

[0482] On the 4th day, the culture medium of endoderm cells induced in Example 1 was replaced with stage2 medium: containing 50ng / ml FGF7, 0.25mmol / L ascorbic acid, 3g / L sodium bicarbonate, 2mmol / L (100×) glutamine Amide, 20mmol / L glucose, 2% BSA (fetal bovine albumin) MCDB131 medium, replace fresh culture medium every day, induce 2 days, and obtain original intestinal tube cells.

[0483] After the above 2-day induction, the cells became denser and thicker, and grew in layers. It can be seen that the nuclear-to-cytoplasmic ratio of the cells almost disappeared, and the gaps between the cells showed a raised shape, and cord-like and spherical structures began to appear. ( Image 6 ).

[0484] The induced cells are detected with FOXA2 and HNF1B as markers, and the positive rate of HNF1B of the obtained endoderm cells should not be lower than 80%, and the positive rate of FOXA2 should not be lower than 90%...

Embodiment 3

[0488] Stage 3: Differentiation of protogut tube to posterior foregut

[0489] On the 6th day, the culture medium of endoderm cells induced in Example 2 was replaced with stage3 medium: containing 50ng / ml FGF7, 0.25mmol / L ascorbic acid, 0.25μmol / L SANT-1, 1μmol / L RA, 100nmol / L LDN193189, 2mmol / L ITS-X, 200nmol / L TPB, 3g / L sodium bicarbonate, 2mmol / L (100×) glutamine, 20mmol / L glucose, 2% BSA (fetal bovine albumin) of MCDB131 Medium. Fresh culture medium was replaced every day, induced for 2 days, and hind-foregut cells were obtained.

[0490] After the above 2-day induction, it can be clearly observed that the flat cells at the bottom layer gradually become thinner, and the spherical and ellipsoidal structures gradually increase. Figure 8 .

[0491] In order to control the quality of the obtained cells, the positive rate of PDX1 of the obtained posterior foregut cells should not be lower than 50%, and the positive rate of SOX9 should not be lower than 50%.

[0492] At the...

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Abstract

The invention relates to the technical field of stem cells, and especially relates to culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets. In the provided differentiation methods, a differentiation process from iPS stem cells to pancreatic progenitor cells needs 14 days (2 weeks); and the process mainly includes following four steps:directional endoderm - archenteric canal -foregut / hindgut - pancreatic progenitor cells. The differentiation process from the pancreatic progenitor cells to functional islets needs about 31 days (1 month); and the process is mainly divided into following three steps: endocrine progenitor cells - early islets - islets in late functional maturity. Each of the above steps needs specific induction culture media; and the culture media can be formed by adding corresponding inducing factors into basic media, and the corresponding culture media need to be daily replaced for culture. Feeder layer cells are not needed during the whole induction of the method; and the adopted culture media have no animal-derived components, so that the risks of alloimmunization can be reduced.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a culture medium and its application and a method for differentiating induced pluripotent stem cells to islets. Background technique [0002] Diabetes Mellitus (DM) is an endocrine and metabolic disorder characterized by insufficient insulin production or functional defects caused by multiple genetic and environmental factors, and characterized by abnormally elevated blood sugar. Due to the excessive accumulation of glucose in the blood, various pathogenic factors will act on the major target organs of the body, leading to the formation of a series of complications. Diabetes is one of the world's most recognized chronic diseases and has become the third largest chronic disease and the fifth leading cause of death in the world. [0003] According to the classification recommended by the World Health Organization, diabetes can be divided into two types: type I diabetes with abs...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K35/39A61P3/10
CPCC12N5/0676C12N5/0678A61K35/39A61P3/10C12N2506/45C12N2501/10C12N2500/32C12N2500/34C12N2500/05C12N2501/998
Inventor 吴理达顾雨春张会远安翠平
Owner ALLIFE MEDICAL SCI & TECH CO LTD
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