Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells

A technology for pluripotent stem cells and stem cell differentiation, which is applied in the field of inducing the directional differentiation of human induced pluripotent stem cells into pancreatic cells, which can solve the problem that the differentiation efficiency is only 16.9%.

Active Publication Date: 2015-06-24
深圳市诺亚起航生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Then, Kunisada et al. used a four-step method to successfully induce human iPS cells to differentiate into Ins + / SST + / Gluc + / Ghre + pancreatic cell population, but its differentiation efficiency is only 16.9%

Method used

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  • Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells
  • Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells
  • Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Substances inhibiting LSD1 gene expression promote the differentiation of hiPSCs into endoderm cells HiPSCs derived from human skin fibroblasts (international common code: hNF1④C11).

[0074] The four kinds of virus liquids packaged with different shRNA-LSD1 sequences are all different virus liquids obtained by introducing different shRNA-LSD1 into hNF1④C11 cells (hereinafter referred to as hiPSCs) through the lentiviral vector pGLV2-U6-Puro. Prepared by Pharmaceutical Co., Ltd.

[0075] The above shRNA-LSD1 sequence is specifically as follows:

[0076] LSD1-homo-863 5'-GCAGTTGTGGTTGGATAATCC-3' (SEQ ID NO: 1)

[0077] LSD1-homo-1086 5'-GCAGCTCGACAGTTACAAAGT-3' (SEQ ID NO: 2)

[0078] LSD1-homo-927 5'-GCACCTTATAACAGTGATACT-3' (SEQ ID NO: 3)

[0079] LSD1-homo-2495 5'-GGGCTCTTATTCCTATGTTGC-3' (SEQ ID NO: 4)

[0080] Scrambled sequence 5'-GTCAAGTCTCACTTGCGTCAC-3' (sequence 5)

[0081] wxya TM 1 Culture medium set (stem cell technologies, Cat. No.: 05850, Can...

Embodiment 2

[0177] Example 2. Highly efficient and directed differentiation of hiPSCs cells inhibited by LSD1 gene expression into IPCs in vitro

[0178] 1. Induction and differentiation of hiPSCs

[0179] Conventional induction group: normal hiPSCs were directly induced by the four-step induction method;

[0180] Experimental group (RNAi induction group):

[0181] A. Inhibition of LSD1 gene expression

[0182]LSD1-homo-927 was transfected into hiPSCs cells by LSD1-homo-927-packaging virus to obtain transfected shRNA-LSD1-927 cells. After screening with Puro at a concentration of 1 μg / ml for 72 hours, the survival was positive transfection of shRNA-LSD1-927 The cells are LSD1-homo-927.

[0183] B. LSD1-homo-927 cells were induced by the following four-step induction method:

[0184] 1. The first stage of differentiation: differentiation to definitive endoderm

[0185] Connect hiPSCs and LSD1-homo-927 cells to Matrigel-coated culture flasks or culture plates, add mTeSR TM 1 culture m...

Embodiment 3

[0283] Example 3, Observation of curative effect of IPCs transplantation in treating diabetes

[0284] The experimental mouse SCID-Beige (male) was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and was kept in the SPF animal room of Shenzhen Center for Disease Control and Prevention.

[0285] 1. Preparation of diabetic model mice

[0286] (1) After adaptive feeding of SCID-Beige mice for 1 week (the mice had reached 9 weeks of age at this time, and their body weight was between 20g and 25g), they were randomly divided into 2 groups according to their body weight: Group 1 (5 mice ) was used as the follow-up normal group; the second group (35 rats) was used for modeling.

[0287] (2) The above 35 mice were fasted (without water) for 12 hours, intraperitoneally injected with STZ solution once at a dose of 175 mg / kg, and fed normally immediately after injection;

[0288] (3) The fasting blood glucose was monitored by tail vein blood collection ev...

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Abstract

The invention discloses a method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells. The invention provides an application of a substance for inhibiting LSD1 gene expression in inducing the directional differentiation of stem cells into endoderm cells or an application of the substance for inhibiting LSD1 gene expression in inducing the directional differentiation of the stem cells into the pancreatic cells. Experiments prove that an LSD1 inhibitor or shRNA is to be adopted in the study to inhibit or silence the LSD1 expression level in hiPSCs at different degrees, the shRNA is screened out so that the transformation of the hiPSCs from proliferation into entoderm differentiation can be better promoted to prepare for promoting the further uniform differentiation state of the hiPSCs; and next, an established four-step induced differentiation system is adopted to induce efficient directional differentiation into IPCs. The theoretical basis is laid for optimizing or establishing an efficient and directional differentiation scheme for the hiPSCs to the IPCs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing human induced pluripotent stem cells to differentiate into pancreatic cells. Background technique [0002] Diabetes mellitus is a group of metabolic diseases characterized by chronically elevated blood glucose levels, which are caused by defects in insulin secretion and / or insulin action. Islet transplantation has been considered the most promising cure for diabetes. However, islet transplantation requires 2-3 donors to meet the needs of a patient, and the serious shortage of islet donors limits the wide clinical application of islet transplantation. Therefore, finding the regeneration source (or replacement cells) of islet cells is a problem to be solved. [0003] With the rapid development of related research in the field of stem cells and tissue engineering, people have focused their attention on stem cells to find the source of islet cell regeneration. Cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/867C12N5/10
Inventor 李富荣周淑艳闫红杰齐晖
Owner 深圳市诺亚起航生物科技有限公司
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