Method for differentiating inducible pluripotent stem cells into testicular interstitial cells by small molecule induction

A technology of pluripotent stem cells and Leydig cells, applied in the field of Leydig cells

Active Publication Date: 2018-11-13
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, there has not been a method for inducing the differenti

Method used

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  • Method for differentiating inducible pluripotent stem cells into testicular interstitial cells by small molecule induction
  • Method for differentiating inducible pluripotent stem cells into testicular interstitial cells by small molecule induction
  • Method for differentiating inducible pluripotent stem cells into testicular interstitial cells by small molecule induction

Examples

Experimental program
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Embodiment 1

[0034] Embodiment 1: the cultivation method of human-derived inducible pluripotent stem cell iPSC

[0035] Inoculate the iPSCs derived from the reprogramming of cloned human urine cells on a 6-well plate that has been incubated with 1% Matrigel (incubate at least 37°C for more than 1 hour), culture in a 37°C carbon dioxide incubator, and invert every day The growth status was observed under a microscope, and fresh E8 medium was replaced in full every day. Once differentiated cells were found, they were picked out in time and passaged every 6 days at a dilution ratio of 6:1. In order to reduce the damage to iPSCs, use 0.25% EDTA for 3-5 minutes during passaging. When the edge of the colony begins to roll up and is about to leave the bottom of the culture dish, wash it once with deionized PBS, and then wash it with 1 Gently pipette mL of E8 medium (no more than 10 times), collect the cells and inoculate them on a new 6-well plate that has been pretreated with 1% Matrigel in mas...

Embodiment 2

[0042] Example 2: Method for Inducing Human-derived Inducible Pluripotent Stem Cells iPSCs to Differentiate into Leydig Cells iPSC-LCs

[0043] Implementation process: when the clonal mass-like iPSCs cultured in Example 1 reached 70% confluence, this time point was defined as day -2, at which time the cell culture medium was replaced with E7 medium without bFGF for culture, pretreatment 2 days, define this time point as the 0th day. Then the medium was changed into differentiation medium iPSC-DIM: DMEM / F12, 1% bovine serum albumin BSA by volume, 5mM ITS and 5 ng / mL LH. From day 0 to 7, add 0.2 μM SAG, 5 μM 22R-OHC, and 5 mMLi to the differentiation medium for early differentiation induction. From day 7 to 10, add 5 ng / mL PDGF-AA and 5 ng / mLFGF2 to the differentiation medium for early pro-proliferation induction. From day 10 to 17, add 5 ng / mL PDGF-AA, 5 nMIGF-1, and 10 μM Androgen to the differentiation medium for induction of metaphase differentiation. From day 17 to 20, 1...

Embodiment 3

[0045] Embodiment 3: the method for measuring serum testosterone content by radioimmunoassay

[0046] Said sample refers to the cell sample differentiated and formed in Example 2.

[0047] Prepare TBS-G solution: 1 g gelatin, 4.4 g Trizma HCl, 2.65 g Trizma Base, 5.84 g NaCl and 1 g Na Azide are dissolved in 1 L double distilled water. Add 500 μL of TBS-G to the labeled TC tube for complete radioactivity measurement without activated carbon adsorption. Add 500 μL TBS-G to the labeled NBS tube used to measure non-specific binding value without adding antibody. Add 200 µL of TBS-G and 100 µL of sample in a labeled sample tube. Add 300 μL of standards of different concentrations to the tube marked with the standard tube: 10-2000 pg / 100 μL, 8 concentrations. Add 200 μL of testosterone antibody to each tube except TC tube and NBS tube. Add 300 μL Tracer to each tube, that is, H-testosterone-TBS-G solution, shake, and leave overnight at 4°C. Except for TC tubes, add 200 μL of a...

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Abstract

The invention discloses a method for differentiating inducible pluripotent stem cells into testicular interstitial cells by small molecule induction. The method comprises the following steps: firstly,obtaining human inducible pluripotent stem cells; secondly, carrying out pretreatment culture on the human inducible pluripotent stem cells by using an E7 culture medium without basic fibroblast growth factors; thirdly, inducing the human inducible pluripotent stem cells to differentiate by using a differential medium and combining with a small molecule inducer, wherein the small molecule inducercomprises a DHH agonist SAG, 22R-OHC, lithium chloride, human platelet-derived growth factor AA, fibroblast growth factor 2, insulin-like growth factor 1, androgen, luteinizing hormone, retinol and 8-bromo-cyclic adenosine monophosphate; fourthly, manually removing clonal cluster-like parenchyma cells; fifthly, continuously culturing the remaining cells after the parenchyma cells are removed by using an enriched medium, replacing the enrichment medium regularly, and finally obtaining the target testicular interstitial cells.

Description

technical field [0001] The invention relates to a method for inducing inducible pluripotent stem cells to differentiate into Leydig cells, in particular to a method for inducing human-sourced inducible pluripotent stem cells to differentiate into Leydig cells by small molecules. Background technique [0002] Male reproductive health is inseparable from the functioning of the reproductive system. Currently, the only treatment for androgen deficiency is androgen replacement therapy. However, long-term androgen therapy can cause complications such as liver and kidney damage, decreased immunity, water and sodium retention, and is not regulated by its own circadian rhythm. Although Leydig cell transplantation can avoid some complications caused by exogenous androgen replacement therapy, the insufficient source of Leydig cells and allogeneic transplantation may lead to host immune rejection, which limits the clinical application of Leydig cells . [0003] At present, the differ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0683C12N2500/12C12N2500/38C12N2500/40C12N2501/105C12N2501/115C12N2501/135C12N2501/31C12N2501/392C12N2506/45
Inventor 郭晓令葛仁山李超陈显武陈勇李晓珩
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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