Comparison of the method establishing embryonic stem cell lines from five different mouse strains

Area of Science:

• * Stem Cell Biology
• * Developmental Biology
• * Mouse Genetics

Background:

• * Establishing embryonic stem cell (ESC) lines is crucial for research.
• * Traditional methods have variable success rates across different mouse strains.
• * Optimization of culture conditions and dissociation techniques is needed.

Purpose of the Study:

• * To develop and evaluate an improved method for establishing mouse ESC lines.
• * To compare the efficacy of the new method with routine techniques.
• * To identify optimal conditions for ESC derivation and maintenance.

Main Methods:

• * Utilized a novel culture medium containing 70% rat heart cell-conditioned medium (RH-CM).
• * Employed primary murine embryo fibroblasts as feeder cells.
• * Developed a "series dispersed method" using a specific digestive enzyme buffer concentration (0.05% Trypsin-0.008% EDTA).
• * Assessed optimal timing for inner cell mass (ICM) dispersion based on proliferation.

Main Results:

• * Achieved significantly higher ESC line establishment rates: KM (12%), ICR (42.1%), 129/ter (33.3%), C57BL/6J (13.3%), BALB/c (19.4%).
• * Identified optimal ICM dispersion times for different mouse strains (e.g., 4-6 days for 129/ter).
• * Determined optimal digestive enzyme buffer concentration for BALB/c ESCs.
• * Demonstrated the superiority of the "series dispersed method" over "once dispersed method" for ICM dispersion and clone formation.
• * RH-CM effectively inhibited ESC differentiation, maintained diploid karyotype, and promoted cell attachment and growth compared to mLIF.

Conclusions:

• * The improved method significantly enhances ESC line establishment efficiency across diverse mouse strains.
• * RH-CM is a superior alternative to mLIF for ESC culture, promoting pluripotency and viability.
• * Optimized dissociation timing and enzyme concentrations are critical for successful ESC derivation.