Effect of embryo cryopreservation on derivation efficiency, pluripotency, and differentiation capacity of mouse embryonic stem cells

Abstract

Mouse embryonic stem cells (mESCs) are pluripotent cells that have the capability for self‐renewal. One of the most important factors that affect the efficiency of their isolation is the condition of the mouse embryos. The main objective of this study is to isolate mESCs from C57BL/6 frozen/thawed eight‐cell mouse embryos using serum‐free culture. We generated mESCs from blastocysts that developed from frozen/thawed embryos of C57BL/6 mice by the 3i\u2009+\u2009LIF medium. Assessments of the isolated mESC lines (MUKF‐1, MUKF‐2, and MUKF‐3) included simple karyotype analysis; polymerase chain reaction of the testis‐determining gene (Sry); determination of alkaline phosphatase (ALP) activity; expressions of pluripotent transcription factors Oct4, Rex1, Sox2, and Nanog by reverse transcription polymerase chain reaction; and immunocytochemistry assessment of OCT4 and SSEA‐I expressions at the protein level. We evaluated the ability of these mESC lines to differentiate into three germ layers by embryoid body (EB) formation. The cell doubling time (DT) of isolated mESCs was determined. The 2‐C57 cell line was served as control. Germline competence of the male mESC line (MUKF‐3) was tested through chimeric mouse production. Three independent mESC lines (MUKF‐1, MUKF‐2, and MUKF‐3) were established from five cryopreserved embryos. The MUKF‐1 and MUKF‐2 lines were female, whereas MUKF‐3 was a male mESC line. Karyotype analysis showed that MUKF‐3 had a diploid karyotype, whereas MUKF‐1 and MUKF‐2 had abnormal karyotypes. All three lines had ALP activity and expressed Oct4, Rex1, and Nanog. Immunocytochemistry assessment for OCT4 and SSEA‐I was positive for all three lines. The DT differed in the three mESC lines. MUKF‐1 and MUKF‐3 could form EB and express developmental genes after spontaneous differentiation. These data demonstrated that probably cryopreservation affected the efficiency of derivation, karyotype, DT, expression of pluripotency, developmental genes, and differentiation capacity of the independent mESC lines.