Comparative analysis of nuclear transfer embryo-derived mouse embryonic stem cells. Part I: cellular characterization

Abstract

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology.

FIG. 1.

Alkaline phosphatase (ALP) activity of ESCs. Representative pictures of ALP enzymatic assay of ntESCs and control ESCs. Names of the cell lines are given on each picture. Scale bar represents 100 μm.

FIG. 2.

Comparison of the growth efficiencies of ntESCs and control ESCs. Population doubling times (PDT) were calculated after trypsinization and hemocytometer counting performed every 12 h over a 72-h culture period by using the online software of Doubling Time–Several Time Points calculator. Exponential curves were adapted to the data of both control (HM1 and B6D2) and the PGA ESC line to make visible the tendency of growth efficiencies.

FIG. 3.

In vitro differentiation of ntESCs. Gene expression of (A) Nkx2.5 (cardiac lineage) and B) Pax6 (neural lineage) of in vitro differentiated ESCs with RT-PCR. no RT: pooled samples of a cell line, without reverse transcriptase. (C) Representative immunocytochemistry of HM1 ntESC during neural and cardiac differentiation. Images were taken at day 20 of differentiation, except for GATA-4 and FORSE-1 immunocytochemistry, which was performed on day 10 (nuclear staining), and Cx43 on day 15. Neural markers: NCAM, GFAP, NESTIN, FORSE-1, and NFL; Cardiac markers: GATA-4, Troponin T, α-actinin, Cx43. Scale bar represents 50 μm.