198 REPROGRAMMING FIBROBLAST CELL CULTURES WITH EMBRYONIC STEM-CELL EXTRACTS

Abstract

Reprogramming differentiated cells could be a effective method to dedifferentiate and transdifferentiate cells and thereby to replace or treat tissues. The goal of our study was to examine the effectiveness of embryonic stem (ES) cell extract on reprogramming fibroblast cell cultures and to use these cells as nucleus donors in subsequent cloning experiments. Based on the method of Collas and Hakelien (2003 Trends Biotechnol. 21, 354-361), cell extracts were produced from ES cells after homogenization and sucrose gradient centrifugation. The ES cells were suspended in one volume of 0.3 M sucrose then one volume of 2 M sucrose was added, and cells were homogenized. Finally 5.6 volumes of deionized H₂O were added to the suspension, and the nucleus compartments were separated with centrifugation (2000 rpm, 10 min; Janetzki K24 angle rotor). The supernatant was separated (cytosol fragment), the precipitate was rehomogenized in the same way (nuclei fraction), and both fractions were centrifuged (8000 rpm, 1 h, Janetzki K24 angle-rotor). The supernatants were ultracentrifuged (40 000 rpm, 30 min; Beckman 40 angle rotor) and used immediately or stored at -80°C. Fibroblast cells were cultured and treated in 24-well plates. Cell extracts were administered to culture media [DMEM + 10% fetal bovine serum (FBS)] directly. Cell numbers were calculated after MTT-assay (Roche, Budapors, Hungary). Cell divisions were monitored by BrDU-tests (Roche) and RT-PCRs (Cdk2 and p21; Invitrogen, Budapest, Hungary). Results were statistically analyzed with Student's t-test. In our experiments the crude cell fractions killed 98% (P < 0.005) of cells after 1-h treatment, thus the administration of protease inhibitors was necessary. The supplemented extracts were tested as to dosage and treatment time: 4-h treatment (P < 0.05) and 10⁵ or 10⁶ cells/mL (P < 0.05; cell number in 1 mL of starting material) were the most appropriate combination. When 5 mM CaCl₂ was administered, the cell cycle-arresting effect was increased using either the nuclear (+24%, P < 0.05) or the cytosol extract (+30%, P < 0.05), but the effect of the nuclear extract was more intensive. When steptolysin O (0.1 µg, 5 min) treatment of fibroblast was used prior to addition of ES cell extracts, 1-h treatment and 10⁴ cells/mL were the most effective combination, as higher concentration or longer treatment was toxic. Further experiments are needed to test the effect of treatment on nuclear transfer procedures.

This research was supported by MEXT-CT-1003-19582; Wellcome Trust (Grant No. 070246); OTKA T046171; and NKTH BIO-10017/2002.