293 EMBRYONIC STEM-LIKE CELLS DERIVED FROM PORCINE INNER CELL MASS CELLS ISOLATED BY DIFFERENT METHODS

Abstract

The aim was to examine isolation methods and culture conditions for the establishment of embryonic stem-like cells derived from the inner cell mass (ICM) of porcine embryos. A total of 83 zona pellucida-enclosed (ZPE) and 88 hatched (ZPH) porcine in vivo Days 5–7 blastocysts were assigned for ICM isolation by: (A) manual dissection by needles (ZPE: n = 10; ZPH, n = 15), (B) immunosurgical isolation (ZPE: n = 30; ZPH: n = 10), (C) immunosurgery and manual cleaning (ZPE: n = 11; ZPH: n = 40), and (D) culture of whole blastocysts (ZPE: n = 42; ZPH: n = 23). Culture was done on mouse embryonic fibroblasts (MEF) at 5% O₂ (for A–C) and 20% O₂ (for D) in DMEM with fetal calf serum (FCS), serum replacement, and leukemia inhibitory factor (LIF). Outgrowth colonies (OC) were evaluated by phase contrast and subjected to either (1) physical passage and RT-PCR for Oct-4, Nanog, and Sox2; or (2) immunocytochemical localization of Oct-4 at Days 6–7. Five OC categories were defined: (I) epiblast-like colonies (multilayered ICM-like with homogeneous nuclear Oct-4 staining), (II) ES-like colonies with few surrounding cells (ES-like cells with homogeneous nuclear Oct-4 staining surrounded by few cells), (III) clearly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4 staining and clear demarcation to many differentiated cells), (IV) poorly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4-staining and poor demarcation to many differentiated cells), and (V) differentiated colonies (heterogeneous cell populations lacking nuclear Oct-4 staining). Oct-4 staining was supported by expression of Oct-4, often associated with Nanog and Sox2. The attachment rates were similar for methods A, B, and C, being higher for ZPH (75–80%) than for ZPE (40–50%) blastocysts. Method D gave 42% attachment for ZPE, but only 23% for ZPH blastocysts at 5% O₂, whereas the figures at 20% O₂ were 63% and 80%, respectively. Methods B and C gave the highest proportion of OCs in categories II–IV (ES-like cell-containing), and the ZPE-derived OCs exhibited a more homogeneous nuclear Oct-4 staining than the ZPH-derived. Method D gave the highest proportion of category II colonies. Passage was performed from OC categories II–IV (with ES-like cells). For method C, 24 OCs resulted in 18 (75%) passage 1 (P1) colonies. In 11, 6, 3, and 2 cases, they were passed to P2, P3, P4, and P5, respectively, maintaining ES-like morphology before they went into quiescence or differentiation. However, most colonies attained cytoplasmic Oct-4 staining and lost Oct-4, Nanog, and Sox2 expression at P1 or P2; only a single colony maintained Nanog and Sox2 expression up to P3. For isolation method D, 10 OCs resulted in 3 (30%) P1 colonies, but only one continued to P2 before differentiation. In conclusion, all methods consistently gave ES-like OCs. Whole blastocysts at 20% O₂ gave the highest attachment rates. However, immunosurgery, eventually followed by manual cleaning, tended to result in the highest proportion of OCs presenting ES-like cells, with the ZPE-derived OCs exhibiting the most homogeneous Oct-4 staining. When subjected to passage, a few colonies maintained ES-like morphology up to P5, but expression of pluripotency markers was lost during the initial passages.