204 SPONTANEOUS DIFFERENTIATION OF PORCINE INNER CELL MASS-DERIVED CELLS INTO CELLS DISPLAYING NEURAL AND GLIAL MARKERS

Abstract

The pig is needed as a model for human embryonic stem (ES) cell therapy. Our aims were to isolate, culture, and characterize porcine inner cell mass (ICM) derived cells. Porcine blastocysts (n = 22) were flushed from sows on Day 8 after insemination and two blastocysts were immediately fixed. A total of 18 (90%) ICMs were microsurgically isolated from the remaining blastocysts and cultured on mitomycin-inactivated mouse embryonic fibroblasts (SLN cells) in a human ES cell medium without (n = 9) or with 10⁶ U/mL leukemia inhibitory factor (LIF, n = 9). The colonies were inspected by stereomicroscopy every second day. After approximately one week, ES-like portions of the primary outgrowth colonies were passaged physically. Subsequently, passages were performed approximately once a week. Colonies from different passages were fixed in 4% paraformaldehyde and processed for immunocytochemistry together with the blastocysts. LIF had no apparent effect on rates of attachment and growth during initial outgrowth and after passages, or on differentiation patterns. A total of 15 (83%) ICMs attached during the outgrowth culture and 12 (80%) of these developed ES-like portions, i.e. compact masses of small tightly packed cells. In blastocysts, nuclei of the ICM stained exclusively for Oct-4. After 6 days of culture, however, Oct-4 staining was lacking in outgrowth colonies. After 14 days of culture, 6 out of 13 passage (P) 1 colonies had developed morphological characteristics compatible with neural differentiation, i.e. large bipolar perikarya and long axon-like structures; after one month, 15 out of 17 P2-4 colonies displayed neural differentiation. Cells in such colonies displayed cytoplasmic staining for ²-III-tubulin in the perikarya and axon-like extensions. After 2-3 months of culture, cell populations in colonies with neural differentation displayed cytoplasmic staining for the intermediate filaments nestin (marker for neural progenitor cells) and vimentin (in the nervous system a marker for glial cells), and cytoplasmic staining for ²-III-tubulin and TUC-4 (markers of post-mitotic neurons). Double-immunostaining revealed a co-localization pattern suggesting the existence of a heterogeneous neural cell population that included neural progenitor cells (staining for nestin only), maturing neural progenitor cells (staining for nestin throughout the cytoplasm combined with ²-III tubulin in the axon-like extensions only), early neurons (staining for ²-III-tubulin, TUC-4, and nestin in the complete cytoplasm), and glial cells (staining for vimentin alone or in combination with nestin). At no time point studied so far were the neural transcription factors Pax6 and Sox2 detected. In conclusion, ICMs were efficiently isolated from Day 8 porcine blastocysts and attached to feeder cells. However, during initial outgrowth culture they lost their Oct-4 expression, and over the subsequent passages they developed into a heterogeneous population of cells at different stages of neural differentiation.