THE EFFECTS OF HUMAN LEUKEMIA INHIBITORY FACTOR (HLIF) AND CULTURE-MEDIUM ON IN-VITRO DIFFERENTIATION OF CULTURED PORCINE INNER CELL MASS (PICM)

Isolation and maintenance of porcine embryonic stem (pES) cells have b een hindered by the inability to inhibit differentiation of the porcin e inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed, from mouse ES cell culture and are not effective for maintaining the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in the differentiation status of in vitro cultured pICM. Porcine ICMs (Da y 7) were isolated by immunosurgery and cultured for 4 d in either Dul becco's modified Eagle's medium (DMEM)-based medium (D medium) or DMEM /Ham's F-10 (1:1)-based medium (DM medium) with or without human Leuke mia inhibitory Factor (hLIF, 1000 iu/ml). Colonies were photographed d aily for morphological analysis, pICMs were categorized into one of tw o types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated pICM different iation using standarzied differentiation profiles. Each pICM series wa s graded on a scale of 1 (fully undifferentiated) to 5 (fully differen tiated) for each time point. Differentiation was;as verified by alkali ne phosphatase activity, cytokeratin staining, and scanning electron m icroscopy. Neither hILF nor culture medium delayed differentiation of pICMs (P = 0.08 and P = 0.25, respectively). The grading system employ ed was an effective tool for detecting treatment effects on differenti ation of the developing pICM. These results demonstrate that hLIF cann ot significantly inhibit differentiation of the pICM, and is unlikely to assist in porcine ES cell isolation. Future experiments utilizing h omologous cytokines may prove more beneficial.