43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS

Abstract

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% ± 4.6 vs. 52.2% ± 5.4 and 50.8% ± 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% ± 2.5 and 18.5% ± 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% ± 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 ± 5.2 and 0.38 ± 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 ± 5.5 and 0.18 ± 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 ± 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 ± 1.8) and in IVF embryos (4.6 ± 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos.

This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.