Isolation, Characterization, Gene Modification, and Nuclear Reprogramming of Porcine Mesenchymal Stem Cells1

Abstract

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

“…Multilineage differentiation potential of isolated porcine MSCs was evaluated by culturing the second-passage MSCs (3 · 10 4 cells/cm 2 in 35-mm dish) with osteogenic or adipogenic induction medium for 3 weeks, as previously described (Bosch et al, 2006). Briefly, for osteogenic induction, differentiation media were replaced every 3-4 days; for adipogenic differentiation, cells were first exposed to induction medium for 3 days and then cultured in maintenance medium for another 1 days.…”

“…The transgenic efficiency was 0.37% (data not reported yet), which is slightly lower than that associated with the Yorkshire MSCs showed in this study. Two previous studies indicated that cultured MSCs can undergo transient as well as stable genetic modification by nonviral and viral vectors (Bosch et al, 2006;Colleoni et al, 2005), but production of genetically engineered cloned pigs from MSCs was not reported in these two studies. During the preparation of this manuscript, a research group reported that they used MSCs to generate gene-targeted pig models through the SCNT technique Leuchs et al, 2012).…”

Oct4 and Sox2 overexpression improves the proliferation and differentiation of bone mesenchymal stem cells in Xiaomeishan porcine

“…These stem cells are capable of differentiating into various cellular lineages, including the osteogenic, chondrogenic, adipogenic, and myogenic lineages (Pittenger et al, 1999;Jiang et al, 2002;Imabayashi et al, 2003;Bosch et al, 2006), and are widely used in clinical investigations (Friedenstein et al, 1966;Horwitz et al, 2002;Kadivar et al, 2006;Yang et al, 2008). Before human clinical trials can be approved, scaled-up cell production and delivery into an animal model with cell doses (number of cells) comparable to those anticipated for human trials are often required in order to satisfy regulatory safety concerns.…”

Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells

“…Previous works have reported the characterization and differentiation of porcine somatic stem cells [1][2][3][4][5][6][7][8][9][10]. The generation of oocytes from somatic stem cells in vitro may provide a valuable model for identifying factors involved in germ cell formation and oocyte differentiation.…”