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BOR - Papers in Press, published online ahead of print September 14, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.045138
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BIOLOGY OF REPRODUCTION 74, 46–57 (2006)
DOI: 10.1095/biolreprod.105.045138
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

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

Pablo Bosch 3, Scott L. Pratt 4, and Steven L. Stice 2 3

Department of Animal and Dairy Science,3 University of Georgia, Athens, Georgia 30602 ViaGen, Inc.,4 Austin, Texas 78727

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.

developmental biology

INTRODUCTION

Mesenchymal stem cells (MSCs) are pluripotent precursor cells that localize to the stromal compartment of the bone marrow (BM), where they support hematopoiesis and differentiate into mesenchymal lineages. The potential of MSCs to form bone, cartilage, and adipose tissues both in vivo [1–3] and in vitro [4] has been well documented. Their plasticity, however, is not limited to those mesenchymal derivatives. Recent reports have suggested that MSCs can differentiate into neurons [5, 6], myoblasts [7], and cardiomyocytes [8]. Cells with features of mesenchymal precursors have been isolated from the BM of many mammals, including laboratory rodents [9, 10], humans [4], cats [11], dogs [12] and pigs [13]. The MSCs from all species studied to date proliferate in vitro as adherent fibroblastic cells, a feature that has been exploited to enrich MSCs from hematopoietic cells that normally remain in suspension. In humans, pluripotent stem cells derived from marrow stroma proliferate ex vivo to form a phenotypically homogeneous population of cells that express several surface markers, such as THY1 (also known as CD90), CD44, and TFRC (also known as CD71), but that do not express the hematopoietic markers PTPRC (also known as CD45) and ITGAM (also known as CD11b) [4]. Like MSCs from other species, porcine MSCs (pMSCs) were capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into adipose, bone, and cartilage tissues in vitro [13]. To our knowledge, however, surface marker expression and culture requirements for ex vivo expansion of MSCs in this species have not been yet defined.

Because of the ability of MSCs to proliferate extensively ex vivo while maintaining their pluripotent differentiation capabilities (in vivo and in vitro), they are regarded as a particularly attractive cell type for cell-based therapies in humans. Of particular interest is the use of intact or genetically engineered MSCs for the treatment of skeletal disorders like osteogenesis imperfecta [14, 15]. Moreover, MSCs have attracted much attention as tools for targeted delivery of anticancer agents into tumors [16, 17]. Before human clinical trials are approved, scaled-up cell production and delivery into a large-animal model in which cell doses (number of cells) comparable to those that will be used in human trials often is required to satisfy regulatory safety concerns. Beyond safety issues, the reprogramming of pMSCs via somatic cell nuclear transfer (SCNT) lays the foundation for future isogenic comparisons between adult pMSCs and reprogrammed embryonic cell sources (therapeutic cloning) in porcine disease models. Kato et al. [18] recently reported the birth of the first calf originated from a bovine MSC, demonstrating that bovine MSCs can be reprogrammed to drive term development after SCNT.

Development of SCNT has provided a new and faster way to create transgenic animals. It now is possible to introduce genetic modifications in cultured cells that later can be used as donor cells to produce cloned animals bearing the genetic transformation (for review, see [19, 20]). Genetic manipulations of cultured cells can range from simple, random integration of the gene of interest to targeted homologous recombination to abolish (knock out) or modulate gene function. In combination with electroporation or a particular transfection compound, DNA plasmids have been used to genetically modify cultured cells for use in nuclear transfer (NT) [21, 22], and transgenic animals have been generated with these cells [23, 24]. In addition, transient expression of proteins in donor cells could open new opportunities, from basic studies of donor cell physiology and nuclear reprogramming to more applied studies aimed to improve cloning efficiencies by conditioning donor cells before NT.

The use of viruses as vectors has emerged as a promising alternative to the classic, mechanical methods of gene delivery. The ability of retroviruses to integrate randomly into the host genome has been exploited to stably introduce the green fluorescent protein (GFP) reporter gene in pig cell lines later used to produce embryos [25] and transgenic cloned pigs by SCNT [26, 27]. Lentivirus, which is a complex retrovirus, is considered to be a promising alternative to the original oncogenic retroviral vectors because of their ability to infect nondividing mammalian cells and to resist methylation-dependent gene silencing. Lentiviral infection of bovine fibroblasts followed by SCNT has resulted in the production of transgenic animals [28].

Adeno-associated virus (AAV) is an integrating, nonpathogenic human virus that requires coinfection with a helper virus, such as adenovirus or herpesvirus for productive infection. In the absence of a helper virus, AAV integrates in a site-specific manner in the host genome, where it remains as latent infection. Vectors derived from AAV are attractive candidates for transgenesis by virtue of their nonpathogenicity, integration capability, infectivity of dividing and nondividing cells, and ability to infect a wide variety of cell types. The AAV vectors have been used to insert small (<20 bp) and large (>1 kb) transgenes by homologous recombination in human cells in culture [29]. More recently, Hirata et al. [30] demonstrated that AAV vectors can efficiently disrupt one allele of the PRNP gene in cultured bovine fibroblasts, expanding the use of AAV vectors to animal transgenesis.

Targeted homologous recombination also has been accomplished in mammalian cells with adenovirus vectors [31]. Because adenovirus very rarely integrates into the host genome by nonhomologous recombination, replication-defective recombinant adenoviral vectors are used as efficient expression vectors. Transient expression of endogenous or even foreign proteins in cultured cells by adenovirus vectors would represent a potential tool to manipulate donor cells in culture. Furthermore, silencing of endogenous genes by adenovirus-mediated expression of small interfering RNA is now a reality [32]. These novel, adenovirus-based approaches could open new possibilities for controlling cell processes, such as cycle progression, DNA methylation, or apoptosis in SCNT donor cells.

In the present study, we have isolated and established adult pMSC lines from live animals using a minimally invasive BM aspiration technique. The mesenchymal identity of isolated cells was determined by expression of surface markers and multilineage differentiation potential. We then designed experiments aimed a) to optimize in vitro culture conditions of pMSCs, b) to compare transfection/transduction efficiencies of pMSCs and isogenic skin fibroblasts (SFs) exposed to integrating and nonintegrating vectors, and c) to examine the ability of pMSCs to drive development of SCNT embryos to the blastocyst stage. We have shown that adult pMSCs can be genetically modified and used to produce SCNT embryos. This is significant in that it prepares us for future large-animal autologous cell/gene therapy modeling comparing the adult cells to embryonic cells derived through SCNT.

MATERIALS AND METHODS

Bone Marrow and Skin Collection

Blood marrow aspirates were obtained from anesthetized, young adult, female pigs (age, ~6 mo). General anesthesia was induced with a combination of ketamine (10 mg/kg body weight i.m.) and xylazine (2 mg/kg body weight i.m.) and maintained with inhalation anesthesia (halothane). Aspirates of BM (~20 ml) were collected from the humeral head with an 11-gauge biopsy-aspiration needle (Medical Device Technologies, Inc.) attached to a heparinized syringe. An ear-skin sample was obtained from the same animal by punch biopsy. Bone marrow and skin samples were immediately transported to the laboratory for further processing. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Georgia.

Isolation of BM pMSCs and SFs

Mononuclear cells were separated by centrifugation of BM aspirates through a solution of polysucrose and sodium diatrizoate (Histopaque; density, 1.077; Sigma) as indicated by the manufacturer. Briefly, 5 ml of Dulbecco phosphate-buffered saline (D-PBS) were added to 3 ml of marrow aspirate and mixed in a 15-ml centrifuge tube. The cell suspension was deposited over 3 ml of Histopaque and centrifuged at 400 x g for 30 min at room temperature. Mononuclear cells were recovered with a Pasteur pipette from the opaque interface and washed twice with D-PBS. For SF isolation, cartilage tissue was removed from the ear-skin sample, followed by scratching the surface of it with a scalpel to partially remove the dermis. Then, specimens were finely chopped with a scalpel blade and digested by treatment with 10 ml of digestion solution containing 0.2% trypsin (catalog no. T4799; Sigma) and 0.2% collagenase (catalog no. C9263; Sigma) in D-PBS containing 5 mg/ml of BSA at 37°C with agitation. At 10-min intervals, the supernatant containing cells was removed and replaced by fresh digestion solution. Approximately 30 min were required to digest the tissue corresponding to a skin sample. Cells recovered from one skin sample were washed in D-PBS and plated in approximately eight 75 cm2 flasks in Minimum Essential Medium (MEM) Alpha medium (catalog no. 12000-022; Invitrogen Corporation) supplemented with 10% fetal bovine serum (FBS).

Culture of pMSCs

After washing, mononuclear cells were resuspended in MEM Alpha medium supplemented with 10% FBS and plated on plastic flasks at a density of approximately 500000 cells/cm2. After 24 h, unattached cells were washed off the flask during medium exchange. Adherent fibroblast-like cells were allowed to grow for 10–14 days, with media replacement every third day. Cells were passaged at 80%–90% confluence by trypsinization (0.25% trypsin-EDTA solution; Sigma) and reseeded at a density of 5000–6000 cells/cm2 in plastic flasks.

Expression of Surface Markers

Expression of surface markers in MSC cultures for phenotypic characterization was performed by indirect immunofluorescence and flow cytometric analysis. For immunocytochemistry, cells grown in glass chamber slides were fixed with 2% formaldehyde for 5 min, washed in D-PBS, and blocked with 3% goat serum in D-PBS for 30 min. Then, cultures were incubated with either a 1:500 dilution of the primary antibody (anti-THY1 or anti-ITGAM) or isotype control (Mouse IgG1, clone, MOPC-31C; BD Biosciences, Pharmingen) or D-PBS (negative control) for 1 h and 15 min. Primary antibody used was anti-human THY1 monoclonal antibody that cross-react with pig antigens (Clone 5E10; BD Biosciences, Pharmingen) or anti-pig ITGAM monoclonal antibody (Clone 2F4/11; BD Biosciences, Pharmingen). After washing in D-PBS, cell monolayers were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:100 dilution; Molecular Probes) for 1 h. Cells were washed in D-PBS, stained with 4',6'-diamidino-2-phenylindole (DAPI; 1 µg/ml; Calbiochem), and mounted with Vectashield mounting medium (Vector Laboratories). Specimens were examined under an epifluorescent inverted microscope (Nikon Eclipse TE2000-S; Nikon Corporation) equipped with a digital camera (Qimaging Ratiga 1300; Qimaging).

The same basic staining procedure described for immunocytochemistry was used to prepare the cells for flow cytometric analysis with minor modifications. Cell cultures were trypsinized, washed in D-PBS, and fixed with 2% formaldehyde solution for 3 min. Nonspecific binding was prevented by incubating the cells in 3% goat serum for 30 min. Cells were incubated in 15 µg/ml of the primary antibody (anti-THY1 or anti-ITGAM) or isotype control for 45 min at room temperature. After washing, cells were incubated with a 1:500 dilution of Alexa Fluor 488 goat anti-mouse IgG. Fluorescent cell analysis was performed with FACSCalibur cytometer (Becton Dickinson Immunocytometry System) and data analyzed by FlowJo software (Tree Star, Inc.).

Lineage Differentiation of MSCs

The pMSC cultures were exposed to chondrogenic, lipogenic, or osteogenic conditions for 14–20 days to determine multipotency. Lipogenic and osteogenic induction was applied to cells growing in monolayers. Chondrogenic and osteogenic differentiation was induced on cell masses as described previously in human MSCs [33] and in pMSCs [13]. Briefly, aliquots of 200000 cells were distributed in 15-ml conical tubes and centrifuged for 5 min at 600 x g. Sedimented cells were cultured in the tubes with loosened caps to allow gas exchange. Cells formed a spherical mass on the bottom of the tube by 24 h of culture. Composition of differentiation media is shown in Table 1. Differentiation media were replaced every 3–4 days. For lipogenic differentiation, cells were first exposed to induction medium for 2–3 days and then cultured in maintenance medium (Table 1) for another 2–3 days. This alternating treatment was repeated three or four times to achieve full lipogenic differentiation.


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  		TABLE 1. Composition of differentiation media and methods used to assess lineage differentiation

Histochemical stains were used to assess cell differentiation into specific lineages in adherent cell cultures and histological cryosections of cell masses. Cell masses were embedded in a water-soluble embedding medium, frozen in liquid N2, and sectioned (thickness, 10 µm) with a Leica CM3050 cryostat (Leica). Accumulation of phosphates and carbonates indicative of osteogenic differentiation was demonstrated by the von Kossa silver reduction method [4]. Cultures or cryosections were fixed with 4% formaldehyde, exposed to 5% silver nitrate solution, and immediately exposed to direct ultraviolet (UV) light for 45–60 min. Specimens were then washed with distilled water and incubated for 2–3 min in 5% sodium thiosulfate solution. Expression of alkaline phosphatase (AP) was assessed by a commercial kit (Vector Red Alkaline Phosphatase Substrate Kit I; Vector Laboratories). Intracellular accumulation of neutral lipids was demonstrated by Oil Red O staining [4]. For this assay, monolayers were fixed and stained with Oil Red O working solution for 1 h. The working solution was made fresh each time by mixing one part distilled water with 1.5 parts of a saturated Oil Red O solution (0.5% w/v Oil Red O in 99% isopropyl alcohol). Acidic mucopolysaccharides in cartilage tissue were stained with alcian blue 8GX (Sigma). Briefly, cryosections were fixed with 3% acetic acid and stained with alcian blue solution (1% w/v alcian blue in 3% acetic acid, pH 2.5) for 30 min. After washing with distilled water, slides were mounted with 90% glycerol and inspected with a transmitted-light microscope. Photographs were taken with a digital camera (Qimaging Ratiga 1300; Qimaging) mounted on the microscope.

Optimization of Culture Conditions for pMSCs

Colony-forming unit fibroblast assay Passage 2–3 MSCs growing in flasks were trypsinized and plated at density of 10 cells/cm2 in 100- x 20-mm dishes. Cells were cultured for 14 days under different experimental conditions. Medium was replaced every third to fourth day. Cell colonies were washed with D-PBS and stained with 3% Crystal Violet (Sigma) in methanol for 15–20 min and the number and size of colonies recorded for each experimental group. Number of colonies as well as major and minor axes of each colony were measured with the aid of an ocular micrometer. The averages of the major and minor axes are reported as colony diameter. For each experimental condition, the best treatment is the one that induces the highest number of colonies with the larger diameter.

Cell proliferation assay Passage 2–3 MSCs growing in flasks were trypsinized and plated at a density of 800 cells/well of 96-well assay plates (black plate with clear bottom; Corning, Inc.). Cells were grown under different experimental conditions for 4 days. Media were removed and the plates stored at –80°C until the cell proliferation assay was performed following the manufacturer's instructions (CyQUANT; Molecular Probes). Cells cultured in medium containing 2% or 20% FBS were included as low- and high-proliferation controls, respectively. Fluorescence in the samples, reported as relative fluorescence units (RFUs), was measured with a fluorescence microplate reader (SPECTRAmax GEMINI; Molecular Devices Corporation) with filters appropriate for 485 nm (excitation) and 538 nm (emission).

The CyQUANT proliferation assay kit also was used to investigate cell adhesion efficiency. Cells were plated in two plates at a density of 13000 cells/well and incubated under different experimental conditions at 37°C in 5% CO2 in air. After a 5-h incubation, one plate was centrifuged at 600 x g for 15 min to pellet the cells, and the media were carefully removed. This plate was used to determine total cell numbers. Media from the second plate were removed, and the wells were washed three times with D-PBS. All plates were stored at –80°C until the CyQUANT proliferation assay was performed according to manufacturer's instructions.

Experiment 1: Effect of FBS concentration on proliferation of pMSCs The objective of this experiment was to study the effect of media containing increasing concentrations of FBS (2.5%, 5%, 10%, 20%, or 30%) on growing characteristics of pMSCs in the colony-forming unit fibroblast (CFU-F) assay.

Experiment 2: Effect of media and oxygen tension on proliferation of MSCs The ability of different media to support pMSC grow in vitro under an atmosphere of low or high oxygen concentration was investigated using the CFU-F and CyQUANT proliferation assays. The pMSCs were cultured in either MEM Alpha, low-glucose Dulbecco modified Eagle medium (DMEM) containing 2.2 g/L of sodium bicarbonate (DMEM 2.2; catalog no. 31600-034; Invitrogen Corporation), low-glucose DMEM containing 3.7 g/L of sodium bicarbonate (DMEM 3.7; catalog no. 31600-034; Invitrogen Corporation), or DMEM/Ham F12 (DMEM/F12; catalog no. D0547; Sigma). All media were supplemented with 10% FBS. Culture was carried out in low oxygen concentration (5% O2, 5% CO2 and 90% N2) or high oxygen concentration (5% CO2 in air). In the CyQUANT proliferation experiment, cells were seeded in 96-well plates in MEM Alpha media with 10% FBS and allowed to attach for 12 h. After this, media were removed and treatments applied. The design of the CFU-F experiment was slightly different, because cells were directly seeded in 100-mm dishes in the treatment media.

Experiment 3: Effect of ascorbic acid supplementation and oxygen tension on proliferation of pMSCs The potential stimulatory effect of increasing concentrations of ascorbic acid 2-phosphate (0 [control], 5, 50, 500, or 5000 µg/ml; catalog no. A8960; Sigma Chemical) added to the culture medium (low-glucose DMEM), and proliferation of pMSCs was investigated. The design also included the effect of culture under an atmosphere with low or high oxygen concentration on proliferation of pMSCs across all ascorbic acid treatments.

Transient Genetic Modification

Transduction with a human adenovirus A chimeric adenovirus type 5 that contains the adenovirus type 35 fiber and carries the GFP gene (Ad5F35-eGFP; 5 x 1012 particles/ml, 3.45 x 1010 pfu/ml) was obtained from the Vector Development Laboratory at Baylor College of Medicine, Houston, Texas.

Passage 2–3 pMSC and matching isogenic SF cell lines were seeded at a density of 43000 cells/cm2 in 12-well plates (150500 cells/well). Twenty-four hours after plating, cultures were infected with 100 multiplicity of infection (defined as pfu/cell) in 500 µl of MEM Alpha with 10% FBS. The percentage of GFP-positive cells, relative fluorescence intensity (RFI) of the GFP-positive cell population and cell viability by exclusion of propidium iodide (50 µg/ml; Roche Applied Science) was determined 24 h after viral exposure by flow cytometric analysis using a FACSCalibur cytometer and FlowJo software.

Transfection with GFP plasmid Early passage (2–3) pMSCs and isogenic SFs were plated in 12-well plates (120000 cells/well) and, 20 h later, were transfected with a plasmid containing enhanced GFP gene under control of cytomegalovirus promoter and neomycin-resistant gene under control of an SV40 promoter that allows selection using geneticin (EGFP-N1; Clontech Laboratories). Transfection was carried out in the presence of a polyamine-based transfection reagent (GeneJammer; Stratagene) according to the manufacturer's recommendations (2 µg plasmid DNA/well). Transfected cells were sorted based on GFP fluorescence 72 h after transfection using a MoFlo fluorescence-activated cell sorting (FACS) set (DakoCytomation) to sort one cell per well of 96-well plates (three plates per cell line). Cells were cultured in MEM Alpha with 15% FBS for 14 days, and development of GFP expressing colonies was determined at this point by inspection under a microscope equipped with UV light.

Stable Genetic Modification

Transfection with GFP plasmid Passage 2–3 MSCs and isogenic SFs were plated in four 100- x 20-mm plastic dishes per cell line at 1.2 x 106 cells/dish. Cultures were transfected with EGFP-N1 plasmid (12 µg plasmid DNA/dish) using GeneJammer Transfection Reagent according to manufacturer's specifications. Selection for transgenic cells was initiated in three dishes per cell line 72 h after transfection by culturing the cells in medium containing Geneticin (250 µg/ml; G418; Sigma). Number of GFP-expressing colonies was determined 14 days after transfection. The remaining 100-mm dish was passaged and propagated in MEM Alpha with 15% FBS. After 8–9 days, cells were trypsinized and stained with 50 µg/ml of propidium iodide (Roche Applied Science). Viable GFP-expressing cells were sorted with a MoFlo FACS cytometer (DakoCytomation) as single cells in 96-well plates (three plates per cell line) containing culture medium supplemented with 20% FBS, and colonies were allowed to grow for 14 days (with media changed at Day 7). At the end of the culture period, colonies were graded according to their development as follows: category 1, colony covering all or almost all the surface of the well; category 2, colony covering approximately half the well; and category 3, colony covering one-fourth of the well. Colonies also were graded as GFP positive (high, medium, or low florescence intensity) or as GFP negative.

AAV transduction Human AAV vector carrying the GFP gene was kindly provided by Vector Development Laboratory at Baylor College of Medicine. The pMSC and SF cultures (passage 2–3) were seeded in four-well plates (40000 cells/well). Cell cultures were transduced 24 h after plating with 3 x 108 viral particles/well in MEM Alpha with 2% FBS. Serum concentration was adjusted to 10% by adding FBS 3.5 h after transduction. Transduced cells were passaged and expanded for 9–10 days in MEM Alpha supplemented with 15% FBS before sorting viable, GFP-positive cells in 96-well plates (one cell per well, three plates per cell line) using a FACS cytometer (MoFlo; DakoCytomation). Cells were cultured for 14 days in MEM Alpha containing 15% FBS (replaced at Day 7 of culture), and colony development was evaluated as described above.

Somatic Cell Nuclear Transfer

Confluent (passage 2) MSC and SF cultures exposed to roscovitine (15 µM; Sigma) during the last 24 h of culture [34] were used as a source of karyoplasts to produce NT embryos. In vitro-matured oocytes were enucleated, and a single-cell (MSC or SF) was transferred into the perivitelline space. Cell-oocyte couplets were fused in Zimmerman medium with a single electric pulse (250 V/mm for 20 µsec) delivered through a needle-type electrode. The NT units were electrically activated (two pulses of 75 V/mm for 60 µsec separated by 5 sec) in a chamber 1 h after fusion and transferred to drops of NCSU-23 medium. Embryos were examined for cleavage and blastocyst formation at 2 and 7 days, respectively, after NT.

Statistical Analysis

The CFU-F data from experiment 1 were analyzed by one-way ANOVA using the general linear model (GLM) procedure of the Statistical Analysis System [35] followed by the protected least-significant-difference (LSD) test. The CFU-F and proliferation data from experiments 2 and 3 were analyzed by two-way ANOVA using the GLM procedure of SAS under a completely randomized factorial design. The model included variation caused by treatment (media in experiment 2 or ascorbic acid in experiment 3), oxygen tension (high or low), and their interaction. When a significant effect was detected with the ANOVAs, treatment means were compared by protected LSD. Student t-test was used for comparing data from two groups (i.e., pMSC vs. SF). All values are presented as the mean ± SEM from at least three replicates. Differences were considered to be significant at P < 0.05.

RESULTS

Isolation of Cell Lines

Mesenchymal stem cell lines were established successfully from BM collected from 10 anesthetized gilts (n = 10). The number of mononuclear cells per BM aspirate (~20 ml) recovered from the density gradient was 2.33 ± 0.5 x 108 mononuclear cells, enough to plate approximately six 75-cm2 flasks. Most of the nonadherent cells were removed during the first media change at 24–48 h. Discrete colonies of fibroblast-like cells attached to the plastic were evident at Days 4–5 after initial seeding. Most cell lines were composed of cells with a characteristic spindle shape, whereas others had cells with polygonal morphology. The number and size of the colonies increased progressively to reach 80% confluency by Days 14–15 after seeding (Fig. 1, A and B).


Figure 1
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  		FIG. 1. Morphology of adherent fibroblast-like cells, later identified as pMSCs, isolated from pig bone marrow after 14 days from initial plating (A). Same cell line at higher magnification showing detailed fibroblastic morphology of pMSCs (B). Bar = 200 µm (A) and 100 µm (B)

Expression of Surface Markers

Immunocytochemistry revealed that MSCs from pig BM were positive for the cell surface marker THY1 (Fig. 2A) and negative for ITGAM (Fig. 2B). Flow cytometric analysis confirmed that 99.4% ± 0.20% of the cells expressed THY1 antigen (Fig. 2C), and virtually the entire population was negative for ITGAM (Fig. 2D).


Figure 2
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  		FIG. 2. Immunofluorescence and flow cytometry for the surface markers THY1 and ITGAM in pMSCs. Specific immunoreactivity for THY1 was observed in pMSCs growing on chamber slides (A). ITGAM immunoreactivity was absent in pMSC monolayers (B; green, immunoreactive THY1; blue, DAPI). These results were later confirmed by flow cytometry (C and D). The pMSC suspension was fixed and immunostainied for THY1 (C) or ITGAM (D) expression. Histograms show frequency distribution and fluorescence intensity data. Blue curves represent the distribution of cells incubated with anti-THY1 or anti-ITGAM primary antibody, whereas red curves represent the distribution of cells incubated with the immunoglobulin isotype control. In the histogram (C) from a representative pMSC line, 99.6% of total cells were positive for THY1. In D, ITGAM (blue) and isotype (red) curves overlap, indicative of absence of immunoreactive sites for ITGAM on pMSCs. Bar = 100 µm

Lineage Differentiation of MSCs

Results indicated that BM mesenchymal cells acquire morphological and histochemical characteristics of adipose, cartilage, or bone tissues when exposed to specific differentiation-inducing conditions (Fig. 3). Conversely, isogenic SFs exposed to identical induction conditions failed to differentiate (Fig. 3). Cells with discrete, although small, lipid droplets were present as early as Days 4–5 of culture. The number of cells with lipid accumulation and the size of the lipid droplets increased until Days 8–9 and then plateaued until the end of culture period (Days 12–14). The percentage of cells undergoing lipogenic differentiation was highly variable among cell lines, ranging from approxiamtely 1% to 15%. Oil Red O confirmed the presence of neutral lipid accumulation in differentiated pMSCs (Fig. 3A). Lipogenesis was not evident in pMSCs maintained in culture medium alone (control) (Fig. 3B) or isogenic SFs exposed to lipogenic medium (Fig. 3C). Alcian blue staining revealed acidic mucopolysaccharides in sections of pMSC masses cultured in chondrogenic medium for 14–17 days (Fig. 3D). Cell morphology also was compatible with chondrocytes. The pMSC controls and SFs cultured in differentiation media were negative for alcian blue stain (Fig. 3, E and F). Extensive osteogenic differentiation, as evidenced by black deposits with von Kossa stain (Fig. 3G) and AP activity (data not shown), was noticeable only in pMSCs exposed to osteogenic conditions. The pMSC controls and SFs cultured in osteogenic differentiation media were both negative for von Kossa stain (Fig. 3, H and I) and AP activity (data not shown).


Figure 3
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  		FIG. 3. Histochemical stains of SFs and pMSCs exposed to lipogenic, chondrogenic, osteogenic, or control media. The pMSCs underwent lipogenic (A), chondrogenic (D), and osteogenic differentiation when exposed to specific induction media. The pMSCs cultured in control medium (B, E, and H) and isogenic SFs (C, F, and I) exposed to differentiation conditions failed to differentiate. Bar = 100 µm (A–C) and 0.5 mm (D–I)

Optimization of Culture Conditions for pMSCs

Experiment 1: Effect of FBS concentration on proliferation of pMSCs The percentage of FBS in the culture medium greatly influenced both the number of colonies per dish and the mean colony diameter. The number of colonies per dish increased with increasing FBS concentrations up to 10%, when a plateau was reached (Fig. 4A). A similar positive effect of FBS on colony diameter also was evident; however, a plateau was not reached with 30% FBS (Fig. 4B).


Figure 4
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  		FIG. 4. Effect of culture conditions on proliferation of pMSCs as determined by CFU-F or CyQUANT assays. Effect of supplementation of culture media with different FBS concentrations on the number of colonies (A) and colony diameter (B). Response of pMSCs to medium type in the CFU-F (C and E) and CyQUANT proliferation assays (D). Effect of increasing concentrations of ascorbic acid on proliferation of pMSCs in the CyQUANT assay (F). Treatments of 2% and 20% FBS were included as low- and high-proliferation controls, respectively (D and F). Effect of low or high oxygen tension on colony formation in the CFU-F assay (G) and proliferation in the CyQUANT assay (H). Values are presented as the mean ± SEM of at least four independent replicates. Bars with different letters are statistically different at P < 0.05 (ANOVA followed by LSD)

Experiment 2: Effect of media and oxygen tension on proliferation of pMSCs The effect of media and oxygen tension on CFU-F assay was replicated with four cell lines (obtained from four different animals). In three of four cell lines, the number and diameter of colonies were markedly smaller in DMEM/F12 medium compared with those in the other media studied, whereas in the remaining cell line, the response to DMEM/F12 medium was similar to that in the other treatments. Because the inclusion of data from this cell line in the statistical analysis would mask the negative effect observed for DMEM/F12 medium in three of four cell lines, we decided to exclude it from the statistical analysis. The number and diameter of colonies was not different for MSCs grown in MEM Alpha, DMEM 2.2, or DMEM 3.7 (Fig. 4, C and E). Cells cultured in DMEM/F12 responded with fewer and smaller colonies compared to those cultured with the other media studied (Fig. 4, C and E). Despite the fact that oxygen tension affected neither the number nor the mean diameter of colonies in the CFU-F assay (Fig. 4G), we observed darker colonies in cultures maintained under low oxygen tension (5%), suggesting higher number of cells per colony.

The effect of media type on proliferation of pMSCs in two different oxygen tensions (low or high) was investigated with the CyQUANT proliferation assay. Significant effect of medium type (P < 0.0001) and oxygen tension (P < 0.0001), but not interaction (P = 0.49) between these variables, was observed. The number of RFUs, which is correlated to the amount of DNA, was higher in DMEM/F12-treated cells compared with the other media types studied (Fig. 4D). Low oxygen tension had a positive effect on cell proliferation, as evidenced by a higher RFU value compared with cells maintained in high-oxygen atmosphere (Fig. 4H).

The ability of MEM Alpha and DMEM/F12 to induce adhesion of pMSCs to plastic was compared with the CyQUANT assay. The MEM Alpha had a better ability to induce pMSC attachment compared with the DMEM/F12 medium (MEM Alpha, 189.33 ± 10.05 RFU; DMEM/F12, 164.66 ± 2.96 RFU; P < 0.05).

Experiment 3: Effect of ascorbic acid supplementation and oxygen tension on proliferation of pMSCs Significant effects of ascorbic acid concentration (P < 0.0001) and oxygen tension (P < 0.0001), but not of the interaction (P = 0.56) between these variables, were observed. Supplementation of the culture medium with 5–500 µg ascorbic acid/ml did not affect cell proliferation compared with the control (Fig. 4F). Addition of 5000 µg ascorbic acid/ml, however, impaired pMSC proliferation (Fig. 4F). Coinciding with results from experiment 2, low oxygen tension significantly improved (P < 0.05) pMSC proliferation rate.

Transient Genetic Modification of SFs and pMSCs

Transduction with a human adenovirus Microscopic inspection of cultures under UV light 24 h after infection revealed a superior transduction efficiency in pMSCs compared to SFs (Fig. 5, E and F), a finding later confirmed by flow cytometry. The percentage of cells expressing GFP was approximately 15% higher in pMSCs than that in isogenic SFs (pMSCs, 70.25% ± 5.45%; SFs, 55.31% ± 6.83%; P = 0.02) (Fig. 5, A–C). Relative fluorescence intensity also was higher in pMSCs compared with that in SFs (pMSCs, 959.66 ± 73.25 RFI; SFs, 585.75 ± 19.32 RFI; P = 0.005) (Fig. 5A). Percentage of propidium iodide-positive cells was higher in pMSCs (5.35% ± 0.38%) compared with that in SFs (3.45% ± 0.24%, P = 0.01) (Fig. 5A).


Figure 5
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  		FIG. 5. Transient transduction/transfection of SFs and pMSCs with a human adenovirus (Ad5F35-eGFP) or a plasmid carrying the GFP gene. The SFs and pMSCs were transfected with Ad5F35-eGFP and, after 24 h, were characterized by flow cytometry. The percentage GFP positive, mean fluorescence intensity, and proportions of viable cells (A) were estimated from the flow cytometric data. Representative dot plots of transduced isogenic SFs (B) and pMSCs (C) showing distribution of cell populations based on GFP intensity (x-axis) and propidium iodide staining (y-axis). Very low proportions of cells are nonviable (top left quadrants in B and C). A larger percentage of cells are viable and GFP positive in pMSCs (74.6%; bottom right quadrant in C) compared with that in SFs (44.1%; bottom right quadrant in B). The SFs and pMSCs were transfected with GFP plasmid and characterized by flow cytometry 72 h after transfection (D). The percentage of GFP-positive and nonviable cells were estimated from the flow cytometric data. Photomicrographs of SFs (E) and pMSCs (F) taken under UV light 24 h after transduction with Ad5F35-eGFP also are shown. Different symbols within each variable denotes significant difference at P < 0.05 (Student t-test). Bar = 200 µm

Transfection with GFP plasmid No difference was observed in the proportions of SFs and pMSCs expressing GFP (SFs, 3.99% ± 0.95%; pMSCs, 8.44% ± 2.33%; P = 0.22) (Fig. 5D). Viability also was similar between the two experimental groups (Fig. 5D). The GFP-positive cells were sorted individually in three 96-well plates per cell line and checked for GFP-expressing colonies after 14 days of culture. Only 7 of 648 colonies that developed in SF and pMSC plates were GFP positive.

Stable Genetic Modification

We used two different vectors, namely a GFP plasmid and AAV, to obtain cell populations displaying extended expression of the transgene, which normally is associated with integration of the transgene into the host DNA. Comparison of proportions of cells that remained GFP positive after transfection/transduction and proliferation in vitro (8–10 days) revealed a higher proportion of GFP-expressing cells in the AAV- than in the GFP plasmid-transfected group, irrespective of cell line (Fig. 6A). No difference was found in transfection/transduction efficiency between SFs and pMSCs (Fig. 6A).


Figure 6
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  		FIG. 6. Stable genetic modification of SFs and pMSCs with a GFP plasmid or an AAV vector. The SFs and pMSCs were transfected with a GFP plasmid or AAV, expanded in vitro for 9–10 days, and sorted as single GFP-positive cells by flow cytometry. The percentage of GFP-positive SF- and pMSC-transfected/transduced cells after propagation in vitro is shown (A). The GFP-expressing cells sorted in 96-well plates were cultured in vitro for 14 days, and the percentage of GFP-negative and GFP-positive colonies were determined for cells transfected with GFP plasmid (B) or AAV (D). Colonies were classified according to degree of development (1, 2, or 3) and fluorescence intensity (high, medium, or low; C and E). Photomicrographs (taken under UV light) of GFP-positive pMSC colonies generated from a single cell transfected either with GFP plasmid (F) or AAV (G) also are shown. *P < 0.05 (Student t-test). Bar = 200 µm

Transfection with GFP plasmid The mean number of colonies per 100-mm dish expressing GFP after 14 days of G418 selection was 74.32 ± 8.34 in SFs and 74.38 ± 4.58 in pMSCs (P > 0.05).

We implemented an approach to reduce the number of cells with transient expression of GFP from episomes, which consisted in propagation of transfected cell lines for 10 days and then sorting individual cells in 96-well plates based on GFP fluorescence. Two weeks after plating, approximately 35% of plated cells formed a colony (Fig. 6B). Irrespective of cell line (SF or pMSC), the vast majority of these colonies (~92%) did not express GFP. Colony development was similar between SFs and pMSCs (Fig. 6C). A higher percentage of high-fluorescence SF colonies was found compared with the same category in pMSCs (Fig. 6C) (P < 0.05). A pMSC colony originated from one cell transfected with the GFP plasmid and positive for GFP after 14 days of culture is shown in Figure 6F.

AAV transduction The proportion of plated cells that formed a colony was not different between cell lines, ranging from 26.39% ± 1.31% in pMSCs to 40.04% ± 7.79% in SFs. Contrasting with results obtained with GFP plasmid-transfected cells, 90.2% of SF colonies and 96.2% of pMSC colonies expressed GFP (Fig. 6, compare B with D). No difference was observed in the proportion of colonies in category 1, 2, or 3 between cell lines (Fig. 6E). The pMSCs had a larger proportion of high-fluorescence colonies and a lower percentage of low-fluorescence colonies compared to SFs (P < 0.05) (Fig. 6E). A pMSC colony positive for GFP after 14 days of culture is shown in Figure 6G.

Somatic Cell Nuclear Transfer

Both pMSCs and SFs synchronized with roscovitine were used in two replicates as nuclear donors to produce cloned porcine embryos. Cleavage rates were 44.5% (65/146) for pMSC and 53.1% (60/113) for SF NT embryos. Development to blastocyst stage was 4.1% (6/146) in the MSC group and 1.77% (2/113) in the SF group. The Yates chi-square test revealed no statistical difference in cleavage and blastocyst development rates between pMSC- and SF-derived embryos (P = 0.21 and P = 0.47, respectively). The number of cells per blastocyst ranged from 7 to 23 cells/blastocyst.

DISCUSSION

Since they were first identified by the pioneering work of Friedenstein et al. [36, 37] in the early 1970s, MSCs, also known as marrow somatic cells or CFU-F cells, have been the subject of numerous studies aimed at deciphering the roles of these stem cells in the complex marrow physiology. The MSCs are considered to be nonhematopoietic precursor cells that support hematopoiesis and can differentiate down the lipogenic, chondrogenic, osteogenic, and tenogenic pathways (for review, see [38]). The broad differentiation potential (along with the extensive ex vivo proliferative capacity) makes these stromal precursors attractive candidates for autologous and allogeneic cell therapy and, potentially, for SCNT transgenesis. We have isolated and characterized the growing properties of pMSCs under different culture conditions. Then, we examined the ability of pMSCs and isogenic SFs to undergo transient and stable genetic modifications using a combination of GFP plasmid with a transfection reagent and viral vectors. Finally, the present study suggests that pMSCs can undergo nuclear reprogramming to generate cloned blastocysts.

We were able to establish a primary MSC line from each of 10 individual animal BM aspirations. Imunocytochemistry and flow cytometric analysis revealed that most cells expressed the mesenchymal marker THY1 and did not express ITGAM, a hematopoietic marker in granulocytes, monocytes, natural killer cells, subsets of T cells, and subsets of B cells [39]. Expression of cell surface markers in BM-derived cells isolated in the present study supports the mesenchymal origin of these cells and agrees with the results of previous work. These markers are conserved across species, because human and rat MSCs also express THY1 and lack ITGAM expression [4, 6]. Furthermore, analysis of forward and scatter light data from pMSCs revealed homogenous cell populations (size and granularity; data not shown) coinciding with flow cytometric results from human MSCs [4]. The MSCs, but not the fibroblasts, were capable of differentiating down mesenchymal lineages [4, 10, 11, 13], demonstrating that isolated cells from pig BM were truly MSCs. Morphology, surface antigen profile, and pluripotency characteristics provide convincing evidence that the BM cells isolated in the present study are pMSCs.

Because little is known about culture conditions that support pMSC proliferation in vitro, we first sought to characterize the growth properties of pMSCs under different culture conditions. Development of MSC colonies depended entirely on the growth factors in FBS (Fig. 4, A and B). No colonies were present when FBS was omitted, and a clear, positive dose-response relationship was observed between colony numbers and FBS concentrations. A similar response to FBS was reported for human MSCs [40]. Overall, it is apparent that addition of 10%–20% of serum to pMSC culture medium provides adequate support for pMSC expansion. In three of four pMSC lines, the number and diameter of colonies was not different for pMSCs cultured in MEM Alpha, DMEM 2.2, or DMEM 3.7 but was significantly lower for cells growing in DMEM/F12. The DMEM/F12 medium, however, enhanced proliferation of pMSCs in the CyQUANT assay (Fig. 4D). This inconsistent response may have arisen from inherent differences between the assays. The CFU-F assay measured both plating efficiency and proliferation capacity of media, whereas the CyQUANT assay only measured proliferation capability. We found that DMEM/F12 medium did reduce rates of cell attachment in the CyQUANT plating assay. Different cell concentrations between CFU-F (very low cell density) and CyQUANT (high cell density) assays also may have contributed to the differential outcomes observed.

The effect of oxygen tension during culture on colony formation was investigated. Despite the fact that oxygen tension did not affect the number and size of the colonies (Fig. 4G), pMSCs growing in low oxygen (5%) proliferated faster than cells cultured in higher oxygen tension (21%) (Fig. 4H). Culture of cells in reduced oxygen tension has been reported to cause inhibition [41, 42] or stimulation of cell growth in vitro [43–47]. Our results demonstrated an increased proliferation rate of pMSCs in an oxygen concentration (5%) that more closely resembles in vivo conditions. Therefore, exogenous control of oxygen tension may have important implications for in vitro propagation of pMSCs and, possibly, differentiation [47, 48].

Ascorbic acid or vitamin C, a primary antioxidant for cells, has been associated with enhancement of cell proliferation [49–51], and it contributes to collagen synthesis in mesenchymal cells [52–54]. One difficulty associated with supplying ascorbate to cultured cells is the instability of this vitamin under standard culture conditions (neutral pH, 37°C, and aerobic environment) resulting from its autoxidation [55]. To overcome this problem, we used an esterified ascorbate (ascorbic acid 2-phosphate), which is more resistant to autoxidation [50] and, therefore, more stable in aqueous solutions. Addition of ascorbic acid to a vitamin C-free medium over a wide range of concentrations (5–500 µg/ml) did not affect the proliferation rate of pMSCs. There may be several reasons why we did not observe an effect. The amount of free ascorbate available for the cells is dependent on the rate of conversion of ascorbate 2-phosphate to ascorbate in a given cell type and culture condition. Therefore, the rate of conversion of ascorbate from its esterified precursor in our culture system might not have been optimal. Additionally, the method used to estimate cell numbers may not have been sensitive enough to detect subtle effects of ascorbic acid on cell proliferation. The impaired cell proliferation observed when ascorbate was added at 5000 µg/ml of medium is consistent with the idea that at high concentrations, ascorbate favors the generation of free radicals, promoting in this way a pro-oxidative rather than an antioxidative state [56].

Transient gene transfer into cultured cells with subsequent expression of the transgene has become a valuable tool for physiological studies, functional genomics, and gene therapy. Furthermore, ectopic expression of signaling molecules and transcription factors has been useful in manipulating the differentiation fate of stem cells [57, 58]. In the present study, we used a recombinant adenoviral vector carrying the GFP gene to transiently transduce pMSCs and SFs in combination with flow cytometric analysis to determine expression of the reporter gene. We have shown that both cell types can be infected with the nonintegrating human adenovirus vector. A higher proportion of pMSCs, however, expressed GFP compared with isogenic SFs (Fig. 5), and GFP intensity data suggest that a larger number of adenoviral particles entered into MSCs than SF cells (Fig. 5A). Because adenoviral entry into the host cells is mediated through membrane receptors, particularly MCP (also known as CD46) for Ad5F35 [59], the pMSCs likely possess higher density of adenoviral receptors. We also observed a higher proportion of nonviable pMSCs in these cultures (Fig. 5A), which could be the result of viral cytopathic effects associated with the higher viral infection achieved in this group. Nonetheless, overall cell viability was very high (>95%) in both cell lines. When pMSCs and SFs were transfected with the GFP plasmid, both cells types transiently expressed GFP, but far fewer cells (ninefold lower) expressed the reporter gene compared to that in adenovirus vector transduced cells.

The recent development of SCNT to produce cloned animals has provided a new method for generating transgenic livestock [20]. Besides being adaptable to in vitro proliferation conditions, the donor cells used in this process should be amenable to stable genetic manipulation and undergo nuclear reprogramming. We demonstrated stable transgene expression in pMSCs and SFs using a GFP plasmid and a viral vector (AAV). Plasmid integration was confirmed by selection of transgenic cells with the antibiotic G418; approximately 1 in every approximately 16000 treated cells integrated the transgene. We then used FACS for clonal propagation of GFP-positive cells in 96-well plates. At sorting, the percentage of GFP-positive cells transduced with AAV vector doubled that of cells transfected with the plasmid (Fig. 6A). After 14 days in culture, approximately 8% of the clonal colonies were GFP positive in the plasmid-transfected group versus approximately 93% in the AAV vector-transduced groups (pMSC and SF). Both plasmid- and AAV vector-derived GFP-positive colonies were expanded in vitro up to approximately 1 x 106 cells without losing GFP expression (~21 cell doublings). These data indicate clonal selection for stable transgene integration and propagation of transgenic donor cells. Furthermore, the AAV vector used in the present study clearly was superior to a conventional GFP plasmid. Viral vectors have been used previously to create transgenic cell lines later used to generate SCNT transgenic embryos [25] and animals [26, 27]. Recently, Hofmann et al. [28] obtained high transduction rates of bovine fibroblasts in culture with a lentivirus vector, and these transgenic cells were able to drive development to term. Therefore, the use of integrating viral vectors like lentivirus and AAV, as we demonstrated in the present study, is a highly effective alternative method to deliver DNA into cells.

Less differentiated cells may be more amenable than terminally differentiated cells to nuclear reprogramming on NT (for review, see [60]). For example, postimplantation survival of clones originated from mouse embryonic stem cells was higher than that of adult somatic cells [61], and enhanced in vitro development of preimplantation pig embryos reconstructed with fetal skin-derived stem cells has been reported [62]. Bovine MSCs were able to undergo nuclear reprogramming after SCNT and supported development to term [18], but SCNT using other somatic cell types were not included in that study for comparison purposes. In the present study, NT results suggest that pMSCs can support blastocyst development after being transferred to enucleated metaphase II oocytes. Although both the pMSCs and SFs produced low cleavage and blastocyst rates, this does indicate that pMSCs can undergo nuclear reprogramming at least in support of development to the blastocyst stage. Additional replicates will be necessary to establish any potential differences in reprogramming ability between cell lines and types. Having shown that pMSC donor cells can develop into initial SCNT blastocyst-stage embryos, future studies might investigate the differentiation potential or differences between pMSCs and embryonic cells derived from the same donor cells. In addition, this work can lead to studies that determine the effects of stable (AAV) or transient (Ad5-F35 vector) expression of certain exogenous genes and their effect on nuclear reprogramming.

In conclusion, we have been able to establish adult pMSC lines from live animals using a minimally invasive BM aspiration technique. These adult stem cells can proliferate extensively in vitro (~30 cell doublings until senescence; data not shown) and undergo transient and stable genetic modification with nonviral and viral vectors. Of particular interest is the highly efficient transduction of MSCs with a nonintegrating human adenovirus and AAV vectors. All these characteristics along with favorable clonal cell propagation properties make pMSCs an attractive source of cells for large-animal preclinical testing. These significant findings will lead to future autologous cell/gene therapy studies comparing easily cultured, genetically modified, adult pMSCs to isogenic embryonic cells derived via SCNT, thus addressing cell rejection issues in nonmurine models for disease and tissue repair.

ACKNOWLEDGMENTS

We would like to thank Julie Nelson from the Center for Tropical and Emerging Global Diseases Flow Cytometry Facility at the University of Georgia for her assistance with flow cytometric analysis.

FOOTNOTES

1 Supported by ViaGen, Inc. Back

2 Correspondence: Steven L. Stice, Edgar L. Rhodes Center for Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771. FAX: 706 542 7925; sstice{at}uga.edu Back

Received: 30 June 2005.

First decision: 23 July 2005.

Accepted: 7 September 2005.

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