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CLONING AND STEM CELLS Volume 7, Number 3, 2005 © Mary Ann Liebert, Inc.
Establishment, Differentiation, Electroporation, Viral Transduction, and Nuclear Transfer of Bovine and Porcine Mesenchymal Stem Cells S. COLLEONI,1 G. DONOFRIO,2 I. LAGUTINA,1 R. DUCHI,1 C. GALLI,1,3 and G. LAZZARI1
ABSTRACT Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro—approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.
INTRODUCTION
T
is a specialized microenvironment that allows both the proliferation of the undifferentiated hematopoietic and mesenchymal stem cells, and the complex processes of differentiation of the several cell types of the hematopoietic system. In vivo, mesenchymal stem cells, as hematopoietic stem cells, support hematopoiesis, producing growth factors comprising interleukins (IL-6, IL-7, IL-8, IL-11, IL-12), leukeHE BONE MARROW
mia inhibitory factor (LIF), stem cell factor, and macrophage colony–stimulating factor (Pittenger et al., 1999; Pittenger and Marshak, 2001). In vitro, the bone marrow microenvironment can be mimicked by long-term bone marrow cultures in which stromal cells and hematopoietic stem cells are co-cultured and can recapitulate the hematopoietic process (Marsicano et al., 1997). Similarly, bone marrow–derived mesenchymal stem cells (MSCs) can be grown in vitro in the undifferentiated state or can be differentiated with adequate
1Laboratorio
di Tecnologie della Riproduzione, CIZ srl, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy. 2Dipartimento di Salute Animale, Sezione di Malattie Infettive, Facoltà di Medicina Veterinaria, Università di Parma, Parma, Italy. 3Dipartimento Clinico Veterinario, Università di Bologna, Bologna, Italy.
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stimuli (Dennis and Charbord, 2001; Sekiya et al., 2002; Hung et al., 2002). In humans, but also in other species such as mouse, rat and cat, mesenchymal stem cells can proliferate in vitro for many passages, displaying a stable fibroblast-like phenotype and can be differentiated, not only to classical mesenchymal derivatives such as bone, fat, cartilage, muscle, and cardiomyocyte (Bruder et al., 1997; Jaiswal et al., 2002; Pittenger et al., 1999; Ferrari et al., 1998; Wakitani et al., 1995; Makino et al., 1999), but also to other lineages such as oval hepatocytes and neurons (Myiazaki et al., 2002; Sanchez-Ramos et al., 2000; Woodbury et al., 2002). In the mouse, a selected population of bone marrow cells that co-purify with MSCs, called multipotent adult progenitors (MAPCs), displays even wider pluripotentiality, giving rise to chimeric fetuses following blastocyst injection of MAPCs transduced with a lentiviral vector carrying green fluorescent protein (Jiang et al., 2002). Recent studies have analyzed gene expression of mesenchymal stem cells by RT-PCR, microSAGE, immunocytochemistry, or flow cytometry, showing that MSCs express mRNAs of multiple cell lineages (Tremain et al., 2001), including germline, endodermal, mesodermal, and ectodermal genes, indicating that they are molecularly heterogeneous (Woodbury et al., 2002). In this paper, we describe a method for the isolation of bovine and porcine MSCs and their culture in vitro in the undifferentiated state and following differentiation. We also provide data on the morphological and molecular characterization of the undifferentiated state and their derivatives following induction of differentiation. As further characterization, we demonstrate that bovine and porcine MSCs can be both electroporated and virally transduced with vectors carrying the GFP marker gene. We show that, following genetic modification, MSCs retain the ability to differentiate. Finally, we used undifferentiated and differentiated bovine and porcine MSCs as nuclear donors in nuclear transfer experiments, obtaining consistently high rates of embryo reconstruction and blastocyst formation.
MATERIALS AND METHODS Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (Milan, Italy) and plastic-ware from Nunc (Roskilde, Denmark).
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Isolation and culture of mesenchymal stem cells Bone marrow cells were obtained by aspiration from the iliac crest of two 1-month-old calves with an 11-g bone marrow biopsy/aspiration needle or by flushing the femurus and tibias of three 1-week-old piglets, in all cases immediately after slaughter. Twenty-milliliter syringes containing 3 mL of aspiration medium (TCM199/ DMEM, 1:1) with 5000 IU of heparin were used, and about 40 mL of bone marrow were removed from each animal. After multiple PBS washings, the cell suspension was loaded onto 70% percoll gradient and centrifuged at 1100 g for 30 min. Cells were collected from the interface and washed. Residual erythrocytes were removed, resuspending the cell pellet in 1 mL of a solution of 0.83% NH4Cl in Tris HCl 10 mM for 1 min; then 9 mL of culture medium were added, and the suspension was washed three times. Following counting, cells were plated at 200,000 cells/cm2 and cultured in TCM199/DMEM (1:1) 10% fetal bovine serum (FBS) at 38.5°C in 5% CO2. Medium was replaced at 24 and 72 h, and then twice weekly. Cells were subsequently subcultured at 5000 cell/cm2 for at least 15 passages. Part of cells from first passage was expanded and frozen. Analysis of karyotype was performed at passage 10.
Osteogenic differentiation Cells from all passages fresh, frozen-thawed, or frozen-thawed and electroporated were plated at 3000 cells/cm2, and treated with osteogenic medium containing dexamethasone 100 nM, ascorbic acid 0.25 mM, and -glycerolphosphate 10 mM. The medium was changed three times per week. At 8–12 days after beginning of the culture in osteogenic medium, the cells were stained with BCIP/NBT (LabVision Corp., Fremont, CA) to detect alkaline phosphatase activity, and at the end of the culture, after 21 days, they were treated with silver nitrate (Von Kossa staining) to assay for the accumulation of calcium. Samples of cells were frozen before and after differentiation for RT-PCR analysis.
Differentiation with 5-azacytidine Subconfluent mesenchymal stem cells from early passages were treated with 5-azacytidine at 3, 6, or 9 M for 24 h; then the medium was re-
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placed with normal culture medium (TCM199/ DMEM, 1:1, supplemented with 10% FBS), and the cells were cultured for at least 4 weeks. The cultures were monitored for morphological changes during the culture. Formation of lipid vacuoles was detected by Red Oil staining. Samples were analyzed by RT-PCR before and after differentiation.
Analysis of gene expression Gene expression of undifferentiated cells from passage 2 or of differentiated cells was studied by RT-PCR (cells to cDNA; Ambion, Austin, TX). Total RNA was extracted and then reverse transcribed with random decamers according to the protocols of the supplier. The samples obtained were immediately used for PCR or frozen for subsequent experiments. Primers and annealing temperatures are indicated in Table 1; -actin was used as positive control.
Herpes virus infection Infection of MSCs was performed with 1 MOI (multiplicity of infection) of BoHV-4EGFPTK, which was propagated as previously described (Donofrio et al., 2002). A BT cell line (bovine turbinate cells, ATCC, CRL-1390) was used to propagate the virus, because of its high sensitivity towards infection of BoHV-4 (Donofrio et al., 2002). BT cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO2. BoHV-4EGFPTK was propagated by infecting confluent monolayers with one of 50% tissue culture infective dose (TCID50) per cell. After 4 days, cultures were frozen and thawed three times to release the virus. After removal of cell debris by low-speed centrifugation, the virus stock was frozen at 80°C. The TCID50 was determined by limiting dilution.
Electroporation and drug selection Five micrograms of plasmid DNA (pEGFP-C1) were electroporated (EquiBio, Easy-Get apparatus, 270 V, 960 F) in DMEM without serum into MSCs from a confluent 25-cm2 flask. Cells were returned to a new 25-cm2 flask, fed with complete medium, and analyzed for EGFP expression under a fluorescence microscope at 48 h post-electroporation. To obtain stably transfected bovine and porcine MSCs, the samples were incubated in complete medium for 48 h and then supplemented with 200 g/mL of G418 (Invitrogen). After 14 days of selection, stable clones were obtained and analyzed for EGFP expression and differentiation.
TABLE 1. Gene Bovine actin (Lonergan et al., 2000) Porcine actin (Dozois et al., 1997) Bovine osteopontin (Gronthos et al., 2003) Porcine osteonectin (Ringe et al., 2002) Bovine myosin HC (Tanabe et al., 1998) Porcine myosin HC (Tanabe et al., 1999)
PRIMERS
FOR
Lentiviral vector preparation and infection Third generation of lentiviral vector was prepared as previously described (Follenzi and Naldini, 2002). Seventy-five-cm2 flasks of 293T cells were transfected by calcium-phosphate precipitation-based method. At 2 h before transfection, the medium (DMEM, 10% FBS, 25 U/mL penicillin and 25 U/mL streptomycin) was refreshed, and four-plasmid DNA transfection mix was prepared by adding 9 g of envelope plasmid (pMD2-VSVG), 12.25 g of core packaging plasmid (pMDLg/pRRE), 6.25 g of REV plasmid (pRSV-REV), and 25 g of transfer vector (pCCLsin18.PPT.Prom.GFP.Wpre). The plasmid
ANALYSIS
OF
GENE EXPRESSION
Sequence of primers
T.annealing
5-GAGAAGCTCTGCTACGTC-3 5-CCAGACAGCACCGTGTTGG-3 5-GGACTTCGAGCAGGAGATGG-3 5-GCACCGTGTTGGCGTAGAGG-3 5-TTGCTTTTGCCTCCTAGGCA-3 5-GTGAAAACTTCGGTTGCTGG-3 5-TCCGGATCTTTCCTTTGCTTTCTA-3 5-CCTTCACATCGTGGCAAGAGTTTG-3 5-TCTGAGTTCAGCAGCCATGAGTTCCGACCAAG-3 5-ATCCAGGCTGCGTAACGCTCTTTGAGGTTGTA-3 5-CACTTGCTAAGAGGGACCTCTGAGTTC-3 5-ATCCAGGTCGCGTAACGCTCTTTGAGGTTGTA-3
60°C 54°C 60°C 55°C 55°C 60°C
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solution was made up to a final volume of 500 L with 450 L of H2O and 50 L of 2.5 M CaCl2. The precipitate was formed by drop-wise addition of 500 L of the 2 HBS solution (280 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4, pH 7.12) to the 500-L plasmid DNA-CaCl2 mix and finally added to the 293T cells. The calcium phosphate–DNA precipitate was allowed to stay on the cells for 14–16 h, after which the medium was replaced with 10 mL of fresh medium. Forty-eight hours after changing the medium, the cell supernatant containing viral particles was collected, centrifuged at 1500 rpm for 5 min, filtered through a 0.22-m nitrocellulose filter, aliquoted, and stored at 80°C. Transducing units per milliliter (TU/mL) were determined by limiting dilution on 293T cells. MSCs were plated on 25-cm2 flasks and grown until they reached 80% (106 cells) of confluence. The lentiviral vector was added onto each flask at a MOI (multiplicity of infection) of 1. MSCs were incubated overnight at 37°C, and then the medium was replaced with 5 mL of fresh medium. After 2 days of incubation, the cells were analyzed for EGFP expression under a fluorescence microscope.
Nuclear transfer experiments Oocyte maturation. Ovaries were collected from slaughtered cows and sows. Oocytes were aspirated from follicles 3–8 mm in diameter and transferred to maturation medium. Bovine oocytes were matured in TCM199 and porcine oocytes in DMEM/F12. In both species, the maturation media were supplemented with 10% (v/v) FBS, ITS Media Supplement (cat. no. I1884, 1 L/mL), 1 mM sodium pyruvate, 0.5 mM L-cystein, 10 mM glycine, 100 M -mercaptoethanol, gonadotropins (0.05 IU/mL FSG and 0.05 IU/mL LH; Pergovet 75, Serono). Bovine and porcine oocytes were cultured at 38.5°C in 5% CO2 in humidified air for 20–21 h and 44 h, respectively. Preparation of donor cells for nuclear transfer. Two days before nuclear transfer, the donor cells were passaged, and the day after were serum starved (0.5% FBS in the growth medium) for 24 h. For nuclear transfer, we used adult fibroblasts, mesenchymal stem cells, and their derivatives following induction of osteogenic differentiation. Porcine osteocytes were used on day 8 (early osteocytes) of differentiation when alkaline phosphatase—the first marker of osteogenic induction—is expressed
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at the highest level, and on day 21, when the cells show terminal differentiation characterized by calcium accumulation and formation of aggregates (Von Kossa staining positive). Bovine osteocytes were used only on day 21. Preparation of enucleated oocytes and NT–embryo construction. The oocytes were denuded of granulosa cells by vortexing in the presence of hyaluronidase. Zona-free method (Oback et al., 2003) of NT–embryo construction was used. Zona pellucida of oocytes with extruded polar bodies was digested with 0.5% pronase in PBS. Zona-free oocytes were stained with Hoechst 33342 in the presence of cytochalasin B (5 g/mL) and enucleated under UV light by enucleation pipette with perpendicular break. All manipulations were performed in SOF-Hepes with 10% FBS. Zona-free cytoplasts were individually washed for several seconds in 300 g/mL phytohemagglutinin P in PBS and then quickly dropped over a single donor cell (Vajta et al., 2003) that settled to the bottom of the drop of SOF-Hepes. Formed cell couplets were washed in 0.3 M mannitol (100 M Mg) solution and fused by double DC-pulse of 1.2 Kv/cm applied for 30 sec. Activation. Oocytes and NT–embryos (2–3 h after fusion) were activated at 27–28 h after the onset of maturation for bovine and 50 h for porcine. For activation, bovine NT–embryos were treated with 5 M ionomycin in SOF-Hepes for 4 min, followed by 4-h culture in 2 mM 6-DMAP in medium SOF, supplemented with MEM essential and non-essential aminoacids and 4 mg/mL BSA (m-SOFaa). Porcine zona-intact oocytes and NT–embryos were washed in 0.3 M mannitol (1 mM Ca and 100 M Mg) solution (Cheong et al., 2002) and activated by double DC-pulse of 1.2 Kv/cm applied for 30 sec, followed by 4-h culture in m-SOFaa with cytochalasin B (5 g/mL). Oocytes were cultured in 20-L drops, and zonafree NT–embryos were cultured individually in 3-L drops under mineral oil at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryo culture. Oocytes and zona-free embryos were cultured, respectively, in 20-L culture drops and individually in wells in 20-L drops (modified Well Of the Well method of Vaita et al., 2000) of m-SOFaa with 4 mg/mL BSA in 5% CO2 and 5% O2 in humidified air at 38.5°C under mineral oil. Half of the medium was renewed on day
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3 with fresh m-SOFaa and on day 5 (pig) or 6 (bovine) with TCM199 supplemented with 16 mg/mL BSA (day 0 was the day of fusion and activation). Cleavage was assessed 48 h after activation, the rate of compacted morula was recorded on day 5 and day 6, and the rate of blastocyst (BL) at day 6–7 and day 7–8, respectively, for porcine and bovine embryos. All experiments were done in four replicates.
Statistical analysis Differences between the experimental groups were verified using 2 test or Student’s t-test. A value of p 0.05 was considered significant.
RESULTS Isolation and culture of MSCs On average, we obtained 50 million nucleated cells from 40 mL of bovine bone marrow aspirate
and 100 million from swine bone flushing. The cells were plated at 200,000 cells/cm2 and incubated for 24 h; at this point, the medium was changed to remove hematopoietic cells that have less capability to attach to the plastic surface than mesenchymal cells. At 72 h, another change of medium was performed. Microscopical observation of adherent cells led us to estimate that, out of 5 million cells plated per 25-cm2 flask, only 80–100 were attached and were growing as colonies. These cells displayed a fibroblastic shape, and 10 days after plating when 90% confluence was reached (Fig. 1A,B), they were subcultured until exhaustion of growth potential. As described in previous studies performed in others species, the cells grew with regular pace until passage 10, and then they started to slow down and finally arrested growth at passage 15 (about 50 population doublings for bovine and 40 for porcine; Fig. 2). Reduction of growth was accompanied by increasing cellular dimension and by changing morphology from spindle-shaped to flat. Cytogenetic analysis demonstrated that cells
FIG. 1. Early passages of bovine (A) and porcine (B) undifferentiated mesenchymal stem cells show fibroblastic shape and homogeneous morphology. After 2 days in osteogenic medium containing dexamethasone, ascorbic acid and -glicerolphosphate both bovine (C) and porcine (D) cells acquire cuboidal morphology.
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FIG. 2. Growth curves of undifferentiated bovine and porcine mesenchymal stem cells show constant replication for ten passages and then slow down until exhaustion of growth potential.
retained normal diploid karyotype at up to 10 passages of culture (data not shown).
Osteogenic differentiation Following exposure to osteogenic medium, the MSCs changed their morphology from spindle shaped to cuboidal (Fig. 1C,D) already at 24 h after treatment and started to grow more rapidly, forming multilayered structures. Assay with BCIP/NBT showed that alkaline phosphatase was more active from day 8 to 10 (Fig. 3A,B). From week 2 to week 3, they formed aggregates (von Kossa staining positive), indicating the accumulation of calcium (Fig. 3C,D). All differentiated cells showed the same characteristics; no difference was observed in relation to passage, except the speed of growth. Also, when cells were derived from frozen cultures, they differentiated as cells from fresh cultures. Moreover, electroporated cells maintained the capacity to differentiate as parental cells.
Differentiation with 5-azacytidine Treatment with 5-azacytidine at 3, 6, and 9 M of porcine and bovine MSCs, and subsequent
culture in medium with 10% of FBS led to an unspecific differentiation and to loss of control of replication, even if there was a preferential differentiation into muscle and adipocytes revealed by expression of the muscle marker myosin HC and by red oil staining. In particular, several days after treatment, we observed increasing in growth rate and formation of multilayered cultures (Fig. 4A). After 3–4 weeks, about 20% of cells changed their shape (becoming round), few became bi- or multinucleated, and in some cells visible lipid vacuoles were identified by Red Oil staining (Fig. 4B); no differences were observed between different concentrations of 5-azacytidine.
Analysis of gene expression RT-PCR was performed to assess change in gene expression at the undifferentiated state and following differentiation (Fig. 5). When cultured in osteogenic medium, bovine MSCs express ostepontin and swine MSCs express osteonectin (characteristic osteocyte markers), whereas both bovine and porcine cells treated with 5-azacytidine became positive for muscular marker myosin HC. None of these markers
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FIG. 5. Analysis of gene expression of bovine (left panel) and porcine (right panel) in the undifferentiated state (A) and following differentiation in osteogenic medium (B) and by 5-azacytidine treatment (C). Mesenchymal stem cells become positive to osteopontin (404 bp, bovine lane 2) and to osteonectin (187 bp, porcine lane 2) when cultured in osteogenic medium (B). Cells of both species expressed myosin HC (359 bp, bovine lane 3; and 377 bp, porcine lane 3) if treated with 5-azacytidine (C). -Actin was used as positive control (245 bp, bovine lane 1; and 233 bp, porcine lane 1).
was expressed in undifferentiated MSCs of both species.
Electroporation and viral transduction To initially test the MSC’s capability of expressing a reporter gene under the control of a specific promoter, MSCs were transfected by elec-
troporation with a plasmid carrying an EGFP expression cassette under the control of the CMV immediate early promoter (pEGFP-C1, Clontech; Fig. 6). The efficiency of transfection obtained was approximately 58% for bovine and 67% for porcine MSCs. The CMV/EGFP expression cassette was well transcribed, as shown by the EGFP-expressing
FIG. 3. Staining with BCIP/NBT of osteodifferentiated bovine (A) and porcine (B) demonstrate alkaline phosphatase staining on day 10 of culture. After 21 days of culture in osteogenic medium bovine (C) and porcine (D) mesenchymal stem cells form aggregates that result positive to Von Kossa staining demonstrating accumulation of calcium. FIG. 4. Mesenchymal stem cells treated for 24 h with 5-azacytidine and then cultured in control medium gave rise to multilayered culture of spindle-shaped and round cells (A). After at least 5 weeks of culture, they form lipid vacuoles revealed by Red Oil staining (B). FIG. 6. Culture of electroporated and BoHV-4EGFPTK transduced MSC. (A) Diagram showing the structure of the CMV/EGFP expression cassette integrated in pEGFP-C1 plasmid. Cytomegalovirus Immediate Early promoter (CMV) followed by the Enhanced Green Fluorescent Protein open reading frame (EGFP) and the Poly-Adenylation sequence (PA) of the bovine growth hormone. (B) Fluorescence microscope image (40) of BoMSC electroporated cells. (C) Phase contrast microscope image. (D) Diagram showing the structure of the BoHV-4EGFPTK, generated by homologous recombination with a CMV/EGFP expression cassette inserted into the TK locus of the DN599 BoHV-4 strain. Polyrepetitive DNA (pr DNA) is indicated to the right and left end of the viral genome. (E) Fluorescence microscope image (40) of BoMSC BoHV-4EGFPTK transduced cells. (F) Phase contrast image. FIG. 7. Culture of lentiviral transduced MSC. (A) Fluorescence microscope observation (40) of lentiviral transduced BoMSC expressing EGFP. (B) Phase contrast microscope observation. (C) MSC in phase contrast combined with fluorescence (40), where a transduced cells expressing EGFP emerge from background. (D) Scheme of the lentiviral vector construct used for this study. HIV-based third generation lentiviral vector containing an internal Immediate Early Cytomegalovirus promoter (CMV) driving the Enhanced Green Fluorescent Protein (EGFP), a central Poly-Purine Tract (cPPT), a Woodchuck Post-transcriptional Regulatory Element (WPRE) and the two Long Terminal Repeat (LTR) sequences. The left-end LTR has a deletion in the U3 region for the self-inactivation (SIN) of the vector. FIG. 8. Electroporated and drug-selected bovine (A,C) and porcine (B,D) mesenchymal stem cells maintain their capability to differentiate. Those cultured for 21 days in osteogenic medium cells show the same kinetics of those not electroporated culture expressing alkaline phosphatase on day 10 (A,B) and accumulating calcium at the end of the culture period (C,D).
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FIG. 4.
FIG. 3.
FIG. 6.
FIG. 7.
FIG. 8.
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cells (Fig. 6B,C). In order to obtain stable transfection of bovine and porcine MSCs, we treated the electroporated cells with G418. Clones of stably transfected G418 resistant MSCs were detectable on the plates and showed bright EGFP expression. Both bovine and porcine MSCs stably expressing EGFP exhibited a phenotype characteristic of untransfected MSCs. The pooled clones were expanded, and about 98% of the porcine MSCs expressed EGFP, whereas only 20% of bovine MSC were positive for this marker. Stably transfected porcine and bovine MSCs mantained their multipotential characteristics, as evidenced by their ability to differentiate into osteocytes after selection and expansion. As expected, after 8 days in osteogenic medium, they were positive for alkaline phosphatase, and after 21 days, calcium accumulation was evidenced by Von Kossa staining while EGFP expression was maintained (Fig. 7). For viral transduction, MSCs were initially infected with a green recombinant BoHV-4 (BoHV4EGFPTK) obtained by insertion of the CMV/ EGFP expression cassette into the TK locus of DN 599 BoHV-4 (Donofrio et al., 2002) strain (Fig. 6D). As in electroporated MSCs (Fig. 6B,C), in BoHV-4EGFPTK–infected MSCs (Fig. 6E,F) the CMV/EGFP expression cassette was very well transcribed. The efficiency of transfection was approximately 100% for BoHV-4, although in a few days the cells were lost due to the cytopathic effect of the virus. We then used a safer thirdgeneration self-inactivating, vescicular stomatitis virus-glycoprotein (VSV-G) pseudotyped lentiviral vector, carrying the CMV/EGFP expression cassette in order to obtain stable transduction (Fig. 8A–C). The efficiency was approximately 4% for both bovine and porcine MSCs.
Nuclear transfer experiments The data shown in Tables 2 and 3 indicate that both undifferentiated and differentiated bovine TABLE 2.
Cell type Adult fibroblasts MSC Early osteocytes Osteocytes
and porcine mesenchymal stem cells can be used as nuclear donors and give rise to similar rates of cleavage and preimplantation development to the blastocyst stage. When compared with adult fibroblasts used as control, no difference was observed in developmental rates at all stages examined. Blastocyst rate ranged from 32.9% to 44.7% in pig (day 7) and from 52.9% to 66.7% in bovine (day 8).
DISCUSSION The mesenchymal stem cell lines described in this paper have been grown by applying culture and differentiation protocols previously reported (Pittenger et al., 1999; Jaiswal et al., 1997; Ringe et al., 2002). Similarly to what has been published in other species, we found that the rate of MSCs over the whole mononuclear bone marrow population was about 1 in 50,000 cells, and this fraction could be selected on the basis of their early adhesion to the plastic surface following plating of percoll-separated bone marrow aspirates. In control experiments, where the medium was not changed at 24 and 72 h after plating, we obtained hematopoietic cultures containing hematopoietic stem cells, stromal cells, and adipocytes (data not shown). Our cell lines have growth kinetics typical of MSCs, with constant growth rate until passage 10, followed by progressive reduction and culture senescence until the definitive exhaustion of doubling potential. However, we observed that slow down of growth did not affect the differentiation potential: cells from all passages treated with osteogenic medium maintained the same capability of changing shape, becoming alkaline phosphatase positive and forming aggregates (Von Kossa staining positive), in spite of the different growth rate. This relatively reduced capacity of “self-re-
DEVELOPMENT OF PIG NUCLEAR TRANSFER (NT) EMBRYOS DERIVED FROM ADULT FIBROBLASTS, MESENCHYMAL STEM CELLS (MSC), AND THEIR OSTEOCYTE DERIVATIVES NT embryos, n
Cleavage, n (%)
MC D5, n (%)
BL D6, n (%)
BL D7, n (%)
86 85 82 86
74 (86.0) 77 (90.6) 71 (86.6) 74 (86.0)
41 (47.7) 39 (45.9) 37 (45.1) 42 (48.8)
30 (34.9) 34 (40.0) 26 (31.7) 34 (39.5)
32 (37.2) 38 (44.7) 27 (32.9) 33 (38.4)
MC D5, compacted morulae on day 5; BL D6, blastocyst on day 6; BL D7, blastocyst on day 7.
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Cell type Adult fibroblasts MSC Osteocytes
DEVELOPMENT OF BOVINE NUCLEAR TRANSFER (NT) EMBRYOS DERIVED FROM ADULT FIBROBLASTS, MESENCHYMAL STEM CELLS (MSC), AND OSTEOCYTES, DIFFERENTIATED FROM MSC NT embryos, n
Cleavage, n (%)
MC D6, n (%)
BL D7, n (%)
BL D8, n (%)
63 102 102
62 (98.4) 102 (100). 102 (100).
36 (57.1) 58 (56.9) 52 (51.0)
33 (52.4) 65 (63.7) 55 (53.9)
42 (66.7) 67 (65.7) 54 (52.9)
MC D6, compacted morulae on day 6; BL D7, blastocyst on day 7; BL D8, blastocyst on day 8.
newal” can be considered a limit of MSCs in comparison with embryonic stem cells. It was reported that some particular populations have reached 80 doublings (Jiang et al., 2002), but in other cases the average obtained in cultures from different donors has been 38 doublings (Jaiswal et al., 1997), and in this study we have cultured the MSCs for about 50 population doublings. Senescence, as recently observed, is imputable to progressive loss of telomeres, short sequence (TTAGGG) repeats associated with proteins that are localized at the end of chromosomes and protect them from degradation and recombination. Somatic cells are destined to lose these sequences and to age, but tumoral cells and embryonic stem cells can maintain their telomeres through an endogenous reverse transcription mechanism. This activity has been detected at low or moderate level in stem cells from skin and gut, in hematopoietic stem cells, but never in mesenchymal stem cells. Interestingly, it has been demonstrated that it is possible to increase the proliferative capacity of mesenchymal stem cells to more than 260 doublings, transfecting them with the catalytic subunit of telomerase without interfering with differentiation potential or karyotype stability (Simonsen et al., 2002). Regarding gene expression, our comparative data confirm that gene expression profile of MSCs is not homogeneous between different species. For example, bovine MSCs express muscular marker myosin HC only if treated with 5-azacytidine, while undifferentiated rat cells are positive for the same marker (Woodbury et al., 2002). Leptin gene, characteristic of adipose tissue, a classical mesenchymal derivative, is expressed by undifferentiated rat cells (Woodbury et al., 2002) but not by human mesenchymal cells, if not treated with adipogenic medium (Gronthos et al., 2003); preliminary experiments with our bovine cells show that they are negative for this marker. Gene expression studies in human mesenchymal
stem cells indicate that cells arising from the same colony express genes typical of multiple differentiated cell lineages, including neural and endoepitelial marker. Moreover, cells derived from clonal lines have different capacity of differentiation (Okamoto et al., 2002). These observations suggest that MSCs represent a population of heterogeneous cells and whether they are undifferentiated or multidifferentiated it is still an open question. Nevertheless, the expression of genes characteristic of multiple lineages and the possibility to direct differentiation of a large part of the cells (in our work, almost 100% when treated with osteogenic medium and about 30% for cells treated with azacytidine) towards specific lineages confirm that MSC can respond efficiently to inductive signals displaying a considerable level of plasticity. Treatment with 5-azacytidine alone, an analogue of cytidine that leads to DNA demethylation, caused an initial rapid increase of growth, but following this increase the cells were maintained for long time without detachment from the surface of the culture dish. In contrast with recent studies, in which beating cells were obtained from bone marrow or from adipose tissue mesenchymal stem cells (Makino et al., 1999; Hakuno et al., 2002; Rangappa et al., 2003), we have observed similar ball-like shaped cells, with increased size, but without any sign of beating. Treatment with 5-azacytidine has been preferentially performed on cells from passage 2, but occasional experiments on late passages have shown the same results. Morphological observation, expression of myosin HC and presence of lipid vacuoles indicate that 5-azacytidine promoted unspecific differentiation in our experiments. Genetically modified MSC that express therapeutic proteins could give rise to tissues of mesenchymal origin expressing gene products essential for tissue regeneration and repair. In order to
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evaluate this potential for cell therapy MSCs were assessed for their capability to express a reporter gene (EGFP) under the control of a specific promoter (CMV), through plasmid electroporation and viral transduction. Electroporation was shown here to be an efficient method for stably expressing a transgene in porcine MSCs and stably transfected MSCs were able to retain their differentiation capability after expansion. Similarly we showed efficient transduction using a third generation self-inactivating lentiviral vector, based on a VSV-G pseudotyped lentiviral vector, capable of transducing dividing, growth-arrested as well as post mitotic cells. Since retroviruses can insert their viral DNA into the host genomic DNA, this allows for stable genetic modification for the life of the host cells (Naldini et al., 1996; Consiglio et al., 2004), which can be followed during the differentiation process and integration in reconstituted damaged organs. However, with respect to cell therapy, three major bio-safety concerns need to be underlined: (i) the risk of the vector packaging system, that could give rise to replication competent lentiviruses through recombination between the transfer vector and packaging constructs, (ii) the risk of insertional oncogenesis, if vectors with transcriptionally active long terminal repeats (LTR) are used, and (iii) the risk of mobilization of HIV-1–based transfer vectors from transduced target cells by subsequent (or prior) infection with wild-type HIV-1. In our nuclear transfer experiments, we have demonstrated both in bovine and in pig that MSCs and their differentiated derivatives give rise to cloned blastocysts at similar rates as adult fibroblasts. However, preimplantation development is not in itself an accurate measure of the subsequent ability to undergo postimplantation development, ability that has not been investigated in the present study. In this respect, a recent report in bovine (Kato et al., 2004) suggests that MSCs derived nuclear transfer embryos do not give rise to offspring at a higher rate than other somatic cell types although the scale of the embryo transfer trial was small. More extensive embryo transfer data are probably needed to completely clarify this issue. In pig, our data on the development of nuclear transfer embryos (32.9–44.7% on day 7) indicate that consistently high development can be obtained in vitro according to the most recent reports (22–26%, Hyun et al., 2003; 20–27%, Zhu et al., 2004; 37%, Lee et al., 2003). In particular, the work of Zhu et al.
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(2004) compares porcine skin–derived fetal stem cells with fetal fibroblasts, showing a higher development rate of the former and suggesting that this could be due to the stem cell nature of the cell donor nucleus. In our study, we have found no difference in preimplantation development among mesenchymal stem cells, osteocytes, and fibroblasts. In the mouse, by contrast, it was shown that preimplantation development of nuclear transfer embryos reconstructed with embryonic stem cells was lower as compared to fibroblasts as donor cells, but interestingly, postimplantation development was higher (Wakayama et al., 1999). Therefore, even in the pig, more extensive data on postimplantation development are needed to shed more light on the effect of the stem cell nature of the donor nucleus. Finally, it must be taken into account that all the different steps that comprise the nuclear transfer procedure, including oocyte maturation, embryo culture, and quality of the donor cell culture, play a relevant role in determing the success of the nuclear transfer procedure up to the final step, that is, obtaining live offspring. In conclusion, our study provides evidence that MSCs can be derived both from bovine and porcine bone marrow displaying growth characteristics, morphology, and differentiation ability similar to that reported in other species. Moreover, we demonstrate that bovine and porcine MSCs can be genetically modified both by electroporation and viral transduction, and that modified MSCs retain the ability to differentiate as the parental lines. Finally, nuclear transfer experiments provide evidence that MSCs from both species, in the undifferentiated state and following differentiation, are capable of driving efficiently the preimplantation development of cloned embryos.
ACKNOWLEDGMENTS We thank Dr Alberto Luciano for valuable advice on preparation of the figures. This work was supported by FONDAZIONE CARIPLO, MIURFIRB (project n RBNE01HPMX), Istituto Superiore di Sanità (Programma Nazionale Cellule Staminali, n° CS11).
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