Background: Adipose-derived Stem Cells (ASC) are obtained from adipose tissue. They can be harvested by liposuction under local anesthesia, making these cells particularly desirable for use in tissue engineering or cell transplantation. However, little is known about the in-vivo characteristics of these cells post-transplantation. Methods and Findings: Here we evaluate the potential of ASC to migrate systemically and the potential for accumulation and colonization in ectopic tissues in a pig model. ASC injected via ear vein can travel through the entire vasculature of the pig within 60 seconds and the cells continue to be present in the bloodstream for at least 1-hour posttransplantation. However, labeled cells were not present in the bloodstream at 2 or 4 weeks after ASC injection. The injected ASC appear to travel to areas of induced trauma and are not observed in filtering tissues of the body such as the spleen, liver, lung and liver. Conclusion: These findings suggest that systemic administration of ASC could be a successful method of cell transplantation for tissue regeneration.

Advances in stem cell biology and materials science have provided a basis for developing tissue engineering methods to repair muscle injury. Among stem cell populations with potential to aid muscle repair, adipose-derived mesenchymal stem cells (ASC) hold great promise. To evaluate the possibility of using porcine ASC for muscle regeneration studies, we co-cultured porcine ASC with murine C2C12 myoblasts. These experiments demonstrated that porcine ASC display significant myogenic potential. Co-culture of ASC expressing green fluorescent protein (GFP) with C2C12 cells resulted in GFP+ myotube formation, indicating fusion of ASC with myoblasts to form myotubes. The presence of porcine lamin A/C positive nuclei in myotubes and RTqPCR analysis of porcine myogenin and desmin expression confirmed that myotube nuclei derived from ASC contribute to muscle gene expression. Co-culturing GFP+ASC with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labeled myotubes increased compared to mouse co-cultures. Enhancing myogenic potential of ASC through soluble factor treatment or expansion of ASC with innate myogenic capacity should allow for their therapeutic use to regenerate muscle tissue lost to disease or injury.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires extreme therapy such as bone grafting or tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice thus far for stem cell therapy for bone regeneration. However, adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC, and both types have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species. The use of scaffolds in combination with stem cells and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Before translation to human medicine, regenerative strategies must be developed in animal models to improve effectiveness and efficiency. The pig presents as a useful model due to similar macro- and microanatomy and favorable logistics of use. This review examines data that provides strong support for the clinical translation of the pig model for bone regeneration.

The importance of mesenchymal stem cells (MSC) for bone regeneration is growing. Among MSC the bone marrow-derived stem cells (BMSC) are considered the gold standard in tissue engineering and regenerative medicine; however, the adipose-derived stem cells (ASC) have very similar properties and some advantages to be considered a good alternative to BMSC. The molecular mechanisms driving adipogenesis are relatively well-known but mechanisms driving osteogenesis are poorly known, particularly in pig. In the present study we have used transcriptome analysis to unravel pathways and biological functions driving in vitro adipogenesis and osteogenesis in BMSC and ASC. The analysis was performed using the novel Dynamic Impact Approach and functional enrichment analysis. In addition, a k-mean cluster analysis in association with enrichment analysis, networks reconstruction, and transcription factors overlapping analysis were performed in order to uncover the coordination of biological functions underlining differentiations. Analysis indicated a larger and more coordinated transcriptomic adaptation during adipogenesis compared to osteogenesis, with a larger induction of metabolism, particularly lipid synthesis (mostly triglycerides), and a larger use of amino acids for synthesis of feed-forward adipogenic compounds, larger cell signaling, lower cell-to-cell interactions, particularly for the cytoskeleton organization and cell junctions, and lower cell proliferation. The coordination of adipogenesis was mostly driven by Peroxisome Proliferator-activated Receptors together with other known adipogenic transcription factors. Only a few pathways and functions were more induced during osteogenesis compared to adipogenesis and some were more inhibited during osteogenesis, such as cholesterol and protein synthesis. Up-stream transcription factor analysis indicated activation of several lipid-related transcription regulators (e.g., PPARs and CEBPα) during adipogenesis but osteogenesis was driven by inhibition of several up-stream regulators, such as MYC. Between MSCs the data indicated an 'adipocyte memory' in ASC with also an apparent lower immunogenicity compared to BMSC during differentiations. Overall the analysis allowed proposing a dynamic model for the adipogenic and osteogenic differentiation in porcine ASC and BMSC.

The measurement of end products of metabolism in combination with the dynamic measurement of the transcriptome can be a valuable approach to study complex processes, such as adipogenesis. For this purpose, we have combined the analysis of metabolic pathways using transcriptomics data with in situ lipid imaging obtained by desorption electrospray ionization–mass spectroscopy (DESI-MS). The transcriptome data was obtained from a previous experiment where adipose-derived stem cells (ASC) from 3 individual pigs were induced to differentiate into adipocytes for 21 days. The metabolic pathway analysis of transcriptomics data was performed using the dynamic impact approach (DIA) to determine the directional impact of pathways and gene categories. For the DESI-MS, porcine ASC were induced towards adipogenic differentiation (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33) for 2 weeks in 24-well plates over round coverslips. Direct lipid profile of fatty acids and phospholipids in 3 different locations in undifferentiated and differentiated ASC was performed. Relative values of ion counts in each location were used for statistical analysis by means of a two-tailed t-test with unequal variance and false discovery rate (FDR) correction. The DIA uncovered amino acid and lipid metabolism as being the most affected pathways during adipogenesis. Among lipid-related pathways, the DIA indicated a strong induction during differentiation of unsaturation of fatty acids (FA) and metabolism of lipids, particularly triacylglycerol formation (TAG). The metabolism of tryptophan, phenylalanine, and histidine was strongly induced among amino acid-related metabolism. Among the lipid molecules identified in the mass-to-charge ratio (m/z) range of 250–1000 by the DESI-MS, >200 had significantly (FDR <0.01) lower abundance and ~30 lipids had higher abundance in differentiated compared with undifferentiated ASC. The lipid profile of undifferentiated cells was featured by a large amount of lipid molecules in the lower (m/z 780) mass range but with almost no signal in the intermediate range. The differentiated cells had a consistent large increase in amount of molecules in the range from m/z 500 to 650 and a decreased amount in the lower and upper ranges. Among the lipid molecules that decreased during differentiation, we identified several phospholipids, particularly phosphoserine and phosphoinositol species, but also linoleic, linolenic, arachidic, and eicosapentaenoic acids. The unsaturated FA oleate and docosahexaenate increased >10-fold and 2.2-fold, respectively, due to adipogenic differentiation. High-resolution experiments are being performed in order to annotate more, if not all, lipid species identified in the samples by DESI-MS. The DIA indicated greater unsaturation of FA during adipogenesis, confirmed by DESI-MS, which corresponds to the characteristic increase in lipid fractions with intermediate molecular mass. This can be considered a signature of adipogenesis in porcine ASC and essential for TAG formation. The DESI-MS appears to be a valuable tool for rapid lipid profile of cells and with the potential to be used to identify cell differentiation.

Purpose: This study investigated the effect of adipose-derived mesenchymal stem cells (ASCs) injected locally or systemically on the bone regeneration of a 10-mm-diameter cylindrical noncritical-size defect in the ramus of the pig mandible.Materials and Methods: Fifteen Yorkshire pigs, weighing 60 to 80 kg, received bilateral 10-mm-diameter cylindrical surgical defects in each ramus of the mandible. Pigs received 1) a direct injection into the defect of 2.5 million carboxy-fluorescein diacetate succinimidyl ester-labeled ASCs from 1 of 2 pig donors (n = 6); 2) an ear vein injection of 5 million carboxy-fluorescein diacetate succinimidyl ester-labeled ASCs from 1 of 2 pig donors (n = 6); or 3) an ear vein injection of culture Dulbecco's Modified Eagle's Medium without stem cells (control; n = 3). Pigs from each treatment were sacrificed at 1 hour, 2 weeks, or 4 weeks after surgery. Healing of the defect was evaluated by dual-energy x-ray absorptiometry, micro-computed tomography, fluorescent microscopy, and histology.Results: Bone healing was accelerated in the ASC-injected treatment groups at 2 and 4 weeks after surgery compared with the control pigs.Conclusions: Results from this animal model provide evidence that the injection of ASC locally into a bone defect or systemically can accelerate the healing of bone. (C) 2012 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 70:e193-e203, 2012

The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, <2 mm in diameter) and medium follicles (MF, 3–6 mm) of abattoir-derived prepubertal gilt ovaries. Oocytes were selected by the presence of the first polar body (1pb) after IVM in a chemically defined medium, for sperm penetration, pronuclear formation, cleavage rate, and development to the blastocyst stage. Relative transcript abundance of genes associated with regulation of oocyte maturation (AURKA, AURKB, and MOS), fertilization (ZP3 and ZP4), maternal effect (NALP9 and HSF1), and anti-apoptosis (BCL2) were also examined in oocytes at germinal vesicle (GV) and metaphase-II (MII) stages. In SF, compared with MF, the maturation rate post-IVM was lower (P < 0.05), but there were no differences in sperm penetration rate (78.2% and 68.5% at 6 hours after insemination and 90.8% and 91.9% at 9 hours after insemination, P = 0.51 and P = 0.67, respectively), the percentage of oocytes that formed both female and male pronuclei (27.9% and 25.8% at 6 hours after insemination and 79.4% and 76.1% at 9 hours after insemination), or cleavage rate at 48 hours after insemination (85.9% and 89.7%, respectively, P = 0.46), whereas blastocyst formation rate was lower (P < 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P < 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P < 0.05) in GV oocytes from SF versus MF. In conclusion, mature oocytes from SF and MF of prepubertal gilts with a visible 1pb had similar fertilizability in vitro and relative transcript abundance of nine genes. However, follicle size affected meiotic competence, early embryonic development to the blastocyst stage, and transcript abundance of the MOS gene.

Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

We have previously shown that heterologous transplantation of porcine adipose-derived stem cells (ADSC) enhances bone healing. Freshly harvested ADSC are a heterogeneous population that contains several types of cells other than stem cells. The isolation of highly purified ADSC could be of clinical importance. In this study, we compared the in vitro growth characteristics and in vivo healing potential of ADSC unsorted or separated using CD34 as a marker. The ADSC were extracted from the back fat of 4 male pigs at 6 months of age. For the in vitro experiment, aliquots of the ADSC were sorted by magnetic beads (Miltenyi Biotec, Gladbach, Germany) into CD34-positive (CD34+) and CD34-negative (CD34–) cell populations. The unsorted ADSC (uADSC), plus the CD34+, CD34–, and a 50:50 mixture of CD34+ and CD34– (MIX) were plated in 24-well plates and differentiated into osteocytes. A robotic stage inverted microscope was used to photograph the entire culture well, and then number, dimension, and density of bone nodules were assessed. Alizarin red (AR) staining was performed and quantified. Cells were harvested before cell plating and then on several time points during expansion, at confluence, and at 3, 6, or 18 days [d] of differentiation for cell counting and RNA extraction. Real-time RT-PCR was performed for CD34, COL1A1, and SPARC genes. For the in vivo experiment, freshly isolated ADSC were sorted by flow cytometry into CD34+ and CD34– cell populations. Unsorted and sorted cells were transplanted, in duplicate, into 10- or 25-mm mandible osteoctomies. Mandibles were harvested after 8 weeks for evaluation of healing by DEXA scanning. In vitro data were statistically analysed using a mixed model (SAS) with time and cell type as fixed effect and pig as the random effect. The in vivo data were assessed by ANOVA with cell types as the fixed effect and pig as the random effect. Freshly harvested ADSC contained 42.3 ± 11.0% CD34+ cells. The uADSC reached confluence at 6 days after plating, whereas other cell types reached confluence at 16 days. Expression of CD34 decreased after plating but was similar between cell types. Among osteogenic genes, only expression of SPARC increased during differentiation. The number of osteogenic nodules was higher (P < 0.05) in uADSC than the in other cell types, but the area and nodule density were similar to CD34– and greater (P < 0.05) than CD34+ and MIX. The amount of AR was higher (P < 0.05) in uADSC compared with CD34– and MIX but similar to CD34+. In the in vivo trial, uADSC had a greater (P < 0.05) healing compared with sorted cells. Among those, CD34– cells appeared to increase healing compared with CD34+ cells. Results indicate that CD34+ cells do not differ significantly from CD34– in the in vitro osteogenic differentiation but have lower in vivo healing capacity; however, in vitro data were confused by a lack of pure CD34– cells. The freshly isolated ADSC have a greater healing capacity than sorted cells, as indicated by in vitro and in vivo experiments. Overall our data indicate that the sorting of ADSC CD34+ cells is not of clinical relevance.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice, thus far, for stem cell therapy for bone regeneration. However, it has been shown that adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC. Moreover, ASC are much more abundant, more accessible and have lower donor morbidity, which combined may make ASC a better alternative to BMSC. ASC are also able to migrate to the site of injury and have immunosuppressive abilities similar to BMSC. Further, ASC have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species, greatly enhancing the healing of critical size defects. The use of scaffolds in combination with ASC and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Some critical elements include ASC-scaffold interactions and appropriate three-dimensional design of the porous osteoinductive structures. This review examines data that provides strong support for the clinical translation of ASC for bone regeneration.

Previous data support adipose-derived stem cells as an alternative to bone marrow as a source of adult stem cells for therapeutic purposes. The aim of the present study was to directly compare the transcriptome of adipose-derived (ADSC) and bone marrow-derived (BMSC) mesenchymal stem cells prior to differentiation and during in vitro osteogenic and adipogenic differentiation. The ADSC and BMSC were harvested from 3 adult pigs and differentiated in vitro into adipocytes and osteocytes for up to 4 weeks. Prior to differentiation and at differentiation day 2, 7, and 21, cells were harvested and RNA extracted for transcriptomics analysis by a 13 263 oligo 70-mers array (Sus scrofa AROS V1.0 with extension; Operon). Data were normalized by Lowess and statistical analysis was run using ANOVA with Benjamini-Hochberg false discovery rate (FDR) correction. Data mining was carried out using Ingenuity Pathway Analysis and David. Using an FDR of <0.05 for overall tissue effect and a post-hoc correction of P < 0.001, we observed 65 differentially expressed genes (DEG) between ADSC and BMSC before starting differentiation (0.66% of unique genes in the array). Functional analysis uncovered significant enrichment of extracellular matrix genes with direct roles in cell adhesion, migration, movement, and morphology. When the interaction cell type × differentiation × time was assessed, we observed >2 000 DEG with an FDR <0.05. This large number was mostly due to time effects. When pair-wise comparisons between cell types for each time point during the same differentiation were performed (post-hoc P < 0.001), we observed a strikingly low number of DEG. The number of DEG was lower between cell types in osteogenic (<100 DEG) compared with adipogenic (<200 DEG) differentiation. We observed significant enrichment (FDR-corrected P-value cut-off <0.05) of functions related to metabolism, antigen presentation, angiogenesis, and cell cycle in both differentiation conditions. We also observed an overall greater induction of the enriched functions in ADSC and a decrease in BMSC during adipogenic differentiation and the opposite during osteogenic differentiation except for metabolism, which appeared to be larger in ADSC in all cases. Among the significant enriched functions of DEG between the 2 differentiations, we observed enrichment of genes involved in metabolism, cell death, cell-to-cell signalling, and antigen presentation in ADSC during adipogenic compared with osteogenic differentiation. In BMSC we observed enrichment of functions related to cell death, antigen presentation, and lipid metabolism in osteogenic v. adipogenic differentiation. Overall data uncovered a high similarity at the transcriptional level between ADSC and BMSC both prior to differentiation and during differentiation. Those data support ADSC being particularly similar to BMSC. This work was support by the Illinois Regenerative Medicine Institute (IDPH # 63080017).

Assessment of general risk posed from transgenic (T) animals is important to their future contributions to society. Identification of potentially harmful properties of transgenic livestock is the initial step in a risk assessment. We previously developed and characterized transgenic swine containing a mammary-specific transgene (bovine a-lactalbumin, bALAC) that results in increased milk production in sows. We are currently determining whether bALAC is expressed in tissues of T swine other than the lactating mammary gland and whether the transgene DNA (Tg) crosses into nontransgenic control (C) swine under various physiological and physical conditions. The specific aims addressed in the present study were to determine (1) whether the Tg can be transferred directly by physical association or contact; (2) whether the Tg can be transferred directly via mating; (3) whether the Tg can be transferred directly during gestation and parturition; and (4) whether the Tg can be transferred directly during lactation. The T animals utilized in these studies are in at least generation 10 and have stable incorporation of the Tg. Comparable age- and weight-matched animals, T and C, were housed together allowing general contact that is normal in swine production, for either 180, 220, or 250 days of age after weaning. Swine typically ingest saliva, regurgitated food, and stool or urinary products, as well as other bodily fluids and cells during normal housing. In the second study, vaginal, cervical, uterine, oviductal, and ovarian tissues from C females on 2, 7, or 90 days after mating to T males, and penis, bulbourethral gland, urethra, testis, and epididymis tissues from C males on 2 or 7 days after mating to Tg females were collected. The presence of Tg in tissues from all C animals was tested by using PCR. We have analyzed for the presence of the Tg in various tissues [including mammary gland, salivary gland, skin (sebaceous gland), muscle, lung, liver, kidney, brain, ovary, oviduct, uterus, cervix, vagina, penis, bulbourethral gland, urethra, testis, epididymis, blood, inner and outer placental membranes and intestine]. Results indicate no presence of the Tg in tissues of C animals (n = 28) after co-habitation for 180, 220, or 250 days (n = 305 samples analyzed) or at 2 (n = 7), 7 (n = 16), or 90 (n = 6) days post-mating (n = 72, 192, or 71 samples analyzed, respectively). At Day 112 of gestation, all the samples (n = 78 samples analyzed) from nontransgenic piglets (n = 13) whose dam was aTg female were negative except for the outer placental membrane (n = 13), which screened positive for the transgene. This is not surprising because the outer placental membrane is in close contact with the uterus of the Tg dam. Finally, control piglets (n = 4) that were cross-fostered (3 days after birth) and suckled Tg dams showed no evidence of the transgene in their tissues (n = 20 samples analyzed) at weaning. The present results suggest that there is no horizontal Tg transmission between T and C pigs caused by rearing, mating, gestation, or lactation. This project was supported by USDA BRAG Project #2005-03799.

Bone marrow is one current source of adult stem cells for therapeutic purposes; however, the magnitude and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative. Numerous in vitro studies have been conducted to determine how these cells act in vitro, but it is imperative to determine the vast abilities of these cells in vivo. The objective of this study was to evaluate in vivo migration and bone healing ability after transplanting adipose-derived stem cells (ADSC) in a swine model. Adipose-derived stem cells were isolated from subcutaneous adipose tissue of adult Yorkshire pigs and cultured in vitro. At 80 to 90% confluence/passage 3, the cells were trypsinized and labeled in suspension with carboxyfluorescein succinimidyl ester (CFDA-SE). This project included 20 pigs weighing between 63.5 and 81.7 kg. Bilateral mandibular osteoectomies with 10-mm defects were performed on each pig. Of the 20 pigs, half received a treatment of 2.5 million CFDA-SE labeled stem cells administered directly into each defect (DI), and the remaining half received a treatment of approximately 5 million CFDA-SE labeled stem cells through an ear vein injection via catheter (EVI). The time points were 1 h and 2 and 4 wk, with 2 pigs per time with the DI and EVI treatments. Pigs were slaughtered at each time, and spleen, liver, lung, kidney, ear vein, blood, and mandible tissues were collected. Blood samples were collected from the jugular vein with EDTA and processed via flow cytometry after collection. Tissues were fixed in 10% buffered formalin for histology. Fluorescent microscopy (CFDA-SE excitation/emission is 492/517 nm) has confirmed that transplanted ADSC do indeed migrate to a site of injury or trauma. Labeled cells were also present in blood collected from the 1-h time point group. Currently, we have not seen the presence of labeled ADSC in the other tissues (spleen, liver, lung, and kidney) after the 1-h time point. We did observe that ADSC administered by DI and EVI were able to significantly heal and regenerate bone defects within 4 wk post-surgery (P < 0.05, ANOVA with F-test), in contrast to bone defects in pigs that did not receive cell injections (control). Evidence of ADSC-related healing and bone regeneration was evident by gross visualization, dual-energy x-ray absorptiometry (DXA) and micro computer tomography (microCT) analysis. The clinical implications of these results are significant for treating many diseases in which inflammation or defects exist, such as cardiac disease, neurological disease, or traumatic injuries to both soft and hard tissue. If the adult stem cells can be harvested from fat, encouraged to produce bone or cartilage, and then reinserted into defects, treatment protocols for trauma victims could be developed that would reduce the need for alternate harvesting techniques for bone. This work was support by a grant from the Illinois Regenerative Medicine Institute (IDPH # 63080017).

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitro culture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al. 2007 Theriogenology 68, 709-716; Du et al. 2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions. Table 1.Effects of cell type, pre-activation process and in vitro culture (IVC) medium on development of handmade pig cloned embryos

During winter hibernation, grizzly bears (Ursus arctos horribilis) do not eat but instead rely on internal fat stores as a primary source of metabolic energy. The resulting seasonal fluctuations in appetite and body mass make the grizzly bear a naturally occurring animal model for human conditions such as obesity and anorexia. An in vitro model of hibernating bear stem cells might enhance our understanding of processes such as stem cell proliferation and differentiation. Mesenchymal stem cells, derived from bone marrow and adipose tissue among others, differentiate into adipocytes and might play important roles in energy metabolism. In the current study, we examined the in vitro viability and morphology of mesenchymal stem cells isolated from grizzly bear adipose tissue (ADSC) and bone marrow (BMSC); these ADSC and BMSCs underwent adipogenic differentiation for 0, 7, 14, 21, and 28 days. Bone marrow stem cells and ADSC were isolated using mechanical disaggregation, collagenase digestion, centrifugation, and plating onto tissue culture polystyrene. Cell viability and proliferation was quantified using the colony forming unit assay and a hemocytometer. Both stem cell types were differentiated into adipocytes using 10 μM insulin, 1 μM dexamethasone, and 0.5 mM isobutylmethylxanthine (all Sigma- Aldrich, St. Louis, MO, USA) with the addition of 10% fetal bovine (FBS) or bear serum from the active feeding period. Adipogenic differentiation was confirmed using Oil Red O and quantified using ImageJ. Statistical analysis was performed using an unpaired t-test between treatments of the same time point. All cells were isolated within 28 h of tissue harvest. Adipose-derived stem cells formed an average of 11 colonies (0.011%), whereas BMSC formed 1.5 colonies (0.0015%) per 100 000 cells. Doubling time forADSC was approximately 54 h in 10% FBS. BothADSC and BMSC had an initial spindle-shaped morphology, which gradually became more rounded during adipogenic differentiation. For bear serum at Day 28, ADSC had a significantly (P < 0.01) greater stained area per cell than did BMSC. In summary, both types of mesenchymal stem cells successfully differentiated into adipocytes and maintained viability. In conclusion, grizzly bear mesenchymal stem cells canbesuccessfully isolated, expanded, and differentiated in culture. These results allow for future studies using the bear as an in vitro model for fat metabolism during hibernation and active periods. This work was partially supported by the Carle Foundation Hospital, the Intel Scholar’s Research Program, USDA Multi-State Research Project W1171, and the Illinois Regenerative Medicine Institute (IDPH # 63080017). In addition, the authors would like to thank Agatha Luszpak for support with the analysis.

Animal biotechnology has been practiced in one form or another since the beginning of the domestication of animals. Many of the previously used tools of animal breeding, genetics and nutrition have played an important role in the selection, propagation and management of desirable and economically important characteristics in livestock. Modern livestock production has been dependent on biotechnology for development of improved feedstuffs, feed ingredients, vaccines, biologicals, enzymes, high quality genetics, genetic markers and assisted reproduction. Recently, new technologies including genomics, transcriptomics, proteomics, metabolomics have been applied to livestock production. The term “omics” refers to a broad field of study in biology and stems from ‘’Omes’’, the Greek for ‘all’ or ‘complete’. Therefore, these technologies offer a holistic instead of a reductionist view of the biological phenomena. Several “omics” technologies are readily available for scientists or industry today and in this section we will provide a brief overview of their availability in livestock science. Other “omics” technologies have developed quickly and are available for research or industry in livestock field. The microarray technology for microRNA is available today for bovine, pigs, and chickens. Combined with the use of appropriate bioinformatics tools, they have been of great help in understanding livestock genomics. Large-scale SNP arrays are also available today, but only for bovine among the livestock species. Epigenomics, the study of the non-DNA hereditable factors affecting the phenotype, has been used for large-scale studies, but data have not been generated using this technology in livestock. Systems biology has emerged to investigate “interrelationships of all of the elements in a functioning system in order to understand how the system works”. A systems biology approach is only possible by combining a single or multiple “omics” technique(s) along with bioinformatics for a broad purpose such as to study the whole system, organism or comparison between organisms. In order to take full advantage of the breakthroughs from “omics” techniques efficient animal breeding and reproduction of these rare genetic individuals needs to take place. For decades assisted reproductive technologies (ART), such as artificial insemination (AI), superovulation (SOV), embryo transfer (ET), and in vitro embryo production (IVEP), have contributed to animal breeding programs allowing faster transmission of desirable traits in livestock populations in a shorter period of time compared to classical approaches. The use of transgenic technologies along with ART’s to introduce single or multiple genes into existing genomes of livestock has played an increasingly larger role in the genetic development of our production livestock. Addition of appropriate stem cell technologies to the genetic “toolbox” has further increased our capabilities to enhance and modify livestock genomes and physiology. In the future, livestock production will rely even more heavily on existing and emerging biotechnological advances to produce our food. However, improvements are still needed in product composition and production efficiency, especially in growth, disease resistance, and reproduction. The attainment of such improvements will depend heavily on our ability to quantify desirable traits, to identify markers linked to gene(s) responsible for those traits, to select or redesign populations of superior individuals, and to propagate those animals efficiently, practically and economically.

INTRODUCTION:The objective of this study was to find highly reliable internal-control genes (ICGs) for normalization of qPCR data from porcine adult mesenchymal stem cells induced to differentiate toward adipogenic and osteogenic lineages.METHODS:Stem cells were acquired from subcutaneous back fat and bone marrow of three castrated Yorkshire crossbred male pigs. Adipose and bone marrow-derived stem cells (ADSCs and BMSCs) were cultured in vitro with specific osteogenic or adipogenic differentiation medium for 4 weeks. Total RNA was extract for microarray (13,000 oligonucleotides) and qPCR analyses. Microarray data were used to uncover the most stably expressed genes (that is, potential ICGs). Co-regulation among potential ICGs was evaluated with Ingenuity Pathway Analysis. qPCR was performed on the non-coregulated ICGs candidates and on specific osteogenic (COL1A1) and adipogenic (DBI) genes. geNorm was used to uncover the most reliable ICGs by using qPCR data and the optimal number of ICGs to be used to calculate the normalization factor.RESULTS:Microarray data analysis revealed 27 potential ICGs. Among those, 10 genes without known co-regulation were selected to perform qPCR. geNorm performed on qPCR data uncovered high stability in expression ratio among the selected ICGs. However, especially reliable normalization was obtained by geometric mean of NSUN5, TIMM17B, and VPS4A. The effect of normalization, assessed on specific osteogenic (COL1A1) and adipogenic (DBI) genes, was apparent for the adipogenic and less apparent for the osteogenic differentiation.CONCLUSIONS:The combination of microarray data and pairwise gene analysis allowed identification of novel and highly reliable ICGs for qPCR data normalization of adult porcine stem cells induced to differentiate to adipogenic and osteogenic lineages.

The pig is an important animal model for the study of human diseases. An important step for better use of this species in biomedical research is to obtain genetically identical individuals by procedures such as somatic cell nuclear transfer (SCNT). As the in vitro culture environment is usually sub-optimal for embryo development, the oviductal transfer of cloned embryos at the 1-cell stage may be more efficient for the establishment of pregnancies. However, the transfer at such an early stage usually requires the presence of zona pellucida or agar embedding to protect embryos from the recipient’s immune system (Loi et al. 1999 Livest. Prod. Sci. 60, 281-294). This study aimed to evaluate the developmental viability of 1-cell-stage porcine handmade cloned embryos directly transferred to the oviduct of female recipients without the zona pellucida or agar embedding. After 40 h of IVM in TCM-199 +10% follicular fluid, COCs obtained from sows were denuded, selected for the presence of a polar body (459/689), and submitted to a 0.2% pronase solution in 25% fetal bovine serum (FBS) for partial zona removal, followed by rinses in manipulation medium and pure FBS. Subsequent to oocyte splitting by manual bisection in a 5 μg mL-1 cytochalasin B solution (CCB), hemi-oocytes (87.1%) were screened under fluorescent microscopy using Hoechst 33 342 stain, resulting in 57.6% enucleated halves (461/800). A somatic cell culture established from a fetal clone pig biopsy (Adam et al. 2007 Oncogene 26, 1038-1045) at passage 4 was used for embryo reconstruction, which was done in a 0.05% phytohemagglutinin (PHA) solution, by sticking 2 cytoplasts and a somatic cell in a linear orientation. Reconstructed couplets, rinsed in calcium- and magnesium-free fusion medium, were electrofused in a fusion chamber after exposure to a 30-V AC pulse for 20 s for alignment, followed by two 24-μs-long DC fusion pulses of 1.3 kV cm-1. Fused couplets (154/214) were exposed for 10 min to a solution containing 5 μg mL-1 CCB and 10 μg mL-1 cycloheximide, followed by electrical activation (two 24-μs-long DC pulses of 0.9 kV cm-1) in fusion medium containing calcium and magnesium. Activated embryos were cultured in vitro for 12 h in 500 μL of PZM-3 medium in the well of the well (WOW) system, in a plastic bag filled with gas mixture (90% N2, 5% O2, 5% CO2), at 38.5°C. Then, a total of 70 and 80 non-agar-embedded, zona-free 1-cell-stage cloned porcine embryos were transferred directly to the oviducts of a sow and a gilt, respectively, both synchronous at approximately 12 h before ovulation. The recipient sow was diagnosed pregnant by ultrasonography on Day 66 of gestation. Although the sow was diagnosed open on Day 72, this study demonstrates that the transfer of 1-cell-stage zona-free embryos directly to the oviduct of a synchronous sow can result in pregnancy.

The quantity, accessibility, and abundance of subcutaneous adipose tissue in humans make it an attractive alternative to bone marrow as a source of adult stem cells for therapeutic purposes. Adult adipose-derived mesenchymal stem cells can differentiate into a variety of lineages including adipose, bone, cartilage, and muscle. In addition, the use of adult stem cells for regenerative medicine rather than those from embryos avoids concerns with ethics, safety, and immunology. One important issue is the ability to track the transplanted stem cells during the regeneration process to evaluate the stem cell-mediated healing. The objective of this study was to compare the efficiency, longevity, and intensity of carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and quantum dot nanocrystal (Qtracker™, Invitrogen, Carlsbad, CA, USA) labeled adipose-derived stem cells (ADSC) over an in vitro culture period of 4 weeks. Adipose-derived stem cells (6 x 106) previously isolated and frozen at -196°C were thawed and cultured in 75-cm3 flasks with 14 mL of DMEM. Cells were grown to 80% confluence and trypsinized. After trypsinization, the cells were divided into 4 treatments (3 x 106 cells per treatment). The treatments were (1) unlabeled control, (2) labeled with 30 μM CFDA SE, (3) labeled with 15 nM Qtracker™, and (4) labeled with 15 nM Qtracker™, following the Invitrogen Qtracker™ protocol. Cells (1 x 106) were removed from each treatment every week for 4 weeks and fixed in formalin for later analysis. When all the samples were collected, they were analyzed using flow cytometry. Data were analyzed via chi-square test. The percentage of cells labeled with CFDA SE and Qtracker™ was 99.35 and 98.46%, respectively, immediately after labeling. By 1 wk, the percentage of cells labeled with CFDA SE and Qtracker™ had deceased (P < 0.01) to 0.11 and 1.48%, respectively. The CFDA SE-labeled cell percentages had decreased (P < 0.01) to 0% at 2, 3, and 4 wk, respectively. The Qtracker™-labeled cells also decreased (P < 0.01) to 0.745, 1.69 and 0.45% at 2, 3, and 4 wk, respectively. The high rate of cell division of these cells in vitro might be responsible for the rapid loss of both labels during the first week of culture. Previous results from our lab have shown that the CFDA SE is retained in the cells for up to 6 wk in vivo (Lima AS et al. 2006 Reprod. Fertil. Dev. 18, 208). Similar studies need to be done with the quantum dot-labeled cells to determine the Qtracker™ label’s longevity in vivo. In conclusion, quantum dots can be used to label ADSC, in vitro, for at least 4 wk, albeit at much lower levels than those observed during the week following labeling. Determination of a suitable label for high-percentage porcine ADSC labeling during long-term in vitro culture remains to be completed. This research was supported by the Intel Scholar’s Program and the Illinois Regenerative Medicine Institute.