A. Lima

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.

Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells

IntroductionThe 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.MethodsStem 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.ResultsMicroarray 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.ConclusionsThe 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.

Morphological and Transcriptomic Comparison of Adipose and Bone Marrow Derived Porcine Stem Cells

In the present study we provided a morphological and transcriptomic comparison of adult porcine adipose- derived stem cells (ADSC) and bone marrow-derived stem cells (BMSC) as they differentiated in vitro towards the os- teogenic and adipogenic lineages for up to 4 weeks. The long term goal of this comparison is to assess the possibility of using ADSC as a potential alternative to BMSC as a source of autologous adult stem cells in human therapies. Our data indicated that ADSC can differentiate into osteocytes and adipocytes similar to BMSC but with some differ- ences. During the osteogenic differentiation both cell types went through morphological changes; however, while ADSC formed predominately osteogenic islands (nodules) in the culture dish, BMSC formed a continuous osteogenic sheet of small nodules. Transcriptomic analysis revealed that both cell types responded to the osteogenic induction. However, BGLAP mRNA expression did not increase in ADSC suggesting, together with the percentage area stained observed for Alizarin Red and von Kossa in ADSC, a lesser mineralization of bone matrix in this cell type compared to BMSC. During the adipogenic induction ADSC as well as BMSC were able to achieve the morphological and transcriptome changes characteristic of the adipogenic lineage. After 7 days of differentiation the expression patterns of DGAT2 and ADFP became greater in ADSC versus BMSC, which agreed with the larger lipid droplets formation observed in the ADSC by Oil Red O staining. Our findings represent an important step towards the assessment of using ADSC as an alternative to BMSC in therapeutic applications.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39°C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

202 In vitro differentiation of adult porcine adipose-derived stem cells after labeling with PKH26 and flow cytometry.

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37°C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 µM dexamethasone, 0.5 mM isobutylmethylxantine, 10 µM insulin and 200°M indomethacin) and osteogenic (0.1 µM dexamethasone, 10 mM ±-glycerophosphate, and 50°M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro. This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).