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The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.

Background: Particulated juvenile allograft cartilage (PJAC) has a good short-term clinical efficacy in repairing articular cartilage defects, but the proliferation ability of PJAC and the biological characteristics of transplanted cells after transplantation are still unclear. Purpose: To study the cartilage proliferation ability of PJAC in repairing full-thickness cartilage defects and the reasons for proliferation to provide experimental evidence for its clinical application. Study design: Controlled laboratory study. Methods: Twenty Guizhou minipigs were randomly divided into the experimental group and control group. In all minipigs, an 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of one knee. The experimental group received PJAC transplantation from five juvenile donors of Guizhou minipigs (PJAC group; n = 10) and the control group received transplantation of autologous cartilage chips (ACC group; n = 10). Both groups were followed at 1 and 3 months after surgery, immunohistochemical evaluation of the tissue sections Ki-67 and Lin28 was conducted, the positive rate was calculated according to the staining, and the proliferation ability of PJAC was analyzed. Results: All 20 Guizhou minipigs were followed, and there was no infection or incision healing disorder after surgery. By Ki-67 and Lin28 immunohistochemical tests, the positive rate of Ki-67 was 88.9 ± 0.2% in the PJAC group and 28.3 ± 3.6% in the ACC group at 1 month, and the difference was statistically significant (P < 0.05); the positive rate of Lin28 was 34.6 ± 3.3% in the PJAC group and 7.6 ± 1.4% in the ACC group at 1 month, and the difference was statistically significant (P < 0.05). At 3 months, the positive rates of Ki-67 in the PJAC group and ACC group were 53.6 ± 6.9% and 1.97 ± 0.3%, respectively (P < 0.05); the positive rates of Lin28 were 86.6 ± 3.3% and 1.4 ± 0.3%, respectively (P < 0.01). Conclusion: A large animal model was established with Guizhou minipigs, and the expressions of Ki-67 protein and Lin28 protein detected by immunohistochemistry in the repaired transplanted tissue of the PJAC group were stronger than those of adult cartilage. The proliferation of PJAC within 3 months of transplantation was stronger than that of adult cartilage. The enhanced expression of Lin28 may be one of the mechanisms by which PJAC achieved stronger proliferation ability than adult cartilage. PJAC technology has shown good application prospects for repairing cartilage defects. [ABSTRACT FROM AUTHOR]

Background: Particulated juvenile allograft cartilage (PJAC) has demonstrated good clinical efficacy in repairing articular cartilage defects, but the related repair mechanism after transplant and the biological characteristics of the transplanted cells are still unclear. Purpose: To study the efficacy of PJAC in repairing full-thickness cartilage defects and the specific fate of donor cells to provide experimental evidence for its clinical application. Study Design: Controlled laboratory study. Methods: Twenty female Guizhou minipigs were randomly divided into an experimental group and a control group. An 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of 1 knee in all minipigs. The experimental group received transplant of PJAC from 5 male juvenile Guizhou minipigs (PJAC group; n = 10) and the control group received autologous cartilage chips (ACC group; n = 10). Follow-up assessments were conducted at 1 month and 3 months to track the transplanted cells by the male-specific sex-determining region Y-linked (SRY) gene; tissue sections were hybridized in situ, and O'Driscoll histological scoring was performed according to hematoxylin and eosin staining, safranin O and fast green staining, and toluidine blue O staining, as well as immunohistochemical evaluation of aggrecan and Sry-type HMG-box 9 (SOX9). Results: All 20 Guizhou minipigs were followed; no infection or incision healing disorder occurred after the operation. By SRY in situ hybridization, the SRY signal of the transplanted cells was positive in the repaired tissue of the defect, and the SRY positive signal could still be detected in repaired tissue at 3 months postoperatively. The average number of positive cells was 68.6 ± 11.91 at 1 month and 32.6 ± 3.03 at 3 months (confocal microscope: ×400), and the difference was statistically significant. The O'Driscoll histological scores were 14 ± 0.71 in the ACC group and 9.8 ± 0.84 in the PJAC group at 1 month, and 18 ± 1.20 in the ACC group and 17.4 ± 1.14 in the PJAC group at 3 months. The scores were statistically significant between the ACC group and PJAC group at 1 month. The positive rates of SOX9 in the PJAC and ACC groups at 1 month were 67.6% ± 3.78% and 63.4% ± 5.30%, respectively, and the difference was not statistically significant (P >.05). The positive rates of SOX9 in the PJAC and ACC groups at 3 months were 68.8% ± 2.69% and 17.1% ± 1.26%, respectively, and the difference was statistically significant (P <.05). The positive rates of aggrecan in the PJAC and ACC groups at 1 month were 40.5% ± 2.78% and 42.4% ± 0.54% respectively, and the difference was not statistically significant (P >.05). The positive rates of aggrecan in the PJAC and ACC groups at 3 months were 40.8% ± 1.50% and 30.1% ± 2.44%, respectively, and the difference was not statistically significant (P >.05). Conclusion: An animal model was established with Guizhou minipigs, and the cartilage defect was repaired with PJAC from male minipigs. The SRY gene positive signal could be detected from the repaired tissue by in situ hybridization, indicating that the transplanted cells survived at least 3 months. The key genes of cartilage formation, SOX9 and aggrecan, were expressed at 1 month and 3 months, and SOX9 expression was stronger in the PJAC group than the ACC group at 3 months. Clinical Relevance: This study suggests that it is feasible to study the biological characteristics of transplanted cells in the cartilage region by the sex-determining gene. [ABSTRACT FROM AUTHOR]