In vitro induced pluripotency from urine-derived cells in porcine

BACKGROUND The generation of induced pluripotent stem cells(iPSC)has been a game-changer in translational and regenerative medicine;however,their large-scale applicability is still hampered by the scarcity of accessible,safe,and reproducible protocols.The porcine model is a large biomedical model that enables translational applications,including gene editing,long term in vivo and offspring analysis;therefore,suitable for both medicine and animal production.AIM To reprogramme in vitro into pluripotency,and herein urine-derived cells(UDCs)were isolated from porcine urine.METHODS The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4(OCT4),SRY-box2(SOX2),Kruppel-like factor 4(KLF4),and C-MYC,and cultured with basic fibroblast growth factor(bFGF)supplementation.To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase(AP),OCT4,SOX2,NANOG,TRA181 and SSEA 1 detection.Endogenous transcripts related to the pluripotency(OCT4,SOX2 and NANOG)were analyzed via reverse transcription quantitative realtime polymerase chain reaction in different time points during the culture,and all three lineages formed embryoid bodies(EBs)when cultured in suspension without bFGF supplementation.RESULTS The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming.Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors(mOSKM),but not from human factors(hOSKM).Three clonal lineages were isolated and further cultured for at least 28 passages,all the lineages were positive for AP detection,the OCT4,SOX2,NANOG markers,albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1,similar results were observed in the abundance of the endogenous transcripts related to pluripotent state.All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts.CONCLUSION For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotentlike state,enabling new numerous applications in both human or veterinary regenerative medicine.

Age and Calcium Sources in Laying Hen Feed Affect Calcium Digestibility

The apparent calcium (Ca) digestibility coefficient (ADC) and true digestibility coefficient (TDC) of different inorganic calcium sources were determined in laying hens of different ages. Three Ca digestibility tests were carried out, each assessing 240 Lohmann Brown lineage laying hens distributed in a completely randomized design. Nine dietary treatments were arranged in a 3 × 3 factorial design consisting of three ages (40, 50 and 70 weeks) × three Ca (dicalcium phosphate (DCP) sources, fine (FL) and coarse (CL)) limestone, comprising eight replicates per treatment of six birds per experimental unit. Regarding the DCP, the ADC was higher (P < 0.05) in 40-week-old birds. The DCP ADC for 40-, 50- and 70-week-old birds was 0.889, 0.613 and 0.712, respectively. No effect (P > 0.05) of age on the ADC was noted for either FL or CL. Comparing Ca sources, DCP exhibited a higher (P < 0.05) ADC (0.889), followed by FL (0.699) and CL (0.515), in 40-week-old birds. DCP (0.712) and FL (0.652) presented (P < 0.05) higher ADC compared to CL (0.482), in 70-week-old birds. No effect of Ca sources at 50 weeks on the ADC was observed (P > 0.05). Endogenous loss values of 790, 860 and 930 mg·kg-1 of consumed dry matter were observed at 40, 50 and 70 weeks, respectively. For the TDC, no interaction (P > 0.05) was observed between Ca sources and bird age. The highest TDC value (P > 0.05) was found in birds fed DCP (0.786) followed by FL (0.637) and CL (0.534). In addition, birds at 40 weeks of age (0.714) exhibited higher TDC values (P < 0.05) compared to animals at 50 weeks of age (0.608). The findings reported herein demonstrate that the true digestibility is greater in the youngest birds and that consumed the DCP and the FL in relation to the birds that consumed the CL.