Porcine induced pluripotent stem cells generated in LACID medium
Pigs are becoming increasingly important for disease model generating, xenotransplantation and interspecies organogenesis due to their similarity to human in anatomy and physiological structure. Porcine induced pluripotent stem cells (piPSCs) induced from porcine somatic cells are believed to support porcine application. But the establishment of piPSCs capable of feeder-free culture, robust development potential, and blastocysts generation through SCNT has yet to be achieved. Further investigation is needed to explore porcine pluripotency. And the reprogramming medium for piPSCs is few optimized. We found that the induced iPS chemically defined medium 3 (iCD3) promoted the formation of epithelium-like colonies in porcine reprogramming. Then Culturing medium containing LIF, activin A, GSK3 inhibitor, IWR-1, (S)-(+)-dimethindene maleate (LACID) was utilized to achieve stable piPSCs. The LACID piPSCs were identified to exist strong pluripotent characteristics. (1) LACID piPSCs had fundamental pluripotent characters, displaying flat morphology, expressing pluripotent transcription factors and surface marker. Stable Feeder-free culture for LACID piPSCs were carried out by laminin 511 coating and the addition with LDN-193189. (2) These cells exhibited a strong ability to differentiate and form teratoma in NOD-SCID mice, containing three germ layers. Additionally, cardiomyocytes, PEFs, and blastoids were achieved successfully by differentiation. (3) These piPSCs had a good ability to proliferate and their genome could be edited using CIRSPR/Cas9 for knock-in and knockout.  Blastocysts from piPSCs were also achieved by SCNT, which indicated the application value of piPSCs for transgenic pig generation. (4) LACID piPSCs could form chimeric blastocysts and contribute to intraembryonic parts of chimeric blastocysts. (5) Compare with other reported porcine stem cells, LACID piPSCs had advantage in the expression of pluripotent gene and signaling pathways regulating pluripotency of stem cells. In summary, LACID piPSCs exhibited favorable pluripotency features, which renders them ideal for investigating porcine medical applications and achieving naïve or more advanced porcine stem cells. Overall design: Porcine induced pluripotent stem cells (piPSCs) were induced from porcine embryonic fibroblasts. Three piPSC lines were established from single colonies and maintained in LACID medium. The pluripotency of these piPSCs was thoroughly evaluated. Then three cardiomyocyte lines were differentiated from these piPSCs. For feeder free culturation, LACID piPSCs were seeded on the plate coated with laminin 511 and passaged more than three times to achieve stable piPSCs (Laminin). To optimize the culture medium for feeder-free culture, 100 nM LDN-193189 was added to the LACID medium to culture piPSCs on laminin 511. These piPSCs were passaged more than three times to obtain more compact piPSCs (Laminin-LDN). For the defined culture conditions, the basal medium was prepared by mixing DMEM-F12 and Neural basal medium in a 1:1 ratio, supplemented with 1% N2 and 1% B27. This medium was tested for LACID piPSCs culture. After more than three passages, the piPSCs in N2B27-LACID showed more dome-shaped and compact colony morphology.