Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs

Abstract

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis-or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.apancreatic pig | organ reconstitution | transplantation | somatic cell nuclear transfer | chimera

Urine excretion strategy for stem cell-generated embryonic kidneys

“…Experiment 1. We generated cloned E30 pig fetuses from a line of female fetal fibroblast cells using somatic cell cloning technology, as described previously (8). Metanephroi were dissected from the cloned fetuses under a stereomicroscope (Fig.…”

“…SCNT was conducted as described previously (8,18), using in vitromatured oocytes as the recipient cytoplasts. Primary culture cells of porcine fetal fibroblasts (female) were prepared as nuclear donors after cell-cycle synchronization, which was accomplished using serum starvation (FBS, 0.5% vol/vol) for 48 h. A single donor cell was inserted into the perivitelline space of an enucleated oocyte.…”

“…Pig metanephroi were cryopreserved using the previously reported vitrification method (8,20), with slight modifications. Briefly, metanephroi were initially equilibrated with 7.5% (vol/vol) ethylene glycol (EG) and 7.5% (vol/vol) DMSO in handling medium [HM; 20 mM Hepes-buffered tissue culture medium 199 + 20% (vol/vol) calf serum] for 25 min, followed by a second equilibration in vitrification solution (VS), consisting of 15% (vol/vol) EG and 15% (vol/vol) DMSO in HM, for 20-50 min, on ice.…”

Derivation and application of pluripotent stem cells for regenerative medicine

“…Similar successes in organ generation were achieved for kidney of mouse (Usui et al, 2012) and pancreas of pig (Matsunari et al, 2013). A great development in tissue engineering is represented by 3D bio-printing technology , which opens a new path for regenerative medicine and holds a great promising future.…”

A practical guide to induced pluripotent stem cell research using patient samples

“…They transferred donor pig iPSC into pancreatogenesis-or nephrogenesis-disabled blastocyst stage pig embryos, and demonstrated the embryos were born as chimeras having pancreas or kidney exclusively derived from the donor pig iPSCs. 55 Any blastocyst complementation using human iPSC into animals has not been performed yet because of ethical issues, but theoretically it is feasible to generate whole functional human organs in animals using the same strategy. This humanized animal or hybrid animal approach using patient-derived iPSC would be a next-generation disease model for studying human pathology.…”