Hepatocyte transplantation and sample preparation.

<p>(A) Hepatocyte transplantation. After retrorsine treatment, the parenchyma of the liver became slightly hard and the surface was irregular. After resection of the left lobe and the left side of the middle lobe, hepatocytes were transplanted directly into the liver. The suspended cells spread rapidly according to the blood flow and produced a demarcation line, which disappeared immediately after injection. (B) Isolated hepatocytes. Trypan blue staining identified the viable hepatocytes, which were negative for 7-AAD (red stain) and expressed GFP. GFP-positive hepatocytes lose GFP expression immediately after cell death. The isolated hepatocytes cells were not completely separated. (C) Sample collection. Two weeks after cell transplantation, the remnant liver grew enlarged and swollen. (Upper arm) A small piece of the liver was obtained from the right edge of the middle lobe before perfusion from the portal vein, and then cut into smaller pieces for PCR analysis. The larger part of the liver was fixed with 2% paraformaldehyde for microscopic observation. (Lower arm) The liver was perfused with 2% paraformaldehyde and removed. The right part of the middle lobe was cut into three pieces and further processed for microscopic observation.</p

Disappearance of GFP-positive hepatocytes in wild-type syngeneic rat liver.

<p>(A) Timeline of Experiment 1. Recipient rats received retrorsine treatment twice (black arrow). One week after hepatocyte transplantation (large black triangle), all rats had a liver biopsy under general anesthesia, and then rats were sacrificed on days 14, 28, or 42 (white triangle). (B) Confocal microscopic observation of GFP fluorescence in hepatocytes after transplantation into wild-type Lewis rats. (Upper lane) The liver biopsy taken at day seven shows engraftment of transplanted hepatocytes in all cases. The nodules of GFP-positive hepatocytes after transplantation were less numerous at day 28 than at day 14. GFP-positive hepatocytes at 28 days also showed more heterogeneous GFP expression with an irregular cell shape than at day 14, indicating cytoplasmic degenerative changes. The dissociation of GFP-positive hepatocytes was apparent along with nuclear debris (small green dots). The infiltration of GFP-negative cells with a small nucleus (blue stain) was also apparent. (Lower lane) The progressive growth of GFP-negative hepatocytes in GFP-Tg Lewis rat liver resulted in the size of each cluster of transplanted hepatocytes increasing with time. (C) Higher magnification of the liver tissue at 28 days after hepatocyte transplantation from GFP-Tg rats to wild-type rats. (D) Merged image of immunofluorescent staining for albumin (red) and immunohistochemical staining for GFP (green). The transplanted hepatocytes from wild-type rats proliferate and express albumin in GFP-Tg Lewis rat liver. (E)The percentage of transplanted GFP-positive hepatocytes increased by 14 days after cell transplantation, but significantly decreased by 28 days, and no GFP-positive hepatocytes were observed at day 42. In contrast, the percentage of GFP-negative transplanted hepatocytes increased steadily. (F) PCR analysis of the GFP transgene showed a reduction at 28 and 42 days, suggesting elimination of the GFP transgene.</p

Immunohistochemical staining for GFP and immunofluorescent staining for ED-2.

<p>(A), (B) The liver specimen showed small clusters of GFP-positive hepatocytes at 7 and 14 days after hepatocyte transplantations into wild-type Lewis rat liver. (C), (D) However, there were fewer GFP-positive hepatocytes apparent at 28 days after hepatocyte transplantation, and dissociated, irregular-shape polygonal cells with debris were observed. Small cells with round nuclei (lymphocytes: arrow) were abundant around the GFP-positive hepatocytes. Cells with a triangular crescent cytoplasmic shape (arrowhead) were reminiscent of Kupffer cells. (E) Some of the infiltrating cells are positive for ED-2 (red). (F) Merged image of GFP (green), ED-2 (red), and DAPI nuclear staining (blue) suggests the accumulation of Kupffer cells adjacent to GFP-positive hepatocytes. (G), (H) The transplanted GFP-positive hepatocytes continued to proliferate at 42 days after hepatocyte transplantation from GFP-negative to GFP-Tg Lewis rats.</p

Reconstruction of immune system by bone marrow transplantation and hepatocyte transplantation.

<p>(A) Timeline of experiment 5. Wild-type recipient rats received 11Gy of whole body irradiation and bone marrow transplantation (BMT). Rats in Group 1 received bone marrow cells from GFP-Tg Lewis rats and after retrorsine treatment and hepatectomy, GFP-positive hepatocytes were transplanted and the rats were sacrificed on day 42. Rats in Group 2 received bone marrow cells from wild-type Lewis rats and GFP-positive hepatocytes (*). The rats in Group 3 received bone marrow transplantation from GFP-Tg rats and HBSS with 10% FBS instead of hepatocyte transplantation (**). (B) Flow cytometry of peripheral blood 7 weeks after bone marrow transplantation. More than 99% of leukocytes in the blood from GFP-Tg rats were GFP-positive, as compared to more than 90% of leukocytes after transplantation of GFP-positive bone marrow cells. After bone marrow transplantation from wild-type rats to wild-type rats, practically none of the leukocytes were positive for GFP. (C) Livers from Group 1 rats showed clusters of GFP-positive hepatocytes at 42 days after hepatocyte transplantation and from Group 3, a few GFP-positive large polygonal cells were observed residing within hepatic cords. These cells are similar to megalocytic GFP-positive hepatocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095880#pone.0095880-Maeda1" target="_blank">[27]</a>. Clusters of GFP-positive small hepatocyte-like progenitor cells or mature hepatocytes were not detected.</p

Rapamycin-inspired macrocycles with new target specificity

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.</p