Direct conversion of human endothelial cells to hepatic progenitor cells


 This protocol describes direct lineage reprogramming of human endothelial cells isolated from the umbilical vein and peripheral blood into hepatic progenitor cells. These induced human hepatic progenitor cells (hiHepPCs) proliferate in long-term culture and give rise to hepatocytes and cholangiocytes as descendants. To induce hiHepPCs from endothelial cells, we first established an efficient culture condition, enabling hiHepPC generation and propagation in a monolayer culture. Then, we confirmed the ability of the hiHepPCs to differentiate into mature hepatocytes by formation of cell aggregates in each well of ultra-low attachment 96-well plates. Furthermore, upon culturing in Matrigel, the hiHepPCs differentiated into cholangiocytes and formed cystic epithelial spheroids, similar to human liver-derived cholangiocytes. Direct induction of the expandable and bipotential human hepatic progenitor cells provides a possibility for generating cells such as hepatocytes and cholangiocytes, which will be useful for developing therapeutic strategies for human liver diseases.


Introduction
Presently, direct reprogramming technology can be used to convert somatic cells into other cell types, without going through the pluripotent state. Using the direct cell-lineage reprogramming method, mouse and human hepatocyte-like cells (iHepCs) reportedly could be generated from broblasts [1][2][3][4] . iHepCs will be useful alternatives to hepatocytes in the treatment of liver diseases. However, human iHepCs (hiHepCs) cannot proliferate unless immortalized using the SV40 large T antigen, MYC, and p53-siRNA 3,4 .
Such nonphysiological activation of hiHepC proliferation may increase the risk of transformation into cancerous cells. Thus, in clinical hiHepC applications, secure proliferation induction is required.
Meanwhile, recent progress in direct reprogramming technology enabled the induction of human somatic stem/progenitor cells, including neural stem 5,6 , intestinal progenitor 7 , and blood progenitor 8 cells, while the possibility of human hepatic progenitor cell generation was elusive. In our recent study, we identi ed a speci c combination of transcription factors such as FOXA3, HNF1A, and HNF6 that directly induce the conversion of human umbilical vein endothelial cells (HUVECs) and peripheral blood-derived endothelial cells (HPBECs) into cells with the properties of hepatic progenitor cells. These hiHepPCs can continuously produce their cell population in long-term monolayer cultures and differentiate into both hepatocytes and cholangiocytes after cell aggregate and cystic epithelial spheroid formation, respectively, under threedimensional (3D) culture conditions. Procedure A) Retrovirus production 1. Plate 1.8 × 10 6 Plat-GP cells on poly-L-lysine-coated 10 cm dishes and culture them in DMEM containing 8% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin for 3 days.
3. Add the above mixture to the plated Plat-GP cells, drop-by-drop. 4. After 6 hours of incubation at 37°C, with 5% CO 2 , replace the medium with fresh medium, and continue culturing.
5. Collect supernatants from the transfected cells 24 hours after medium replacement. Replace the medium with fresh medium, and continue culturing. 2. Thaw the cryopreserved HUVECs, seed them at a density of 1.5 × 10 4 cells/well in 12-well gelatincoated plates, and culture them in HUVEC medium. After 6 hours, replace the medium with fresh medium.
3. Twenty-four hours after seeding (5-10% con uency), add the concentrated viral supernatants and 5 µg/ml protamine sulfate to the culture medium. After centrifuging at 800 × g for 10 minutes, incubate the plates at 37°C, with 5% CO 2 , for 6 hours. Then, replace the medium with fresh medium and incubate the plates at 37°C, with 5% CO 2 , for 18 hours.

5.
After the 8th infection, replace the medium with a 1:1 mixture of HPBEC medium and hepato-medium (plus).
3. After cystic epithelial spheroids are observed in the 3D culture, collect the spheroids with the medium and Matrigel from each well of 24-well plates, transfer them to 15 ml tubes, break the spheroids into small pieces by mechanically pipetting, and wash them with the medium to remove the Matrigel. Divide the broken pieces in half, mix them with new Matrigel, and resume the culturing. Troubleshooting 1. A-1: Do not continue to culture Plat-GP cells after they reach 90% con uency.

B-3 & C-2:
If the growth ability of HUVECs and HPBECs is low, they cannot be used for retrovirus infection.

Anticipated Results
Using de ned transcription factors, expandable and bipotential hiHepPCs can be directly induced from non-hepatic lineage cells. hiHepPCs can be used as a source of hepatocytes and cholangiocytes, which are required for the study and treatment of human liver diseases.