Human iPS Cell–based Liver-like Tissue Engineering at Extrahepatic Sites in Mice as a New Cell Therapy for Hemophilia B

Abstract

Instead of liver transplantation or liver-directed gene therapy, genetic liver diseases are expected to be treated effectively using liver tissue engineering technology. Hepatocyte-like cells (HLCs) generated from human-induced pluripotent stem (iPS) cells are an attractive unlimited cell source for liver-like tissue engineering. In this study, we attempted to show the effectiveness of human iPS cell–based liver-like tissue engineering at an extrahepatic site for treatment of hemophilia B, also called factor IX (FIX) deficiency. HLCs were transplanted under the kidney capsule where the transplanted cells could be efficiently engrafted. Ten weeks after the transplantation, human albumin (253 μg/mL) and α-1 antitrypsin (1.2 μg/mL) could be detected in the serum of transplanted mice. HLCs were transplanted under the kidney capsule of FIX-deficient mice. The clotting activities in the transplanted mice were approximately 5% of those in wild-type mice. The bleeding time in transplanted mice was shorter than that in the nontransplanted mice. Taken together, these results indicate the success in generating functional liver-like tissues under the kidney capsule by using human iPS cell–derived HLCs. We also demonstrated that the human iPS cell–based liver-like tissue engineering technology would be an effective treatment of genetic liver disease including hemophilia B.

Transforming the spleen into a liver-like organ in vivo

“…We chose to remodel an existing organ, instead of delivering an engineered scaffold, because the former has inherent advantages such as highly organized vasculature, versatile stromal cells, sophisticated ECM structure, and well-formed communication with the host body, which remain hard to recapitulate in most current scaffolds (25). Our strategy resonates with other excellent studies demonstrating the benefits of using an existing organ for growing different tissue-the liver for pancreatic islets (26), lymph nodes for hepatocytes (27), and, notably, kidney for hepatocytes (28)(29)(30)(31). The present study has further extended the application of this concept, aimed at enhancing both the efficacy and feasibility of this approach.…”

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes

“…isolated from primary hepatocytes was used as a positive control. Primers are described for alpha-fetoprotein (AFP), α-1-antitrypsin (AAT), transthyretin (TTR), cytochrome P450 1A2 (CYP 1A2), cytochrome P450 3A4 (CYP 3A4), cytochrome P450 2C9 (CYP 2C9), hepatocyte nuclear factor 1α (HNF1A), hepatocyte growth factor (HGF), albumin (ALB), and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [13][14][15][16][17][18]. The PCR primer sequences are listed on S1 Table. Conditions for PCR reactions were initial denaturation at 94˚C for 3 min followed by 30 cycles of denaturation at 94˚C for 1 min, annealing for 1 min at 56˚C, and elongation for 1 min at 72˚C.…”

<p>In vitro Differentiation of TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells</p>

“…Primers are described for α-fetoprotein (AFP), α-1-antitrypsin (AAT), transthyretin (TTR), cytochrome P450 1A2 (CYP 1A2), cytochrome P450 3A4 (CYP 3A4), cytochrome P450 2C9 (CYP 2C9), hepatocyte nuclear factor 1α (HNF1A), hepatocyte growth factor (HGF), albumin (ALB), and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). [20][21][22][23][24][25] Primer sequences are shown in Supplemental Table 1. PCR reactions were initiated by denaturation at 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 1 min.…”