Detailed Abstract
[Basic Research Oral Presentation - Basic Research (Basic Research)]
[BR OP 4] DEVELOPMENT OF IMMUNE-EVASIVE META-SOFT ORGAN MODULES THROUGH ADVANCED CELL ENGINEERING AND ASSEMBLY TECHNOLOGY
Hee Ju KIM 1, Jiyoon Byeon BYEON 1, Chae Eun LEE 1, Songwan JIN 2, Gyeong Woon KONG 2, Youngju SON 3, Kangin LEE 3, Jae Young LEE 3, Jinah JANG 4, Dayoon KANG 4, Daekeun KIM 4, Donghwan KIM 4, Ji Hyun SHIN 1, Dongho CHOI 1
1 Biology, Research Institute of Regenerative Medicine And Stem Cells, Hanyang University,, Korea, 2 Biology, T&R Biofab Co. LTD, Korea, 3 Biology, Therapeutics R&D, ToolGen Inc, Korea, 4 3D Organ Printing, Pohang University of Science And Technology (POSTECH), Korea
Background : Immune rejection and donor shortages pose challenges in liver transplantation. This study addresses these barriers by engineering immune-evasive Meta-iPSC via HLA knockout and immune checkpoint molecule knock-in to improve transplantation outcomes and advance regenerative medicine.
Methods : Using CRISPR-Cas9 genome-editing technology, TnRhipsc-4F cells were modified via HLA gene knockout and immune checkpoint molecule knock-in to generate ALC3-3 cells capable of inducing immune tolerance. These cells were differentiated into Meta-iPSC-derived hepatocytes over a 20-day period. Hepatocyte-specific gene expression was validated through RT-PCR, while immunocytochemistry confirmed the protein expression of hepatocyte markers. Albumin secretion was quantified using flow cytometry (FACS). The differentiated hepatocytes were then assembled into three-dimensional organ modules with vascular structures and transplanted into rabbit livers to evaluate in vivo biocompatibility.
Results : Pluripotency markers in the undifferentiated Meta-iPSC ALC3-3 state and hepatocyte-specific markers in Meta-iPSC-derived hepatocytes were verified via RT-PCR and immunocytochemistry. FACS analysis showed albumin and MRP2 expression in over 80% of Meta-iPSC-derived hepatocytes. Post-transplantation analyses, including DAB staining, confirmed the expression of human-specific markers such as hGAPDH and hALB in rabbit liver tissues, demonstrating successful engraftment and functional integration of the immune-evasive cells.
Conclusions : The successful differentiation and characterization of Meta-iPSC-derived hepatocytes, along with their assembly into bioengineered organ modules, were validated through extensive in vitro and in vivo analyses. Transplantation into a rabbit model confirmed their biocompatibility and functionality. This approach marks a transformative step in addressing key barriers in organ transplantation, such as immune rejection, and highlights the potential for future advancements in regenerative medicine.
Methods : Using CRISPR-Cas9 genome-editing technology, TnRhipsc-4F cells were modified via HLA gene knockout and immune checkpoint molecule knock-in to generate ALC3-3 cells capable of inducing immune tolerance. These cells were differentiated into Meta-iPSC-derived hepatocytes over a 20-day period. Hepatocyte-specific gene expression was validated through RT-PCR, while immunocytochemistry confirmed the protein expression of hepatocyte markers. Albumin secretion was quantified using flow cytometry (FACS). The differentiated hepatocytes were then assembled into three-dimensional organ modules with vascular structures and transplanted into rabbit livers to evaluate in vivo biocompatibility.
Results : Pluripotency markers in the undifferentiated Meta-iPSC ALC3-3 state and hepatocyte-specific markers in Meta-iPSC-derived hepatocytes were verified via RT-PCR and immunocytochemistry. FACS analysis showed albumin and MRP2 expression in over 80% of Meta-iPSC-derived hepatocytes. Post-transplantation analyses, including DAB staining, confirmed the expression of human-specific markers such as hGAPDH and hALB in rabbit liver tissues, demonstrating successful engraftment and functional integration of the immune-evasive cells.
Conclusions : The successful differentiation and characterization of Meta-iPSC-derived hepatocytes, along with their assembly into bioengineered organ modules, were validated through extensive in vitro and in vivo analyses. Transplantation into a rabbit model confirmed their biocompatibility and functionality. This approach marks a transformative step in addressing key barriers in organ transplantation, such as immune rejection, and highlights the potential for future advancements in regenerative medicine.
SESSION
Basic Research Oral Presentation
Room 5 3/27/2025 10:50 AM - 11:50 AM