Hypoimmunogenic Human Pluripotent Stem Cells as a Powerful Tool for Liver Regenerative Medicine

Authors:
Piera Trionfini , Elena Romano , Marco Varinelli , Lorena Longaretti , Paola Rizzo , Roberta Giampietro , Annalina Caroli , Sistiana Aiello , Marta Todeschini , Federica Casiraghi , Giuseppe Remuzzi , Ariela Benigni , Susanna Tomasoni 
In:
Source: Int J Mol Sci
Publication Date: (2023)
Issue: 24: 14
Research Area:
Stem Cells
Regenerative medicine
Cells used in publication:
Fibroblast, dermal (NHDF-Neo), human neonatal
Species: human
Tissue Origin: dermal
Platform:
Nucleofector® I/II/2b
Experiment

4.1. iPSC Culture

We used iPSCs derived from Normal Human Dermal Fibroblasts Neonatal (NHDF-Neo, Lonza, Milan, Italy) using the Sendai virus technology (https://hpscreg.eu—IRFMNi001-B, released on 17 September 2020) and its subclone knockout for CIITA that we had recently generated using the CRISPR/Cas9 system (https://hpscreg.eu—IRFMNi001-B-1, released on 25 June 2021). The cells were routinely cultured on Matrigel-coated dishes (Matrigel hESC-Qualified Matrix, Corning, Sial S.r.l., Rome, Italy) in mTeSR Plus medium (Stemcell Technologies, Cologne, Germany), detached using Accutase (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) twice a week, and plated at a density of 20,000–60,000 cells/cm2

4.2. Human iPSC CRISPR/Cas9-Mediated Genome Editing

For B2M knockout, 106 CIITA knockout iPSCs were nucleofected with 10 µg (52 pmol)-Cas9-GFP-protein (Sigma-Aldrich, St. Louis, MO, USA) precomplexed with 390 pmol sgRNA B2M custom (Sigma-Aldrich, St. Louis, MO, USA) (RNP:sgRNA = 1:7.5) according to the manufacturer’s instructions. Nucleofection was conducted using the A023 program with the Human Stem Cell Nucleofector Kit 2 and Nucleofector2b Device (Lonza, Milan, Italy). Transfected cells were plated on Matrigel-coated 6-well plates in mTeSR Plus with 10 µM Y-27632. After 24 h, GFP+ single cells were isolated through cell sorting (FACSAria IIu; Becton Dickinson Italia S.p.A., Milan, Italy), plated on MEF feeder-coated plates with a density of 500 cells/cm2 and then the single-cell-derived colonies were expanded for another 8 days. Thirty emergent clones were picked manually and plated on Matrigel-coated wells in mTeSR Plus medium.

4.8. Differentiation of iPSCs into Endothelial-like Cells

For expansion, CD144+ cells were grown in EGM-2 medium (Endothelial Cell Growth Medium-2, Lonza, Milan, Italy) without hydrocortisone, supplemented with 20% defined fetal bovine serum (FBS HyClone, Euroclone, Milan, Italy) and 10 µM SB431542 (Sigma–Aldrich St. Louis, MO, USA) (EC expansion medium). All experiments described in the manuscript were performed using iPSC-derived ECs between passages three and five.
Abstract

Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the ß2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.