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

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 β2-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.

Keywords: cell therapy; endothelial cells; hepatic stellate cells; hepatocytes; hypoimmunogenic iPSC; regenerative medicine.

Figure 1

Characterization of M HYPO iPSC line: (A) Schematic representation of CRISPR/Cas9 strategy to target the B2M gene. (B) Chromatogram showing the Sanger sequencing of B2M single alleles isolated by TOPO TA cloning and the alignment to the WT sequence using the SnapGene software, confirming the presence of compound heterozygous mutations in the targeted region of B2M. (C) Bright-field image showing M HYPO cell morphology. Scale bar: 100 μm. (D) Karyotype analysis of M HYPO iPSC clone. (E) Analysis of transcript levels of the pluripotency marker genes OCT4, NANOG, and SOX2 using qRT-PCR. Data were normalized using GAPDH and expressed relatively to the H9 cell line. Data are expressed as the mean ± standard deviation (SD) of three independent biological experiments. (F) Immunostaining analysis for the pluripotency markers OCT4, NANOG, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Scale bars: 50 μm. (G) Immunofluorescence for the endodermal marker α-fetoprotein (AFP), the ectodermal marker βIII-tubulin (β-TUBULIN), and the mesodermal marker α-smooth muscle actin (α-SMA). Scale bars: 50 μm.

Figure 2

Differentiation of iPSCs towards endothelial-like cells: (A) Schematic diagram of the experimental protocol. (B) qRT-PCR analysis for pluripotent markers (OCT4, NANOG) and mesoderm marker (T) during EC differentiation. Results were normalized by HPRT and expressed relatively to the undifferentiated control (n  =  3). Data are means ± SD; * p < 0.05 vs. corresponding day 0. (C) Representative immunofluorescence images for the expression of CD144 (vascular endothelial cadherin) and α-SMA on EC-WT (upper panels) and EC-M HYPO (lower panels) pre and post sorting. Scale bars: 50 μm. (D) Expression of CD144 and vWF (von Willebrand factor) by immunofluorescence of EC-WT (left) and EC-M HYPO (right). Scale bars: 50 μm. (E) In vitro tube formation assay of EC-WT (left) and EC-M HYPO (right). Scale bars: 250 μm.

Figure 3

Differentiation of iPSCs towards hepatocyte-like cells: (A) Schematic diagram of the differentiation protocol. (B) qRT-PCR analysis of hepatic markers ALB (Albumin), AFP, and HNF4α (Hepatocyte Nuclear Factor 4 alpha) on iPSC-HLCs at various differentiation time points. Results were normalized using HPRT and expressed as fold versus day 0 (n = 3). Data are means ± SD; * p < 0.05 vs. corresponding day 0, day 3, and day 8; ns: not significative. (C) Immunofluorescence for ASGPR1 (Asialoglycoprotein 1), ALB, HNF4α, and AFP of differentiated cells, HLC-WT (upper panel) and HLC-M HYPO (lower panel). Scale bars: 50 μm. (D) Analysis of ICG uptake (left) and PAS staining (right). Scale bars: 100 μm.

Figure 4

Differentiation of iPSCs towards hepatic stellate-like cells: (A) Schematic representation of the differentiation protocol. (B) qRT-PCR analysis of HSC markers (PCDH7, PDGFRβ, and DES). Results were normalized to HPRT mRNA levels and expressed as fold versus day 0. Data are means ± SD of three independent experiments; * p < 0.05 vs. corresponding day 0. (C) Immunofluorescence analysis for PCDH7, PDGFRβ, NCAM, and VIM in iPSC-HSCs from WT (upper panel) and M HYPO (lower panel) at the end of the differentiation protocol. Scale bars: 10 μm. (D) Representative overlay histogram plots of flow cytometry analysis of iPSC-HSCs showing PDGFRβ-positive cells and vitamin-A-positive cells at the end of a 4-day retinol treatment. The red line divides negative (on the left) from positive (on the right) marker expression.

Figure 5

(A) Representative overlay histogram plots of flow cytometric analysis of HLA-ABC and HLA-DR expression in untreated and IFNγ-treated endothelial cells derived from WT or M HYPO cells. The red line divides negative (on the left) from positive (on the right) marker expression. (B) Proliferation of T cells in resting condition, after coculture with endothelial cells derived from WT or M HYPO iPSCs or after stimulation with Transact, as positive control. # p < 0.05 vs. all. Data are means ± SD of three independent experiments.

Figure 6

(A) Representative overlay histogram plots of flow cytometric analysis of CD47 expression in undifferentiated and differentiated WT (green) and M HYPO (red) iPSCs, and the respective isotype controls (dashed line). The red line divides negative (on the left) from positive (on the right) marker expression. (B) LDH release assay after allogeneic natural killer coculture with EC-M HYPO and EC-WT cells treated or not with IFNγ. Data are means ± SD of three independent experiments. * p < 0.05 vs. corresponding WT.