Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives

Abstract

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue, making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1α)-hESC lines using chEF1α promoter system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology, karyotype, and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers, OCT-3/4 and SSEA-4, in undifferentiated mHLA-G(EF1α)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors, expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1α)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives.

Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients

“…However, according to the 'missingself theory', MHC class I-deficient mouse and human PSCs become susceptible to NK cell killing [23][24][25] . Although isolated expression of HLA-E 26 or HLA-G 27 in human pluripotent stem cells has been used to mitigate NK cell cytotoxicity, we observed that CD47 is a very effective non-MHC ligand to silence all innate immune responses. However, cells eluding immune monitoring may pose the long-term risks of uncontrollable malignant transformation or impaired virus clearance, although for the latter alternative mechanisms have been shown 28 .…”

Concise Review: One Stone for Multiple Birds: Generating Universally Compatible Human Embryonic Stem Cells

“…C) . Such protection has also been demonstrated in hESCs ectopically expressing an optimized form of HLA‐G .…”

“…Since 2013, breakthroughs in genome editing in hPSCs have made it possible to knock out HLA and genes essential for HLA expression, including β ‐2‐microglobulin ( B2M ) for class‐I HLA and class‐II MHC transactivator ( CIITA ) for class‐II HLA expression. In addition, hypoimmunogenic hESCs can be obtained by knocking down B2M or ectopically expressing a modified form of HLA‐G . Recently, it was reported that UC‐hESCs can also be achieved through the disruption of T cell costimulatory pathways with cytotoxic T lymphocyte antigen 4 fused with immunoglobulin (CTLA4‐Ig) and simultaneous activation of the T cell inhibitory pathway with programmed death ligand‐1 (PD‐L1) .…”

Hypoimmunogenic Derivatives of Induced Pluripotent Stem Cells Evade Immune Rejection in Fully Immunocompetent Allogeneic Recipients

“…cell killing [23][24][25] . Although isolated expression of HLA-E 26 or HLA-G 27 in human pluripotent stem cells has been used to mitigate NK cell cytotoxicity, we observed that CD47 is a very effective non-MHC ligand to silence all innate immune responses. However, cells eluding immune monitoring may pose the long-term risks of uncontrollable malignant transformation or impaired virus clearance, although for the latter alternative mechanisms have been shown 28 .…”