Henriette Reventlow S. Frederiksen: Generation and Evaluation of Non-Immunogenic Stem Cell Lines for in vivo Implantation

Email: henriette.r.frederiksen@sund.ku.dk 
 

BACKGROUND
Successful cell therapy and transplantation depend partly on matching of the donor's and host's immune systems. The high variability between individual immune systems makes finding a compatible donor particularly challenging, limiting the application of these treatments. Developing a stem cell line that can evade immune system detection will therefore function as a universal donor. Such a cell line would help the great promise of using cell therapy for treatment.

PURPOSE OF THE PROJECT
The purpose of this project was to genomically modify a stem cell line to evade rejection upon transplantation into an immunologically incompatible host. Specifically, the project focused on generating stem cell lines suitable for in vivo transplantation into pigs and mice. The cell lines were engineered using CRISPR-Cas9 to knock out genes encoding major histocompatibility complexes 1 and 2, in conjunction with the overexpression of CD47.

RESULTS 
Several novel stem cell lines were generated from pigs and human embryonic stem cells. The human stem cell lines were successfully engineered with knockout of the B2M and CIITa genes and with insertion of either the murine or porcine version of CD47. For tracking cell survival in vivo, the human secreted embryonic alkaline phosphatase (hSEAP) gene was inserted, allowing detection both in vitro and in vivo in a mouse pilot study.

Cells expressing murine CD47 were transplanted into immunocompetent BALB/c mice to evaluate survival. The hSEAP signal disappeared after 8 days, confirmed by immunohistochemical staining for human cells at the injection site. Additionally, immunohistochemical staining revealed immune cell infiltration after day 8, indicating that the cells were rejected by the immune system, despite the genomic modifications.