US 12,331,318 B2
Non-immunogenic engineered tissue and methods of producing and using the same
Lothar Germeroth, Goettingen (DE)
Appl. No. 17/259,900
Filed by Lothar Germeroth, Goettingen (DE)
PCT Filed Jul. 15, 2019, PCT No. PCT/EP2019/068944
§ 371(c)(1), (2) Date Jan. 12, 2021,
PCT Pub. No. WO2020/012033, PCT Pub. Date Jan. 16, 2020.
Claims priority of application No. 18183294 (EP), filed on Jul. 13, 2018.
Prior Publication US 2021/0284965 A1, Sep. 16, 2021
Int. Cl. C12N 5/077 (2010.01)
CPC C12N 5/0657 (2013.01) [C12N 2500/90 (2013.01); C12N 2510/00 (2013.01)] 13 Claims
 
1. A method of producing a non-immunogenic engineered heart muscle tissue from pluripotent stem cells, comprising:
modifying ND50039 pluripotent stem cells to provide modified ND50039 pluripotent stem cells comprising (i) a deletion of the β2-Microglobulin (B2M) gene endogenous to wild-type ND50039 cells, thereby preventing cell-surface expression of endogenous HLA-A, B, and C by the modified ND50039 cells, and (ii) introduction of an expression construct configured to drive cell-surface expression of an exogenous immunomodulatory fusion protein comprising at least a portion of B2M covalently linked to at least a portion of an HLA class Iα chain by the modified ND50039 cells;
differentiating the modified ND50039 pluripotent stem cells into at least one first cell type that is essential for the function of the engineered heart muscle tissue, wherein the first cell type is a cardiomyocyte;
differentiating the modified ND50039 pluripotent stem cells into at least one second cell type that forms a supporting component for the first cell type within the engineered heart muscle tissue as a separate cell population from the first cell type, wherein the second cell type is a fibroblast; and
mixing the first cell type and the second cell type at a defined ratio in the presence of a biocompatible extracellular matrix to form the engineered heart muscle tissue,
wherein the engineered heart muscle tissue is non-immunogenic to a recipient of the engineered heart muscle tissue.