Hematopoietic stem cell- (HSC) and induced pluripotent stem (iPS) cell-derived natural killer (NK) cells containing engineered functions, such as chimeric antigen receptors (CAR), offer great promise for the treatment of seemingly incurable oncological malignancies. Today, some of the main challenges of CAR cell-based therapeutics are the long manufacturing time and safety of the cell sources used. Additional challenges include avoiding graft vs host disease (GVHD) and cytokine release syndrome (CRS). Here, we show compelling evidence for the use of NK cell therapeutics as a reliable off-the-shelf option, as they address key issues. Furthermore, we highlight how iPS cells and directed differentiation toward HSC and NK cells address industrial scalability and safety.

Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A, HLA-B, and HLA-C. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.

Fluorescent protein-based Genetically Encoded Voltage Indicators (GEVI) offer a remarkable system for high-throughput screening of membrane potential phenotypes. The GEVI MARINA is a derivative from ArcLight, which conversely to ArcLight increases its fluorescence intensity alongside depolarization. Here we created knock-in reporter human iPS cell lines carrying the MARINA reporter using SpCas9 programmable nuclease and characterize a heterozygous clone.

Immune matching and rejection pose major hurdles in tissue transplantation. Here, we profile HLA-A , HLA-B , and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A , 43.2% of HLA-B , and 59.2% of HLA-C , with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of populations with pre-Neolithic hunter-gatherer ancestry, such as European-American and British groups. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A , HLA-B , and HLA-C . Compatibility modeling showed triple homozygous profiles match 60.5% of Lithuanians (33.3% for double homozygotes), 13.4% of British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B , while preserving HLA-C , producing edited profiles matching over 98.1% of Lithuanians, 95.8% of European-Americans, and 95.6% of British population. Finally, we established 16 fibroblast lines from double and triple homozygotes, offering a resource for immune-compatibility studies and regenerative medicine applications.