In this study, we demonstrate that a small population of pluripotent stem cells, termed adipose multilineage-differentiating stress-enduring (adipose-Muse) cells, exist in adult human adipose tissue and adipose-derived mesenchymal stem cells (adipose-MSCs). They can be identified as cells positive for both MSC markers (CD105 and CD90) and human pluripotent stem cell marker SSEA-3. They intrinsically retain lineage plasticity and the ability to self-renew. They spontaneously generate cells representative of all three germ layers from a single cell and successfully differentiate into targeted cells by cytokine induction. Cells other than adipose-Muse cells exist in adipose-MSCs, however, do not exhibit these properties and are unable to cross the boundaries from mesodermal to ectodermal or endodermal lineages even under cytokine inductions. Importantly, adipose-Muse cells demonstrate low telomerase activity and transplants do not promote teratogenesis in vivo. When compared with bone marrow (BM)- and dermal-Muse cells, adipose-Muse cells have the tendency to exhibit higher expression in mesodermal lineage markers, while BM- and dermal-Muse cells were generally higher in those of ectodermal and endodermal lineages. Adipose-Muse cells distinguish themselves as both easily obtainable and versatile in their capacity for differentiation, while low telomerase activity and lack of teratoma formation make these cells a practical cell source for potential stem cell therapies. Further, they will promote the effectiveness of currently performed adipose-MSC transplantation, particularly for ectodermal and endodermal tissues where transplanted cells need to differentiate across the lineage from mesodermal to ectodermal or endodermal in order to replenish lost cells for tissue repair.

Alu elements are primate-specific short interspersed elements (SINEs), over 1 million copies of which are present in the human genome; thus, Alu elements are useful targets for detecting human cells. However, previous Alu-based techniques for detecting human genomic DNA do not reach the theoretical limits of sensitivity and specificity. In this study, we developed a highly sensitive and specific Alu-based real-time PCR method for discriminating human cells from rodent cells, using a primer and probe set carefully designed to avoid possible cross-reactions with rodent genomes. From 100 ng of mixed human and rodent genomes, 1 fg of human genome, equivalent to 1 human cell in 100 million rodent cells, was detectable. Furthermore, in vivo mouse subrenal capsule xenotransplantation assays revealed that 10 human cells per mouse organ were detectable. In addition, after intravenous injection of human mesenchymal stem cells into NOD/SCID mice via tail vein, the biodistribution of human cells was trackable in the mouse lungs and kidneys for at least 1 week. Our findings indicate that our primer and probe set is applicable for the quantitative detection of tiny amounts of human cells, such as xenotransplanted human cancer or stem cells, in rodents.

Retinitis pigmentosa affects 1 in 4,000 people worldwide. Mutations in over twenty genes cause autosomal dominant retinitis pigmentosa. 30-40% of autosomal dominant retinitis pigmentosa are due to mutations rhodopsin with more than 100 known mutations being identified. Due to this allelic and causal diversity, allele-independent approaches are an attractive option. Here, we demonstrate an allele-independent CRISPR/Cas9 approach; this contrasts with many current allele-specific rhodopsin approaches. A homology-independent transgene is co-delivered with RSV-spCas9 packaged in an AAV5 vector for gene replacement via insertion in the Rho I307N mouse model of autosomal dominant retinitis pigmentosa. First, we establish the safety of this system, in C57BL/6J mice, demonstrating no loss of retinal thickness or function. We further show that outer nuclear layer thickness, electrical response, and rhodopsin expression in heterozygous Rho I307N were significantly preserved six months after treatment. This retention results from a 5.7% transgene integration and 88% indel rate in treated animals.

Retinitis pigmentosa affects 1 in 4,000 people worldwide. Mutations in over twenty genes cause autosomal dominant retinitis pigmentosa. 30-40% of autosomal dominant retinitis pigmentosa are due to mutations rhodopsin with more than 100 known mutations being identified. Due to this allelic and causal diversity, allele-independent approaches are an attractive option. Here, we demonstrate an allele-independent CRISPR/Cas9 approach; this contrasts with many current allele-specific rhodopsin approaches. A homology-independent transgene is co-delivered with RSV-spCas9 packaged in an AAV5 vector for gene replacement via insertion in the Rho I307N mouse model of autosomal dominant retinitis pigmentosa. First, we establish the safety of this system, in C57BL/6J mice, demonstrating no loss of retinal thickness or function. We further show that outer nuclear layer thickness, electrical response, and rhodopsin expression in heterozygous Rho I307N were significantly preserved six months after treatment. This retention results from a 5.7% transgene integration and 88% indel rate in treated animals.