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Adult stem cells possess the ability to differentiate and mature into defined cell types; however, tissue-specificity and donor and culture inconsistencies have presented a challenge in identifying these cells. Adult adherent dermal cell-products have been efficiently utilized for isogenic cosmetic therapies. The purpose of this study is to identify, isolate, and characterize progenitor subsets from adult adherent dermal cells capable of ex vivo differentiation. LAVIV® adult dermal cells were independently immunoselected for CD146, CD271, and CD73/CD90/CD105 to investigate the mesenchymal differentiation capacity and possible enrichment in the purified fractions. After differentiation, the osteogenic, chondrogenic, and adipogenic potential and cell-specific gene expression were evaluated and compared for each phenotype. Adult dermal cells possess the ability to differentiate into the three cell lineages, osteocyte, chondrocyte, and adipocyte that co-express the adult stem cell immunophenotypic markers CD146 and CD271 with independent enrichment of the multipotent capacity for both fractions. We conclude that subpopulations in human dermal primary cultures possess the potential to differentiate into other cell types providing a novel source of multipotent cells for regenerative medicine.

Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse “immunogenicity” genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context.

Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue-engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19-expressing bile ducts and vascular structures with α-smooth muscle actin expression, desmin-expressing stellate cells, and CD31-expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin-expressing cells and identification of β2-microglobulin-expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4-OH-debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics.

Urea cycle disorders (UCDs) are incurable genetic diseases that affect the body's ability to produce urea, leading to hyperammonemia due to a deficiency in any one of six enzymes in the cycle. For arginase deficiency, a mutation in the ARG1 gene, the final step of the cycle, results in hyperargininemia, developmental delays and disabilities, seizures, psychomotor function loss, and in serious cases, death. There is currently no completely effective treatment available. Advances in human induced pluripotent stem cell (hiPSC) research and genome-editing technologies have enabled the genetic modification of stem cells for potential cellular replacement therapies. In this study, we applied such technology to develop a stem cell-based approach for treating arginase deficiency applicable to all arginase deficient patients regardless of their mutation. Methods: Fibroblasts from three patients with arginase deficiency were obtained, defined for their mutation, and reprogrammed into hiPSCs. Selectable, full-length codon optimized human arginase cDNA (coARG) expression cassettes were then developed for site-specific integration into either the HPRT or albumin (ALB) locus. After confirming specificity by Sanger sequencing, genetically corrected hiPSCs were differentiated to hepatocyte-like cells. Results: Fibroblasts were reprogrammed into hiPSCs by applying a STEMCCA lentivirus-based method and were characterized for pluripotency by immunophenotyping for common stemness markers via ICC, alkaline phosphatase staining, and in vivo teratoma formation. Using a site-specific CRISPR/Cas9 nickase-mediated gene transferring system, we inserted coARG by two approaches. First, coARG was inserted into the HPRT locus under the control of constitutive hEF1α promoter (LEAPR); targeting HPRT allowed for positive clonal selection of successful on-target integration by 6-thioguanine treatment. Second, we inserted coARG into the ALB locus for expression under the endogenous ALB promoter (ALB-coARG) as ALB is highly expressed in human liver. After LEAPR and ALB-coARG modification and sequence confirmation, hiPSCs were differentiated to hepatocyte-like cells and characterized by immunophenotyping via ICC and RNA expression via RT-PCR for common hepatic markers; cells demonstrated more fetal-like characteristics. Moreover, LEAPR- and ALB-coARG-modified hepatocyte-like cells demonstrated 41% and 1% functional arginase activity recovery compared to human fetal liver, respectively. Discussion: In this study, we demonstrated the ability to genetically correct mutated ARG1 gene expression in hiPSCs derived from patients with hyperargininemia and restored arginase function in hiPSCs and hepatocyte derivatives by CRISPR/Cas9-based gene addition. As the LEAPR construct demonstrated marked coARG expression, recovery of arginase activity of ALB-ARG-modified hepatocyte-like cells was low; we expect significant arginase recovery after in vivo maturation of transplanted cells as ALB expression increases with hepatocyte maturation. Also, to demonstrate potential in vivo recovery of arginase deficiency pathogenesis, ongoing studies aim to transplant both cohorts of gene-corrected hepatocyte-like cells into an established arginase deficient immunosuppressed mouse model. Successful restoration of enzyme function in patient-specific hiPSCs and relevant cellular derivatives will highlight hiPSCs as a valuable tool in cell replacement therapies and advance applications of genetically modified hiPSCs to treat UCDs and other single enzyme liver deficiencies.