Long-Term Correction of Diabetes in Mice by In Vivo Reprogramming of Pancreatic Ducts.

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic ß cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to ß-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic ß cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced ß-like cells are mono-hormonal, express genes essential for ß cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of ß cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Adult Mouse Liver Contains Two Distinct Populations of Cholangiocytes.

The biliary system plays an important role in several acquired and genetic disorders of the liver. We have previously shown that biliary duct epithelium contains cells giving rise to proliferative Lgr5+ organoids in vitro. However, it remained unknown whether all biliary cells or only a specific subset had this clonogenic activity. The cell surface protease ST14 was identified as a positive marker for the clonogenic subset of cholangiocytes and was used to separate clonogenic and non-clonogenic duct cells by fluorescence-activated cell sorting. Only ST14hi duct cells had the ability to generate organoids that could be serially passaged. The gene expression profiles of clonogenic and non-clonogenic duct cells were similar, but several hundred genes were differentially expressed. RNA fluorescence in situ hybridization showed that clonogenic duct cells are interspersed among regular biliary epithelium at a ∼1:3 ratio. We conclude that adult murine cholangiocytes can be subdivided into two populations differing in their proliferative capacity.

Efficient generation of pancreatic ß-like cells from the mouse gallbladder.

Direct reprogramming is a promising approach for the replacement of ß cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to ß cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional ß cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of ß cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with ß cells and had a metabolic gene expression profile resembling neonatal ß cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.

A universal system to select gene-modified hepatocytes in vivo.

Many genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often subtherapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles could be overcome if hepatocytes that have undergone genetic modification were to be selectively amplified or expanded. We describe a universally applicable system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background. In this system, the therapeutic transgene is coexpressed with a short hairpin RNA (shRNA) that confers modified hepatocytes with resistance to drug-induced toxicity. An shRNA against the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase protected hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate, a small-molecule inhibitor of fumarylacetoacetate hydrolase. To select for specific gene targeting events, the protective shRNA was embedded in a microRNA and inserted into a recombinant adeno-associated viral vector designed to integrate site-specifically into the highly active albumin locus. After selection of the gene-targeted cells, transgene expression increased 10- to 1000-fold, reaching supraphysiological levels of human factor 9 protein (50,000 ng/ml) in mice. This drug resistance system can be used to achieve therapeutically relevant transgene levels in hepatocytes in any setting.

The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.

Pancreatic Lgr5 expression has been associated with organoid-forming epithelial progenitor populations but the identity of the organoid-initiating epithelial cell subpopulation has remained elusive. Injury causes the emergence of an Lgr5(+) organoid-forming epithelial progenitor population in the adult mouse liver and pancreas. Here, we define the origin of organoid-initiating cells from mouse pancreas and liver prior to Lgr5 activation. This clonogenic population was defined as MIC1-1C3(+)/CD133(+)/CD26(-) in both tissues and the frequency of organoid initiation within this population was approximately 5% in each case. The transcriptomes of these populations overlapped extensively and showed enrichment of epithelial progenitor-associated regulatory genes such as Sox9 and FoxJ1. Surprisingly, pancreatic organoid cells also had the capacity to generate hepatocyte-like cells upon transplantation to Fah(-/-) mice, indicating a differentiation capacity similar to hepatic organoids. Although spontaneous endocrine differentiation of pancreatic progenitors was not observed in culture, adenoviral delivery of fate-specifying factors Pdx1, Neurog3 and MafA induced insulin expression without glucagon or somatostatin. Pancreatic organoid cultures therefore preserve many key attributes of progenitor cells while allowing unlimited expansion, facilitating the study of fate determination.

Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming.

Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable ß-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the ß-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true ß-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations.

In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

In vivo selection of transplanted hepatocytes by pharmacological inhibition of fumarylacetoacetate hydrolase in wild-type mice.

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4-6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20-100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes.