Craig Dorrell

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.

Robust expansion of human hepatocytes in Fah −/− / Rag2 −/− / Il2rg −/− mice

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

Stem Cells and Liver Regeneration

One of the defining features of the liver is the capacity to maintain a constant size despite injury. Although the precise molecular signals involved in the maintenance of liver size are not completely known, it is clear that the liver delicately balances regeneration with overgrowth. Mammals, for example, can survive surgical removal of up to 75% of the total liver mass. Within 1 week after liver resection, the total number of liver cells is restored. Moreover, liver overgrowth can be induced by a variety of signals, including hepatocyte growth factor or peroxisome proliferators; the liver quickly returns to its normal size when the proliferative signal is removed. The extent to which liver stem cells mediate liver regeneration has been hotly debated. One of the primary reasons for this controversy is the use of multiple definitions for the hepatic stem cell. Definitions for the liver stem cell include the following: (1) cells responsible for normal tissue turnover, (2) cells that give rise to regeneration after partial hepatectomy, (3) cells responsible for progenitor-dependent regeneration, (4) cells that produce hepatocyte and bile duct epithelial phenotypes in vitro, and (5) transplantable liver-repopulating cells. This review will consider liver stem cells in the context of each definition.

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver

Liver repopulation with bone marrow–derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow–derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah−/− mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1−/− mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow–derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.

Epigenomic plasticity enables human pancreatic α to β cell reprogramming

Insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Using ChIP sequencing and RNA sequencing analysis, we determined the epigenetic and transcriptional landscape of human pancreatic α, β, and exocrine cells. We found that, compared with exocrine and β cells, differentiated α cells exhibited many more genes bivalently marked by the activating H3K4me3 and repressing H3K27me3 histone modifications. This was particularly true for β cell signature genes involved in transcriptional regulation. Remarkably, thousands of these genes were in a monovalent state in β cells, carrying only the activating or repressing mark. Our epigenomic findings suggested that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. Indeed, we show that treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. Thus, mammalian pancreatic islet cells display cell-type-specific epigenomic plasticity, suggesting that epigenomic manipulation could provide a path to cell reprogramming and novel cell replacement-based therapies for diabetes.

Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Transcriptomes of the major human pancreatic cell types

Aims/hypothesisWe sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors.MethodsIslet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor–ligand representation in these populations.ResultsAnalysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand–receptor interactions suggested that EPH receptor–ephrin communication between exocrine and endocrine cells contributes to pancreatic function.Conclusions/interpretationThis is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types—including beta cells—and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Surface markers for the murine oval cell response.

The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence‐activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors. Conclusion: A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed. (HEPATOLOGY 2008.)