Kay Samuel

Generation of functional human hepatic endoderm from human induced pluripotent stem cells

With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient “off the shelf” models of human liver disease. Here, we show the “proof of concept” that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%–90%, using a method mimicking physiological relevant condition. The iPSC‐derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E‐cadherin, as assessed by immunohistochemistry. They also expressed alpha‐fetoprotein, hepatocyte nuclear factor‐4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC‐derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha‐fetoprotein, an essential feature for functional HE. Additionally iPSC‐derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC‐derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models. (HEPATOLOGY 2009.)

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.

Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion

BACKGROUND & AIMS Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. METHODS Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. RESULTS We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. CONCLUSIONS We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.

Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver

Background Stem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling. Objective To examine the composition and formation of stem progenitor cell niches. Methods The composition of the hepatic progenitor cell niche in independent models of liver injury and hepatic progenitor cell activation in rodents and humans was studied. To identify the origin of the progenitor and niche cells, sex-mismatched bone marrow transplants in mice, who had received the choline–ethionine-deficient-diet to induce liver injury and progenitor cell activation, were used. The matrix surrounding the progenitor cells was described by immunohistochemical staining and its functional role controlling progenitor cell behaviour was studied in cell culture experiments using different matrix layers. Results The progenitor cell response in liver injury is intimately surrounded by myofibroblasts and macrophages, and to a lesser extent by endothelial cells. Hepatic progenitor cells are not of bone marrow origin; however, bone marrow-derived cells associate intimately with these cells and are macrophages. Laminin always surrounds the progenitor cells. In vitro studies showed that laminin aids maintenance of progenitor and biliary cell phenotype and promotes their gene expression (Dlk1, Aquaporin 1, γGT) while inhibiting hepatocyte differentiation and gene expression (CEPB/α). Conclusions During liver damage in rodents and humans a stereotypical cellular and laminin niche forms around hepatic progenitor cells. Laminin helps maintenance of undifferentiated progenitor cells. The niche links the intrahepatic progenitor cells with bone marrow-derived cells and links tissue damage with progenitor cell-mediated tissue repair.

Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo

Recent work has indicated that adult bone marrow-derived cells have the ability to contribute to both the haematopoietic system and other organs. Haematopoietic reconstitution by whole bone marrow and selected but not fully characterised cell populations have resulted in reports indicating high-level repopulation of lung epithelia. The well-characterised cells from the side population have a robust ability for haematopoietic reconstitution. We have used freshly isolated side population cells derived from ROSA26 adult bone marrow and demonstrate that despite being unable to contribute to embryos following blastocyst injection, or air liquid interface cultures or denuded tracheal xenografts, they could contribute to the tracheal epithelium in vivo. Epithelial damage is reported to be important in encouraging the recruitment of marrow-derived stem cells into non-haematopoietic organs. Here we demonstrate that mice engrafted with side population cells have donor-derived cells present in the epithelial lining of the trachea following damage and repair. Donor-derived cells were found at a frequency of 0.83%. Widefield and confocal microscopy revealed donor cells that expressed cytokeratins, indicative of cells of an epithelial nature. These results imply that SP haematopoietic stem cells from the bone marrow do not have the ability to contribute to airway epithelia themselves but require factors present in vivo to allow them to acquire characteristics of this tissue.

Phase I/II study of vaccination with dendritic-like leukaemia cells for the immunotherapy of acute myeloid leukaemia.

Twenty‐two patients with acute myeloid leukaemia were recruited into a phase I/II clinical trial investigating the vaccination of patients in complete remission (CR) with autologous dendritic‐like leukaemia cells (DLLC). At trial entry, leukaemia cells were harvested and tested for their ability to undergo cytokine‐induced dendritic cell differentiation. Patients were then treated with intensive chemotherapy. Five patients achieved both CR and had leukaemia cells that successfully underwent differentiation and therefore proceeded to vaccination. Four escalating doses of DLLC were administered weekly by subcutaneous injection. Vaccination was generally well tolerated although one patient developed extensive eczema and an increased antinuclear factor titre possibly indicating induction of autoimmunity. Development of anti‐leukaemic T‐cell responses was assessed by enzyme‐linked immunospot analysis of gamma‐interferon secreting T lymphocytes and by human leucocyte antigen tetramer analysis for WT1‐specific T cells. Increases in anti‐leukaemic T‐cell responses were demonstrated in four patients, but only two of the five remained in remission more than 12 months postvaccination. The study has demonstrated that generation of DLLC is feasible in only a subgroup of patients and is currently neither broadly applicable or clinically effective.

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte‐colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real‐time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow‐derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow‐derived circulating precursor for endothelial cells. STEM CELLS2013;31:338–348

WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

The survival of differentiating embryonic stem cells is dependent on the SCF-KIT pathway

The stem cell factor (SCF)-KIT signal transduction pathway plays a role in the proliferation, differentiation and survival of a range of stem and progenitor cell types but little is known about its function in embryonic stem (ES) cells. We generated ES cells carrying a null allele of Kit as well as a knock-in allele that encodes an SCF-independent hybrid KIT receptor that can be activated by the FKBP binding drug, AP20187. KIT null ES cells die when induced to differentiate upon withdrawal of leukaemia inhibitory factor in monolayer culture. This phenotype is recapitulated in wild-type ES cells treated with a KIT-neutralising antibody and reversed in mutant cells by activation of the hybrid KIT receptor. Differentiating KIT null ES cells exhibit elevated levels of DNA laddering and reduced BCL2 expression, indicative of apoptosis. We conclude that mouse ES cell differentiation in vitro is dependent on the SCF-KIT pathway contrasting with the apparently normal differentiation of KIT null inner cell mass or epiblast cells in vivo. This discrepancy could be explained by the presence of compensatory signals in the embryo or it could lend support to the idea of a phenotypic relationship between ES cells and early germ cells.

The Comparison between Conditioned Media and Serum-Free Media in Human Embryonic Stem Cell Culture and Differentiation

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such, hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However, for their full potential to be realized, both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however, they suffer from some major limitations including lack of definition, xenobiotic nature, batch-to-batch variation, and labor-intensive production. Therefore, hESC culture definition is essential if hESC lines, and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state, for over 30 passages using MT and SP. Additionally, we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture, contributing to the standardization of hESC in vitro models and ultimately their application.