Manufacturing development and clinical production of NKG2D chimeric antigen receptor-expressing T cells for autologous adoptive cell therapy.

Background Aims: Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825).

Translating cell-based regenerative medicines from research to successful products: challenges and solutions.

The Tissue Engineering & Regenerative Medicine International Society-Europe (TERMIS-EU) Industry Committee as well as its TERMIS-Americas (AM) counterpart intend to address the specific challenges and needs facing the industry in translating academic research into commercial products. Over the last 3 years, the TERMIS-EU Industry Committee has worked with commercial bodies to deliver programs that encourage academics to liaise with industry in proactive collaborations. The TERMIS-EU 2013 Industry Symposium aimed to build on this commercial agenda by focusing on two topics: Operations Management (How to move a process into the good manufacturing practice [GMP] environment) and Clinical Translation (Moving a GMP process into robust trials).

Successful isolation of liver progenitor cells by aldehyde dehydrogenase activity in naïve mice.

Unlabelled: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting.

Evaluation of the multipotent character of human adipose tissue-derived stem cells isolated by Ficoll gradient centrifugation and red blood cell lysis treatment.

In the present study, the multipotent potential of two differential isolated human adipose-derived stem cell (hADSC) populations was evaluated. More specifically, hADSC isolated by means of classical Ficoll (F) gradient centrifugation were compared to hADSC isolated by means of red blood cell (RBC) lysis treatment and subsequent cultivation as 3D spheres. No significant difference in the genotypic expression of the multipotent markers Oct-4, Sox-2, Nanog, Klf-4 and cMyc could be observed between both isolation methods.

Evaluation of the multipotent character of human foreskin-derived precursor cells.

In the present study, the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal, (iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media, keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®.

Hepatic differentiation of mesenchymal stem cells: in vitro strategies.

Recently, evidence has been provided that mesenchymal stem/progenitor cells (MSC) from various sources (bone marrow, adipose tissue, skin, placenta, umbilical cord) could occasionally overcome lineage borders and differentiate into endodermal (hepatocytes) and ectodermal (neural cells) cell types in vitro. Whereas unidirectional differentiation into other mesenchymal cell types, including adipocytes, chondrocytes, and osteoblasts, readily occurs in the presence of a simple cocktail of growth factors and nutrients, successful bypassing of lineage borders mainly depends on multistep processes in a coordinated signaling network. Here, we provide a reproducible basic methodology to differentiate adult MSC into functional hepatocytes in a sequential and time-dependent way.

Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4-Me2N-BAVAH, a hydroxamate-based HDAC-inhibitor.

Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis.

Preliminary in vivo evaluation of [131I]-2-iodo-D-phenylalanine as a potential radionuclide therapeutic agent in R1M-fluc rhabdomyosarcoma tumor-bearing NuNu mice using bioluminescent imaging.

Background: Carrier-added [(123)I]-2-iodo-D-phenylalanine (CA [(123)I]-2-I-D-Phe) was previously found to have a preferential retention in tumors with a high tumor background contrast in animal models. A previous human dosimetry study demonstrated a favorable biodistribution and radiation burden in human subjects.

Aim: The aim of this study was to investigate the potential of CA [(131)I]-2-I-D-Phe as an agent for radionuclide therapy.

DNA methyltransferase 3a expression decreases during apoptosis in primary cultures of hepatocytes.

Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control of liver homeostasis, its role in the occurrence of hepatocyte cell death has yet been poorly documented. We therefore have investigated the expression pattern of the effectors of DNA methylation, namely DNA methyltransferase (DNMT) isoenzymes, during Fas-mediated apoptotic cell death in primary hepatocyte cultures. Cell death was assessed by in situ stainings with Annexin V, Hoechst 33342 and Propidium iodide, and measurement of caspase 3-like activity and lactate dehydrogenase release.

Characterization and hepatic differentiation of skin-derived precursors from adult foreskin by sequential exposure to hepatogenic cytokines and growth factors reflecting liver development.

In the present study, we investigated whether precursor cells isolated from the dermis of infant human foreskin are capable to differentiate into hepatocyte-like cells upon sequential and gradual exposure to hepatogenic factors [fibroblast growth factor (FGF)-4, hepatocyte growth factor (HGF), insulin-transferrin-selenite (ITS), dexamethasone and oncostatin M (OSM)], mimicking the liver embryogenesis in vivo. Undifferentiated human skin-derived precursors (hSKP) are characterized by a fibroblast-like shape. Yet, they already express typical hepatic proteins, including cytokeratin (CK)-18, hepatocyte nuclear factor (HNF)-4 and HNF-1alpha.

Histone deacetylase inhibition and the regulation of cell growth with particular reference to liver pathobiology.

The transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.

Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions.

In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art.

Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm.

Differentiation of neonatal rat epithelial cells from biliary origin into immature hepatic cells by sequential exposure to hepatogenic cytokines and growth factors reflecting liver development.

It was investigated whether cryopreserved rat liver epithelial cells (RLEC) from biliary origin are capable of undergoing hepatic differentiation upon sequential exposure to liver-specific factors (fibroblast growth factor (FGF)-4, hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS), dexamethasone (Dex) and oncostatin M (OSM)), reflecting liver embryogenesis in vivo. As differentiation progressed, cells acquired a hepatic morphology (polygonal-to-cuboidal shaped, binucleated cells), corresponding well with the phenotypic changes observed. Biliary cytokeratin (CK)19 and connexin (Cx)43-expression both gradually decreased; CK19-expression disappeared even completely.

The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.

Histone deacetylase (HDAC) inhibitors show great pharmaceutical potential, particularly in relation to cancer. However, very little is known about their biological outcome on hepatocytes, the major executors of xenobiotic biotransformation in the organism. The current study was set up to investigate the effects of the newly synthesized HDAC inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamate (4-Me(2)N-BAVAH) on hepatocyte gap junctions and adherens junctions, being main guardians of liver homeostasis.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow.

Background: The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.

Results: Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression.

Differential role of epigenetic modulators in malignant and normal stem cells: a novel tool in preclinical in vitro toxicology and clinical therapy.

Adult stem cells are primitive cells that undergo asymmetric division, thereby giving rise to one clonogenic, self-renewing cell and one cell able to undergo multipotent differentiation. Disturbance of this controlled process by epigenetic alterations, including imbalance of histone acetylation/histone deacetylation and DNA methylation/demethylation, may result in uncontrolled growth, formation of self-renewing malignant stem cells and eventually cancer. In view of this notion, several epigenetic modulators, in particular those with histone deacetylase inhibiting activity, are currently being tested in phase I and II clinical trials for their promising chemotherapeutic properties in cancer therapy.

Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.

Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated.

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells.

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked.

Involvement of cell junctions in hepatocyte culture functionality.

In liver, like in other multicellular systems, the establishment of cellular contacts is a prerequisite for normal functioning. In particular, well-defined cell junctions between hepatocytes, including adherens junctions, desmosomes, tight junctions, and gap junctions, are known to play key roles in the performance of liver-specific functionality. In a first part of this review article, we summarize the current knowledge concerning cell junctions and their roles in hepatic (patho)physiology.

Isolation of rat bone marrow stem cells.

Stem cell research has become an important field of study for molecular, cellular, and clinical biology as well as pharmaco-toxicology. Indeed, stem cells have a strong proliferative and unlimited self-renewal potential and are multipotent. In vivo as well as in vitro studies have confirmed the differentiation of adult bone marrow stem cells into muscle cells, adipocytes, cardiomyocytes, neuroectodermal cells, osteoblasts, chondroblasts, and so on.

Hepatocytes in suspension.

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the "gold standard" for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h.

Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures.

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g.

Rat hepatocyte cultures: conventional monolayer and cocultures with rat liver epithelial cells.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended lifespan and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions.

Isolation of rat hepatocytes.

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined.