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Hepatocyte-like cells (HLCs) can be derived from pluripotent stem cells (PSCs) by sequential treatment of chemical cues to mimic the microenvironment of embryonic liver development. However, these HLCs do not reach the full maturity level of primary hepatocytes. In this study, we carried out a meta-analysis of cross-species transcriptome data of in vitro differentiation of human PSCs to HLCs and in vivo mouse embryonic liver development to identify the developmental stage at which HLC maturation was blocked at. Systematic variations were found associated with the data source and removed by batch correction. Using principal component analysis, HLCs from different stages of differentiation were aligned with mouse embryonic liver development chronologically. A “unified developmental time” (DT) scale was developed after aligning in vitro HLC differentiation and in vivo embryonic liver development. HLCs were found to cease further maturation at an equivalent stage of mouse embryonic day (E)13–15. Genes with discordant time dynamics were identified by aligning in vivo and in vitro data set onto a common DT scale. These genes may be targets of genetic intervention for enhancing the maturity of PSC-derived HLCs.

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109-1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. ispartof: Stem Cells and Development vol:26 issue:4 pages:274-284 ispartof: location:United States status: published

Many newly developed inhaled medicines fail to reach the market due to safety or efficacy. One of the main reasons for this is the observation of a foamy alveolar macrophage (AM) responses in rats in pre-clinical studies which limits the dose of these compounds in subsequent studies despite not knowing if these observations are toxic in humans. The aim of this work is to develop an accurate, immune responsive, in vitro cell co-culture model of the respiratory airways to provide more meaningful assessment of lung responses to new inhaled medicines. Human lung-derived monocyte cell line U937, was differentiated to a macrophage phenotype in the presence of 5 nM and 100 nM phorbol myristate acetate (PMA) over 24-96h. The activation, differentiation and functionality characteristics of naive and treated cells were assessed via cell viability and morphological analyses, microarray technology, flow cytometry and phagocytic ability. Protein microarray results showed PMA treated U937 cells were rich in surface markers specific to AM phenotype. Significant differences (p Our work suggests the differentiation protocol for U937 cells is important to consider as it may impact cell response. The differentiated U937 cell line offers the potential to explore macrophage responses to inhaled pharmaceutics in a robust in vitro immunocompetent co-culture model with airway epithelial cells.

Summary Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation., Graphical Abstract, Highlights • SOX10 is sufficient to generate myelinating human OLs from hPSCs in only 22 days • SOX10-induced OLs resemble primary human OLs at the transcriptome level • The methodology allows efficient generation of OLs from MS and ALS patients • OL-neuron co-cultures respond to myelinating drugs in a high-throughput setting, In this article, García-León JA and colleagues demonstrate the generation of functional oligodendrocytes (OLs) from human pluripotent stem cells in a rapid and efficient manner by the single overexpression of SOX10. Generated OLs resemble primary OLs at the transcriptome level and can myelinate neurons both in vivo and in vitro. Neuron-OL co-cultures, adapted to high-throughput screening formats, responded to drugs affecting myelination.

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.

Cell-replacement therapy and tissue regeneration using stem cells are of great interest to recover histological damage caused by neuro-degenerative disease or traumatic insults to the brain. To date, the main intra-cerebral delivery for these cells has been as a suspension in media through a thin needle. However, this does not provide cells with a support system that would allow tissue regeneration. Scaffold particles are needed to provide structural support to cells to form de novo tissue. In this 16-d protocol, we describe the generation and functionalization of poly (D,L-lactic-co-glycolic) acid (PLGA) particles to enhance cell attachment, the attachment procedure to avoid clumping and aggregation of cells and particles, and their preparation for intra-cerebral injection through a thin needle. Although the stem cell-scaffold transplantation is more complicated and labor-intensive than cell suspensions, it affords de novo tissue generation inside the brain and hence provides a significant step forward in traumatic brain repair.

Replacing the tissue lost after a stroke potentially provides a new neural substrate to promote recovery. However, significant neurobiological and biotechnological challenges need to be overcome to make this possibility into a reality. Human neural stem cells (hNSCs) can differentiate into mature brain cells, but require a structural support that retains them within the cavity and affords the formation of a de novo tissue. Nevertheless, in our previous work, even after a week, this primitive tissue is void of a vasculature that could sustain its long-term viability. Therefore, tissue engineering strategies are required to develop a vasculature. Vascular endothelial growth factor (VEGF) is known to promote the proliferation and migration of endothelial cells during angio- and arteriogenesis. VEGF by itself here did not affect viability or differentiation of hNSCs, whereas growing cells on poly(D,L-lactic acid-co-glycolic acid) (PLGA) microparticles, with or without VEGF, doubled astrocytic and neuronal differentiation. Secretion of a burst and a sustained delivery of VEGF from the microparticles in vivo attracted endothelial cells from the host into this primitive tissue and in parts established a neovasculature, whereas in other parts endothelial cells were merely interspersed with hNSCs. There was also evidence of a hypervascularization indicating that further work will be required to establish an adequate level of vascularization. It is therefore possible to develop a putative neovasculature within de novo tissue that is forming inside a tissue cavity caused by a stroke.

Dendritic cells (DCs) are part of the innate immune system with a key role in initiating and modulating T cell mediated immune responses. Coeliac disease is caused by inappropriate activation of such a response leading to small intestinal inflammation when gluten is ingested. Tissue transglutaminase, an extracellular matrix (ECM) protein, has an established role in coeliac disease; however, little work to date has examined its impact on DCs. The aim of this study was to investigate the effect of small intestinal ECM proteins, fibronectin (FN) and tissue transglutaminase 2 (TG-2), on human DCs by including these proteins in DC cultures. The study used flow cytometry and scanning electron microscopy to determine the effect of FN and TG-2 on phenotype, endocytic ability and and morphology of DCs. Furthermore, DCs treated with FN and TG-2 were cultured with T cells and subsequent T cell proliferation and cytokine profile was determined. The data indicate that transglutaminase affected DCs in a concentration-dependent manner. High concentrations were associated with a more mature phenotype and increased ability to stimulate T cells, while lower concentrations led to maintenance of an immature phenotype. These data provide support for an additional role for transglutaminase in coeliac disease and demonstrate the potential of in vitro modelling of coeliac disease pathogenesis.

Aims: Recently, there have been numerous preclinical and human studies investigating the regenerative capacity of cell suspensions following their direct injection into a target organ: the fundamental parameters for successful (clinical) cell therapy. At present, limited data exist in the identification of factors important for the survival of these cells (i.e., morphology, viability and proliferation rates) during and following their ejection via narrow-bore needles. Materials & methods: Primary murine mesenchymal stem cells (mMSCs) were isolated, expanded and processed into a concentrated cell suspension consisting of either HBSS or HBSS supplemented with the antioxidant n-acetyl-cysteine. This suspension was then ejected from a 10 µl Hamilton syringe, via a variety of bore-sized needles, at different ejection rates. Cell characteristics including viability, spreading and attachment, apoptosis and proliferative ability were then assessed. Results: Following manipulation within a syringe, a decrease in the viability and cell spreading of mMSCs and a concurrent increase in the production of the caspase-3 protein, an early regulatory event in apoptosis, occurs. These detrimental effects were found to be increased when the cells were left in the syringe chamber for increased periods of time, and were similar at 5 µl/min and 1 µl/min ejection rates. However, on increasing the needle bore diameter, a significant reduction in these characteristics was observed. By comparison, mMSCs that were left to stand at room temperature (18–20°C), but were not manipulated within a syringe, showed a significantly greater viability compared with manipulated cells. However, cells kept at 4°C demonstrated a decreased viability compared with manipulated cells. When the mMSC were incubated with n-acetyl-cysteine, a known antioxidant, no significant change in caspase-3 production or cell spreading was observed. Conclusions: This study highlights potential parameters, such as minimizing the time period the cells are within the syringe and the use of wider-bore needles, involved in maintaining the high viable cell density required for the delivery of cell suspensions for cell therapy applications.

As the first line of defence, skin is regularly exposed to a variety of biological, physical and chemical insults. Therefore, determining the skin sensitization potential of new chemicals is of paramount importance from the safety assessment and regulatory point of view. Given the questionable biological relevance of animal models to human as well as ethical and regulatory pressure to limit or stop the use of animal models for safety testing, there is a need for developing simple yet physiologically relevant models of human skin. Herein, we describe the construction of a novel immunocompetent 3D human skin model comprising of dendritic cells co-cultured with keratinocytes and fibroblasts. This model culture system is simple to assemble with readily-available components and importantly, can be separated into its constitutive individual layers to allow further insight into cell-cell interactions and detailed studies of the mechanisms of skin sensitization. In this study, using non-degradable microfibre scaffolds and a cell-laden gel, we have engineered a multilayer 3D immunocompetent model comprised of keratinocytes and fibroblasts that are interspersed with dendritic cells. We have characterized this model using a combination of confocal microscopy, immuno-histochemistry and scanning electron microscopy and have shown differentiation of the epidermal layer and formation of an epidermal barrier. Crucially the immune cells in the model are able to migrate and remain responsive to stimulation with skin sensitizers even at low concentrations. We therefore suggest this new biologically relevant skin model will prove valuable in investigating the mechanisms of allergic contact dermatitis and other skin pathologies in human. Once fully optimized, this model can also be used as a platform for testing the allergenic potential of new chemicals and drug leads.

Background Sampling the microenvironment at sites of microbial exposure by dendritic cells (DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation. Methodology/Principal Findings Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H3-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a ‘more immature’ phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation. Conclusion These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.