Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.

BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.

Cell patterning by laser-assisted bioprinting.

The aim of tissue engineering is to produce functional three-dimensional (3D) tissue substitutes. Regarding native organ and tissue complexity, cell density and cell spatial 3D organization, which influence cell behavior and fate, are key parameters in tissue engineering. Laser-Assisted Bioprinting (LAB) allows one to print cells and liquid materials with a cell- or picoliter-level resolution. Thus, LAB seems to be an emerging and promising technology to fabricate tissue-like structures that have the physiological functionality of their native counterparts. This technology has additional advantages such as automation, reproducibility, and high throughput. It makes LAB compatible with the (industrial) fabrication of 3D constructs of physiologically relevant sizes. Here we present exhaustively the numerous steps that allow printing of viable cells with a well-preserved micrometer pattern. To facilitate the understanding of the whole cell patterning experiment using LAB, it is discussed in two parts: (1) preprocessing: laser set-up, bio-ink cartridge and bio-paper preparation, and pattern design; and (2) processing: bio-ink printing on the bio-paper.

Cell patterning technologies for organotypic tissue fabrication.

Bottom-up tissue engineering technologies address two of the main limitations of top-down tissue engineering approaches: the control of mass transfer and the fabrication of a controlled and functional histoarchitecture. These emerging technologies encompass mesoscale (e.g. cell sheets, cell-laden hydrogels and 3D printing) and microscale technologies (e.g. inkjet printing and laser-assisted bioprinting), which are used to manipulate and assemble cell-laden building blocks whose thicknesses correspond to the diffusion limit of metabolites, and present the capacity for cell patterning with microscale precision, respectively. Here, we review recent technological advances and further discuss how these technologies are complementary, and could therefore be combined for the biofabrication of organotypic tissues either in vitro, thus serving as realistic tissue models, or within a clinic setting.

Laser-assisted bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite.

Developing tools to reproduce and manipulate the cell micro-environment, including the location and shape of cell patterns, is essential for tissue engineering. Parallel to inkjet printing and pressure-operated mechanical extruders, laser-assisted bioprinting (LAB) has emerged as an alternative technology to fabricate two- and three-dimensional tissue engineering products. The objective of this work was to determine laser printing parameters for patterning and assembling nano-hydroxyapatite (nHA) and human osteoprogenitors (HOPs) in two and three dimensions with LAB. The LAB workstation used in this study comprised an infrared laser focused on a quartz ribbon that was coated with a thin absorbing layer of titanium and a layer of bioink. The scanning system, quartz ribbon and substrate were piloted by dedicated software, allowing the sequential printing of different biological materials into two and/or three dimensions. nHA printing material (bioink) was synthesized by chemical precipitation and was characterized prior and following printing using transmission electron microscopy, Fourier transformed infrared spectroscopy and x-ray diffraction. HOP bioink was prepared using a 30 million cells ml(-1) suspension in culture medium and cells were characterized after printing using a Live/Dead assay and osteoblastic phenotype markers (alcaline phosphatase and osteocalcin). The results revealed that LAB allows printing and organizing nHA and HOPs in two and three dimensions. LAB did not alter the physico-chemical properties of nHA, nor the viability, proliferation and phenotype of HOPs over time (up to 15 days). This study has demonstrated that LAB is a relevant method for patterning nHA and osteoblastic cells in 2D, and is also adapted to the bio-fabrication of 3D composite materials.

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

Laser-assisted cell printing: principle, physical parameters versus cell fate and perspectives in tissue engineering.

We describe the physical parameters involved in laser-assisted cell printing and present evidence that this technology is coming of age. Finally we discuss how this high-throughput, high-resolution technique may help in reproducing local cell microenvironments, and thereby create functional tissue-engineered 3D constructs.

Differentiation of pre-osteoblast cells on poly(ethylene terephthalate) grafted with RGD and/or BMPs mimetic peptides.

The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2, BMP-7 and BMP-9 play a major role in the bone and cartilage formation. The BMP peptides corresponding to residues 73-92, 89-117, and 68-87 of BMP-2, BMP-7 and BMP-9 respectively as well as adhesion peptides (GRGDSPC) were grafted onto polyethylene terephthatalate (PET) surfaces. We evaluated the state of differentiation of pre-osteoblastic cells. The behavior of these cells on various functionalized surfaces highlighted the activity of the mimetic peptides immobilized on surfaces. The induced cells (observed in the case of surfaces grafted with BMP-2, 7 or 9 mimetic peptides and GRGDSPC peptides) were characterized on several levels. First of all, we focused on the evaluation of the osteoblastic markers such as the transcriptional factor Runx2, which is a critical regulator of osteoblastic differentiation. Secondly, the results obtained showed that these induced cells take a different morphology compared to the cells in a state of proliferation or in a state of extracellular matrix production. Induced cells were characterized by an increased thickness compared to non-induced cells. Thus, our studies prove a direct correlation between cell morphology and state of induction. Thereafter, we focused on characterizing the extracellular matrix formed by the cells on various surfaces. The extracellular matrix thickness was more significant in the case of surfaces grafted with mimetic peptides of the BMP-2, 7 or 9 and GRGDSPC peptides which once again proves their activity when immobilized on material surface. These results demonstrate that GRGDSPC and BMPs peptides, grafted to PET surface, act to enhance osteogenic differentiation and mineralization of pre-osteoblastic cells. These findings are potentially useful in developing engineered biomaterials for bone regeneration.

In vivo bioprinting for computer- and robotic-assisted medical intervention: preliminary study in mice.

We present the first attempt to apply bioprinting technologies in the perspective of computer-assisted medical interventions. A workstation dedicated to high-throughput biological laser printing has been designed. Nano-hydroxyapatite (n-HA) was printed in the mouse calvaria defect model in vivo. Critical size bone defects were performed in OF-1 male mice calvaria with a 4 mm diameter trephine. Prior to laser printing experiments, the absence of inflammation due to laser irradiation onto mice dura mater was shown by means of magnetic resonance imaging. Procedures for in vivo bioprinting and results obtained using decalcified sections and x-ray microtomography are discussed. Although heterogeneous, these preliminary results demonstrate that in vivo bioprinting is possible. Bioprinting may prove to be helpful in the future for medical robotics and computer-assisted medical interventions.

Laser assisted bioprinting of engineered tissue with high cell density and microscale organization.

Over this decade, cell printing strategy has emerged as one of the promising approaches to organize cells in two and three dimensional engineered tissues. High resolution and high speed organization of cells are some of the key requirements for the successful fabrication of cell-containing two or three dimensional constructs. So far, none of the available cell printing technologies has shown an ability to concomitantly print cells at a cell-level resolution and at a kHz range speed. We have studied the effect of the viscosity of the bioink, laser energy, and laser printing speed on the resolution of cell printing. Accordingly, we demonstrate that a laser assisted cell printer can deposit cells with a microscale resolution, at a speed of 5 kHz and with computer assisted geometric control. We have successfully implemented such a cell printing precision to print miniaturized tissue like layouts with de novo high cell density and micro scale organization.

The LIF cytokine: towards adulthood.

The aim of this article is to recapitulate the key features of leukaemia inhibitory factor cytokine (LIF), to review its numerous physiological effects and to comment on the most recent data. We will also present results of transcriptome analyses, which have highlighted different categories of LIF targets, identified in murine embryonic stem (ES) cells and early derivatives. We hope to stimulate new research fields on this puzzling cytokine, which, forty years after its discovery, has still not disclosed all its secrets.

Human primary endothelial cells stimulate human osteoprogenitor cell differentiation.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18alpha-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.