Mette Hemmingsen

The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose-derived Stem Cells

Introduction High cell density is known to enhance adipogenic differentiation of mesenchymal stem cells, suggesting secretion of signaling factors or cell-contact-mediated signaling. By employing microfluidic biochip technology, we have been able to separate these two processes and study the secretion pathways. Methods and results Adipogenic differentiation of human adipose-derived stem cells (ASCs) cultured in a microfluidic system was investigated under perfusion conditions with an adipogenic medium or an adipogenic medium supplemented with supernatant from differentiating ASCs (conditioned medium). Conditioned medium increased adipogenic differentiation compared to adipogenic medium with respect to accumulation of lipid-filled vacuoles and gene expression of key adipogenic markers (C/EBPα, C/EBPβ, C/EBPδ, PPARγ, LPL and adiponectin). The positive effects of conditioned medium were observed early in the differentiation process. Conclusions Using different cell densities and microfluidic perfusion cell cultures to suppress the effects of cell-released factors, we have demonstrated the significant role played by auto- or paracrine signaling in adipocyte differentiation. The cell-released factor(s) were shown to act in the recruitment phase of the differentiation process.

A self-contained, programmable microfluidic cell culture system with real-time microscopy access

Utilizing microfluidics is a promising way for increasing the throughput and automation of cell biology research. We present a complete self-contained system for automated cell culture and experiments with real-time optical read-out. The system offers a high degree of user-friendliness, stability due to simple construction principles and compactness for integration with standard instruments. Furthermore, the self-contained system is highly portable enabling transfer between work stations such as laminar flow benches, incubators and microscopes. Accommodation of 24 individual inlet channels enables the system to perform parallel, programmable and multiconditional assays on a single chip. A modular approach provides system versatility and allows many different chips to be used dependent upon application. We validate the system’s performance by demonstrating on-chip passive switching and mixing by peristaltically driven flows. Applicability for biological assays is demonstrated by on-chip cell culture including on-chip transfection and temporally programmable gene expression.

The MainSTREAM Component Platform: A Holistic Approach to Microfluidic System Design

A microfluidic component library for building systems driving parallel or serial microfluidic-based assays is presented. The components are a miniaturized eight-channel peristaltic pump, an eight-channel valve, sample-to-waste liquid management, and interconnections. The library of components was tested by constructing various systems supporting perfusion cell culture, automated DNA hybridizations, and in situ hybridizations. The results showed that the MainSTREAM components provided (1) a rapid, robust, and simple method to establish numerous fluidic inputs and outputs to various types of reaction chips; (2) highly parallel pumping and routing/valving capability; (3) methods to interface pumps and chip-to-liquid management systems; (4) means to construct a portable system; (5) reconfigurability/flexibility in system design; (6) means to interface to microscopes; and (7) compatibility with tested biological methods. It was found that LEGO Mindstorms motors, controllers, and software were robust, inexpensive, and an accessible choice as compared with corresponding custom-made actuators. MainSTREAM systems could operate continuously for weeks without leaks, contamination, or system failures. In conclusion, the MainSTREAM components described here meet many of the demands on components for constructing and using microfluidics systems.

3D Printed Silicone–Hydrogel Scaffold with Enhanced Physicochemical Properties

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

Hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions and to perform functional comparisons with fresh human precision‐cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis in the hiPSC‐derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels comparable with hPCLS indicating excellent future perspectives for the development of a bioartificial liver system for toxicity testing or as liver support device for patients.

Hepatic differentiation of human induced pluripotent stem cells in a perfused 3d porous polymer scaffold for liver tissue engineering

B design and 3-D printing of scaffold with heterogeneous internal geometry is essential for cell distribution, blood vessel in growth and biomaterial degradation in bone tissue engineering. This study was designed to demonstrate the heterogeneous pores and channels in 3-D printed scaffolds for bone tissue engineering. Scaffolds were prepared using ceramic particles through 3-D printing. Pores and connecting channels with diameters of 200mm-500mm were designed for facilitating cell seeding and cell distribution. Internal pores of 50mm-200mm were designed for bone regeneration. Nano-sized surface topography was designed for enhanced degradation of scaffold. The fabricated scaffolds were evaluated using scanning electronic microscopy. SEM of fabricated scaffolds revealed that 400mm500mm inter-connecting channels crossed over the entire scaffold, that ~200 mm internal pores were scattered over the scaffold and connected to each other and to the interconnecting channels, and that ~200 nm pores showed on the surfaces of inter-connecting channels and internal pores, which would play an important role in increasing the surface ratio of materials and facilitating material degradation. A heterogeneous profile of connecting channels and internal pores was evident in these 3-D printed biomimetic scaffolds. As a conclusion, the biomimetic design and fabrication of scaffolds for bone tissue engineering can be fulfilled by a 3-D printing process. Heterogeneous profiles of inter-connecting channels, internal pores, and nano-sized surface topography can be generated to provide a biomimetic environment suitable for bone tissue engineering.Background: Myocardial infarction (MI) was the leading cause of death in worldwide. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of biological processes, including cell viability, proliferation, development and differentiation. The purpose of this study was to investigate difference of miRNA profiles between infarct zone and border zone in post-MI remodeling using the second generation sequencing.Neia Naldaiz-Gastesi1, Patricia Garcia-Parra1, Maria Goicoechea1, Sonia Alonso-Martin2, Ana Aiastui1, Macarena Lopez-Mayorga3, Paula Garcia-Belda4, Jaione Lacalle1,5, Veronique Le Berre6, Ander Matheu1, Jose Manuel Garcia-Verdugo4, Jaime J. Carvajal3, Frederic Relaix2, Adolfo Lopez de Munain1 and Ander Izeta1 1Instituto Biodonostia, Spain 2Myology Research Center, France 3Centro Andaluz de Biologia del Desarrollo, Spain 4Instituto Cavanilles, Universidad de Valencia, Spain 5University of the Basque Country (UPV-EHU), Spain 6UMR INSA, FranceOur knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONSS cells have been recognized as a potential tool to restore cells damaged by cerebral ischemic injury. Key functions such as the replacement of neural cells have been recently challenged by intrinsic bystander capacities of undifferentiated donor cells. One of opportunity for neurological disorder treatment is the transplantation of mesenchymal stem cells (MSCs) which have neuroprotective, neuroregenerative and anti-inflamatory properties. However, a comprehensive knowledge how transplanted MSCs exert their therapeutic achievements is still lacking. The aim of the project was to analyze the presence, distribution and quantity of human bone marrow mesenchymal stem cells (hBM-MSCs) transplanted into focal brain ischemic rats. The experiments were performed in adult male Wistar rats withbrain focal ischemiainduced with 1μl/50nmol ouabain(sodium-potassium pump inhibitor) injection into right stratium. Then 5x105 hBM-MSC (Lonza) stained with iron nanoparticles and rhodamine (Molday, BioPAL) were transplanted into internal carotid artery, 48 hours after brain insult. At 1, 3, 7 and 14 days rat brains were removed. Immunocytochemical analysis of human markers using different antibodies anti: CD44, STEM121and Ku80 were performed. The preliminary results showed that after intra-arterially injection of hBM-MSC, the donor cells were present in the ipsilateral rat hemisphere between cortical cortex and stratium near the ischemic lesion. The positive staining for Molday particles and human antigens were observed at 1, 2, 3 and 7 days after hBM-MSC transplantation. The further studies relating to the function of transplanted cells are in progress.M stem cells (MSCs) represent a population of multipotent stem cells with immunomodulatory, antiapoptotic and cytoprotective capabilities and thus hold a great promise for treatment of many inflammatory diseases and for use in a regenerative medicine. Numerous studies have shown that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone. However, the exact mechanism of such synergism has not yet been described.