Regulation von Ionenkanälen und Gap Junctions während der Zellentwicklung

Humanes neuronales Zellmaterial wird für die regenerative Medizin sowie physiologische und pharmakologische Studien dringend benötigt, ist aber sehr eingeschränkt verfügbar. Es wurden deshalb bereits verschiedene Ansätze entwickelt, neuronale Zellen aus gut zugänglichen und in vitro vermehrbaren Zellen zu transdifferenzieren und damit ein neuronales Zellmodell zu kreieren. Für ein aussagekräftiges Zellmodell ist es allerdings notwendig, die generierten Zellen, wie auch das Ausgangszellmaterial, exakt zu kennen und den neuronalen Entwicklungsstand nach der Differenzierung möglichst präzise einordnen zu können. Sowohl Ionenkanäle als auch Gap Junctions sind für die Integration, Funktion und Entwicklung von Zellen in ein Gewebe essenziell, da sie die inter- und intrazelluläre Signalgebung und somit auch die Zellkommunikation sowie Differenzierung maßgeblich beeinflussen, so auch in neuronalem Gewebe. Ziel dieser Dissertation war deshalb die Untersuchung der Regulation von Ionenkanälen und Gap Junctions während der Zellentwicklung am in vitro-Modell neuronal transdifferenzierter humaner MSCs aus dem Knochenmark. Die unbehandelten MSCs exprimierten die charakteristischen MSC-Marker CD73, CD90, CD105 und CD166 wie auch den neuronalen Marker Tuj1. Während der neuronalen small molecule-induzierten Transdifferenzierungen wurden die MSC-Marker signifikant herunterreguliert und die Expression von Tuj1 gesteigert. Die neuronal transdifferenzierten Zellen entwickelten zudem eine elektrische Erregbarkeit und konnten einzelne Aktionspotentiale erzeugen. In den MSCs und den neuronal differenzierten Zellen konnten verschiedene Isoformen der Gap Junction-bildenden Connexine nachgewiesen werden, wobei sich das Expressionsmuster durch die Differenzierung veränderte. Die in den MSCs starke Gap Junction-vermittelte Zellkopplung war in den transdifferenzierten Zellen fast vollständig unterdrückt, wobei die Aktivität von Gap Junction-Halbkanälen gesteigert war. Bei einer extrazellulären Zugabe des Neurotransmitters ATP reagierten sowohl die MSCs als auch die induzierten neuronalen Zellen mit intrazellulären Ca2+-Signalen, wobei die Reaktionsfähigkeit der MSCs dabei insgesamt höher war. Das Ca2+-Signal in den MSCs wurde vor allem durch die metabotropen P2Y-Rezeptoren und geringfügig durch die ionotropen P2X-Rezeptoren determiniert. Die Aktivität der P2Y-Rezeptoren wurde durch die neuronale Transdifferenzierung deutlich verringert. Die Analyse verschiedener Marker und die physiologische Charakterisierung der neuronal induzierten Zellen ließen auf eine induzierte Entwicklung unreifer Neuronen oder neuronaler Stamm-oder Vorläuferzellen schließen, die für die Verwendung als neuronales Zellmodell durchaus vielversprechend sind.Human neuronal cell material is highly needed for regenerative medicine as well as for physiological and pharmacological studies but has a very limited availability. Different approaches have been developed to transdifferentiate well-accessible and in vitro expandable cells into neuronal cells, thereby creating a neuronal cell model. To establish a sophisticated model it is imperative to know the evolved as well as the initial cells in detail and to be able to precisely assess the neuronal developmental state after differentiation. Ion channels as well as gap junctions are essential for integration, function and development of cells in tissue, as they strongly influence the inter- and intracellular signalling. Thus, they affect cell communication and differentiation as well, also in neuronal tissue. The aim of this dissertation was to examine the regulation of ion channels and gap junctions during cell development on the in vitro model of neuronal transdifferentiated bone marrow-derived mesenchymal stem/stromal cells (MSCs). Untreated MSCs express the characteristic MSC markers CD73, CD90, CD105 and CD166 as well as the neuronal marker Tuj1. During small molecule-induced neuronal transdifferentiation the MSC markers were significantly down-regulated while Tuj1 was up-regulated. The neuronal transdifferentiated cells developed an electrical excitability and were able to evoke single action potentials. In MSCs as well as in the differentiated cells different isoforms of the gap junction-building connexins could be detected, whereby the expression pattern changed during differentiation. The strong gap junction coupling of the MSCs was nearly completely suppressed in the transdifferentiated cells whereas these cells showed an enhanced gap junction hemichannel activity. Upon extracellular stimulation with the neurotransmitter ATP the MSCs as well as the induced neuronal cells reacted by provoking intracellular Ca2+ signals. In MSCs, the response capacity was higher than in the neuronal cells and the Ca2+ signal was mainly determined by metabotropic P2Y-receptors and only slightly by ionotropic P2X-receptors. The activity of the P2Y-receptors was significantly reduced during neuronal transdifferentiation of the MSCs. Analysing different markers and physiologically characterising the neuronal induced cells leads to the conclusion that immature neurons or neuronal stem and progenitor cells have been developed, which appear promising concerning their use as a neuronal cell model

Gap Junction Dependent Cell Communication Is Modulated During Transdifferentiation of Mesenchymal Stem/Stromal Cells Towards Neuron-Like Cells

In vitro transdifferentiation of patient-derived mesenchymal stem/stromal cells (MSCs) into neurons is of special interest for treatment of neurodegenerative diseases. Although there are encouraging studies, little is known about physiological modulations during this transdifferentiation process. Here, we focus on the analysis of gap junction dependent cell-cell communication and the expression pattern of gap junction-building connexins during small molecule-induced neuronal transdifferentiation of human bone marrow-derived MSCs. During this process, the MSC markers CD73, CD90, CD105, and CD166 were downregulated while the neuronal marker Tuj1 was upregulated. Moreover, the differentiation protocol used in the present study changed the cellular morphology and physiology. The MSCs evolved from a fibroblastoid morphology towards a neuronal shape with round cell bodies and neurite-like processes. Moreover, depolarization evoked action potentials in the transdifferentiated cells. MSCs expressed mRNAs encoding Cx43 and Cx45 as well as trace levels of Cx26, Cx37- and Cx40 and allowed transfer of microinjected Lucifer yellow. The differentiation protocol increased levels of Cx26 (mRNA and protein) and decreased Cx43 (mRNA and protein) while reducing the dye transfer. Cx36 mRNA was nearly undetectable in all cells regardless of treatment. Treatment of the cells with the gap junction coupling inhibitor carbenoxolone (CBX) only modestly altered connexin mRNA levels and had little effect on neuronal differentiation. Our study indicates that the small molecule-based differentiation protocol generates immature neuron-like cells from MSCs. This might be potentially interesting for elucidating physiological modifications and mechanisms in MSCs during the initial steps of differentiation towards a neuronal lineage

A parallelized, perfused 3D triculture model of leukemia for in vitro drug testing of chemotherapeutics

Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictive in vitro drug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creating in vivo-like conditions to enable in vitro drug testing. This system is a step towards drug testing in biomimetic, parallelized in vitro approaches, facilitating the discovery of new anti-leukemic drugs

Rebuilding the hematopoietic stem cell niche: Recent developments and future prospects

Hematopoietic stem cells (HSCs) have proven their clinical relevance in stem cell transplantation to cure patients with hematological disorders. Key to their regenerative potential is their natural microenvironment – their niche – in the bone marrow (BM). Developments in the field of biomaterials enable the recreation of such environments with increasing preciseness in the laboratory. Such artificial niches help to gain a fundamental understanding of the biophysical and biochemical processes underlying the interaction of HSCs with the materials in their environment and the disturbance of this interplay during diseases affecting the BM. Artificial niches also have the potential to multiply HSCs in vitro, to enable the targeted differentiation of HSCs into mature blood cells or to serve as drug-testing platforms. In this review, we will introduce the importance of artificial niches followed by the biology and biophysics of the natural archetype. We will outline how 2D biomaterials can be used to dissect the complexity of the natural niche into individual parameters for fundamental research and how 3D systems evolved from them. We will present commonly used biomaterials for HSC research and their applications. Finally, we will highlight two areas in the field of HSC research, which just started to unlock the possibilities provided by novel biomaterials, in vitro blood production and studying the pathophysiology of the niche in vitro. With these contents, the review aims to give a broad overview of the different biomaterials applied for HSC research and to discuss their potentials, challenges and future directions in the field. Statement of significance Hematopoietic stem cells (HSCs) are multipotent cells responsible for maintaining the turnover of all blood cells. They are routinely applied to treat patients with hematological diseases. This high clinical relevance explains the necessity of multiplication or differentiation of HSCs in the laboratory, which is hampered by the missing natural microenvironment – the so called niche. Biomaterials offer the possibility to mimic the niche and thus overcome this hurdle. The review introduces the HSC niche in the bone marrow and discusses the utility of biomaterials in creating artificial niches. It outlines how 2D systems evolved into sophisticated 3D platforms, which opened the gateway to applications such as, expansion of clinically relevant HSCs, in vitro blood production, studying niche pathologies and drug testing

Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels

Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue