Limited Potential or Unfavorable Manipulations? Strategies Toward Efficient Mesenchymal Stem/Stromal Cell Applications

Despite almost 50 years of research and over 20 years of preclinical and clinical studies, the question of curative potential of mesenchymal stem/stromal cells (MSCs) is still widely discussed in the scientific community. Non-reproducible treatment outcomes or even absence of treatment effects in comparison to control groups challenges the potential of these cells for routine application both in tissue engineering and in regenerative medicine. One of the reasons of such outcomes is non-standardized and often disadvantageous ex vivo manipulation of MSCs prior therapy. In most cases, clinically relevant cell numbers for MSC-based therapies can be only obtained by in vitro expansion of isolated cells. In this mini review, we will discuss point by point possible pitfalls in the production of human MSCs for cell therapies, without consideration of material-based applications. Starting with cell source, choice of donor and recipient, as well as isolation methods, we will then discuss existing expansion protocols (two-/three-dimensional cultivation, basal medium, medium supplements, static/dynamic conditions, and hypoxic/normoxic conditions) and influence of these strategies on the cell functionality after implantation. The role of potency assays will also be addressed. The final aim of this mini review is to illustrate the heterogeneity of current strategies for gaining MSCs for clinical applications with their strengths and weaknesses. Only a careful consideration and standardization of all pretreatment processes/methods for the different applications of MSCs will ensure robust and reproducible performance of these cell populations in the different experimental and clinical settings

The extracellular matrix of hematopoietic stem cell niches

Hematopoietic stem cells (HSCs) are the life-long source of all types of blood cells. Their function is controlled by their direct microenvironment, the HSC niche in the bone marrow. Although the importance of the extracellular matrix (ECM) in the niche by orchestrating niche architecture and cellular function is widely acknowledged, it is still underexplored. In this review, we provide a comprehensive overview of the ECM in HSC niches. For this purpose, we first briefly outline HSC niche biology and then review the role of the different classes of ECM molecules in the niche one by one and how they are perceived by cells. Matrix remodeling and the emerging importance of biophysics in HSC niche function are discussed. Finally, the application of the current knowledge of ECM in the niche in form of artificial HSC niches for HSC expansion or targeted differentiation as well as drug testing is reviewed. © 2021 The Author(s

Untersuchung zum Gesundheitsverhalten von Medizinstudierenden

Stichprobe: Daten von 258 Studierenden aus dem ersten Studienjahr der Humanmedizin fließen in die Auswertung mit ein. Der Rücklauf beträgt 73,11 %. 61,48 % der Befragten sind weiblichen Geschlechts, 38,52 % sind Männer. Das Durchschnittsalter beträgt 21,57 Jahre. Diese demographischen Variablen sind repräsentativ für das gesamte Semester. Methoden: Die Erhebung erfolgt mittels eines fünfseitigen Fragebogens. Darin werden Fragen zum Alkoholkonsum, Rauchen, Obst- und Gemüsekonsum, zur körperlichen Aktivität und bezüglich des Gewichts zur Berechnung des Body-Mass-Index gestellt. Die Befragten werden entsprechend ihrer Angaben zur Häufigkeit von Binge-Episoden(fünf alkoholische Getränke für Männer und vier alkoholische Getränke für Frauen bei einer Gelegenheit) innerhalb der vorangegangenen 30 Tage einer von vier Binge-Gruppen zugeordnet: Nicht-Binger: keine Binge-Episode; Moderate-Binger: 1 bis 2 Binge-Episoden, Binger: 2 bis 5 Episoden; Häufige-Binger: mindestens 6 Binge-Episoden. Die Datenauswertung erfolgt mit SPSS 12.0. Nach einer explorativen Datenanalyse wird mit Hilfe einer binär logistischen Regression nach Risikofaktoren für übermäßigen Alkoholkonsum gesucht. Ergebnisse: 50 % der Befragten geben mindestens eine Binge-Episode innerhalb des letzten Monats an. Männer berichten davon wesentlich häufiger als Frauen. Frauen zeigen durchschnittlich 2,16 Risikofaktoren, Männer 2,57 Risikofaktoren. Folgende Prädiktoren konnten zur Unterscheidung zwischen den Binge-Gruppen als signifikant bestätigt werden: „Geschlecht“, „Rauchen“ und „BMI“. Es lag eine geringe Bereitschaft vor, Gewohnheiten bezüglich des Alkoholkonsum und der Ernährung zu ändern. Eine höhere Motivation zur Verhaltensänderung wurde bei den Risikofaktoren „Rauchen“ und „ungenügende sportliche Aktivität“ festgestellt. Die von den Kommilitonen konsumierte Alkoholmenge wurde deutlich überschätzt. Fazit: Aufgrund der zukünftigen Rolle der Medizinstudierenden im Gesundheitssystem sollte ihr Gesundheitsverhalten gezielt gefördert werden. In Orientierung am Transtheoretischen Modell könnten Veränderungsstrategien eingesetzt werden

Nitric Oxide in the Control of the in vitro Proliferation and Differentiation of Human Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation is the best-studied cellular therapy and successful in vitro control of HSPCs has wide clinical implications. Nitric oxide (NO) is a central signaling molecule in vivo and has been implicated in HSPC mobilization to the blood stream in mice. The influence of NO on HSPC behavior in vitro is, however, largely obscure due to the variety of employed cell types, NO administration systems, and used concentration ranges in the literature. Additionally, most studies are based on murine cells, which do not necessarily mimic human HSPC behavior. Thus, the aim of the present study was the systematic, concentration-dependent evaluation of NO-mediated effects on human HSPC behavior in vitro. By culture in the presence of the long-term NO donor diethylenetriamine/nitric oxide adduct (DETA/NO) in a nontoxic concentration window, a biphasic role of NO in the regulation of HSPC behavior was identified: Low DETA/NO concentrations activated classical NO signaling, identified via increased intracellular cyclic guanosine monophosphate (cGMP) levels and proteinkinases G (PKG)-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and mediated a pro-proliferative response of HSPCs. In contrast, elevated NO concentrations slowed cell proliferation and induced HSPC differentiation. At high concentrations, s-nitrosylation levels were elevated, and myeloid differentiation was increased at the expense of lymphoid progenitors. Together, these findings hint at a central role of NO in regulating human HSPC behavior and stress the importance and the potential of the use of adequate NO concentrations for in vitro cultures of HSPCs, with possible implications for clinical application of in vitro expanded or differentiated HSPCs for cellular therapies

Finding new edges:systems approaches to MTOR signaling

Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult - if not impossible - to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease

Differential control of ageing and lifespan by isoforms and splice variants across the mTOR network

Ageing can be defined as the gradual deterioration of physiological functions, increasing the incidence of age-related disorders and the probability of death. Therefore, the term ageing not only reflects the lifespan of an organism but also refers to progressive functional impairment and disease. The nutrient-sensing kinase mTOR (mammalian target of rapamycin) is a major determinant of ageing. mTOR promotes cell growth and controls central metabolic pathways including protein biosynthesis, autophagy and glucose and lipid homoeostasis. The concept that mTOR has a crucial role in ageing is supported by numerous reports on the lifespan-prolonging effects of the mTOR inhibitor rapamycin in invertebrate and vertebrate model organisms. Dietary restriction increases lifespan and delays ageing phenotypes as well and mTOR has been assigned a major role in this process. This may suggest a causal relationship between the lifespan of an organism and its metabolic phenotype. More than 25 years after mTOR's discovery, a wealth of metabolic and ageing-related effects have been reported. In this review, we cover the current view on the contribution of the different elements of the mTOR signalling network to lifespan and age-related metabolic impairment. We specifically focus on distinct roles of isoforms and splice variants across the mTOR network. The comprehensive analysis of mouse knockout studies targeting these variants does not support a tight correlation between lifespan prolongation and improved metabolic phenotypes and questions the strict causal relationship between them

Potential of electrospun cationic BSA fibers to guide osteogenic MSC differentiation via surface charge and fibrous topography

Large or complex bone fractures often need clinical treatments for sufficient bone repair. New treatment strategies have pursued the idea of using mesenchymal stromal cells (MSCs) in combination with osteoinductive materials to guide differentiation of MSCs into bone cells ensuring complete bone regeneration. To overcome the challenge of developing such materials, fundamental studies are needed to analyze and understand the MSC behavior on modified surfaces of applicable materials for bone healing. For this purpose, we developed a fibrous scaffold resembling the bone/bone marrow extracellular matrix (ECM) based on protein without addition of synthetic polymers. With this biomimetic in vitro model we identified the fibrous structure as well as the charge of the material to be responsible for its effects on MSC differentiation. Positive charge was introduced via cationization that additionally supported the stability of the scaffold in cell culture, and acted as nucleation point for mineralization during osteogenesis. Furthermore, we revealed enhanced focal adhesion formation and osteogenic differentiation of MSCs cultured on positively charged protein fibers. This pure protein-based and chemically modifiable, fibrous ECM model allows the investigation of MSC behavior on biomimetic materials to unfold new vistas how to direct cells’ differentiation for the development of new bone regenerating strategies

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications

The relevance of cellular in vitro models highly depends on their ability to mimic the physiological environment of the respective tissue or cell niche. Static culture conditions are often unsuitable, especially for endothelial models, since they completely neglect the physiological surface shear stress and corresponding reactions of endothelial cells (ECs) such as alignment in the direction of flow. Furthermore, formation and maturation of the glycocalyx, the essential polysaccharide layer covering all endothelial surfaces and regulating diverse processes, is highly dependent on applied fluid flow. This fragile but utterly important macromolecular layer is hard to analyze, its importance is often underestimated and accordingly neglected in many endothelial models. Therefore, we exposed human umbilical vein ECs (HUVECs) and human induced pluripotent stem cell-derived ECs (iPSC-ECs) as two relevant EC models in a side-by-side comparison to static and physiological dynamic (6.6 dyn cm−2) culture conditions. Both cell types demonstrated an elongation and alignment along the flow direction, some distinct changes in glycocalyx composition on the surface regarding the main glycosaminoglycan components heparan sulfate, chondroitin sulfate or hyaluronic acid as well as an increased and thereby improved glycocalyx thickness and functionality when cultured under homogeneous fluid flow. Thus, we were able to demonstrate the maturity of the employed iPSC-EC model regarding its ability to sense fluid flow along with the general importance of physiological shear stress for glycocalyx formation. Additionally, we investigated EC monolayer integrity with and without application of surface shear stress, revealing a comparable existence of tight junctions for all conditions and a reorganization of the cytoskeleton upon dynamic culture leading to an increased formation of focal adhesions. We then fabricated cell sheets of EC monolayers after static and dynamic culture via non-enzymatic detachment using thermoresponsive polymer coatings as culture substrates. In a first proof-of-concept we were able to transfer an aligned iPSC-EC sheet to a 3D-printed scaffold thereby making a step in the direction of vascular modelling. We envision these results to be a valuable contribution to improvements of in vitro endothelial models and vascular engineering in the future

Cardiac PI3K p110 alpha attenuation delays aging and extends lifespan

Phosphoinositide 3-kinase (PI3K) is a key component of the insulin signaling pathway that controls cellular me-tabolism and growth. Loss-of-function mutations in PI3K signaling and other downstream effectors of the insulin signaling pathway extend the lifespan of various model organisms. However, the pro-longevity effect appears to be sex-specific and young mice with reduced PI3K signaling have increased risk of cardiac disease. Hence, it remains elusive as to whether PI3K inhibition is a valid strategy to delay aging and extend healthspan in humans. We recently demonstrated that reduced PI3K activity in cardiomyocytes delays cardiac growth, causing subnormal contractility and cardiopulmonary functional capacity, as well as increased risk of mortality at young age. In stark contrast, in aged mice, experi-mental attenuation of PI3K signaling reduced the age-dependent decline in cardiac function and extended maximal lifespan, suggesting a biphasic effect of PI3K on cardiac health and survival. The cardiac anti-aging effects of reduced PI3K activity coincided with enhanced oxida-tive phosphorylation and required increased autophagic flux. In humans, explanted failing hearts showed in-creased PI3K signaling, as indicated by increased phos-phorylation of the serine/threonine-protein kinase AKT. Hence, late-life cardiac-specific targeting of PI3K might have a therapeutic potential in cardiac aging and related diseases

3D models of the hematopoietic stem cell niche under steady-state and active conditions

Hematopoietic stem cells (HSCs) in the bone marrow are able to differentiate into all types of blood cells and supply the organism each day with billions of fresh cells. They are applied to cure hematological diseases such as leukemia. The clinical need for HSCs is high and there is a demand for being able to control and multiply HSCs in vitro. The hematopoietic system is highly proliferative and thus sensitive to anti-proliferative drugs such as chemotherapeutics. For many of these drugs suppression of the hematopoietic system is the dose-limiting toxicity. Therefore, biomimetic 3D models of the HSC niche that allow to control HSC behavior in vitro and to test drugs in a human setting are relevant for the clinics and pharmacology. Here, we describe a perfused 3D bone marrow analog that allows mimicking the HSC niche under steady-state and activated conditions that favor either HSC maintenance or differentiation, respectively, and allows for drug testing