TY - JOUR A1 - Deng, Zijun A1 - Zou, Jie A1 - Wang, Weiwei A1 - Nie, Yan A1 - Tung, Wing-Tai A1 - Ma, Nan A1 - Lendlein, Andreas T1 - Dedifferentiation of mature adipocytes with periodic exposure to cold JF - Clinical hemorheology and microcirculation : blood flow and vessels N2 - Lipid-containing adipocytes can dedifferentiate into fibroblast-like cells under appropriate culture conditions, which are known as dedifferentiated fat (DFAT) cells. However, the relative low dedifferentiation efficiency with the established protocols limit their widespread applications. In this study, we found that adipocyte dedifferentiation could be promoted via periodic exposure to cold (10 degrees C) in vitro. The lipid droplets in mature adipocytes were reduced by culturing the cells in periodic cooling/heating cycles (10-37 degrees C) for one week. The periodic temperature change led to the down-regulation of the adipogenic genes (FABP4, Leptin) and up-regulation of the mitochondrial uncoupling related genes (UCP1, PGC-1 alpha, and PRDM16). In addition, the enhanced expression of the cell proliferation marker Ki67 was observed in the dedifferentiated fibroblast-like cells after periodic exposure to cold, as compared to the cells cultured in 37 degrees C. Our in vitro model provides a simple and effective approach to promote lipolysis and can be used to improve the dedifferentiation efficiency of adipocytes towards multipotent DFAT cells. KW - Adipocyte KW - dedifferentiation KW - cold KW - lipid Y1 - 2019 U6 - https://doi.org/10.3233/CH-199005 SN - 1386-0291 SN - 1875-8622 VL - 71 IS - 4 SP - 415 EP - 424 PB - IOS Press CY - Amsterdam ER - TY - JOUR A1 - Nie, Yan A1 - Wang, Weiwei A1 - Xu, Xun A1 - Zou, Jie A1 - Bhuvanesh, Thanga A1 - Schulz, Burkhard A1 - Ma, Nan A1 - Lendlein, Andreas T1 - Enhancement of human induced pluripotent stem cells adhesion through multilayer laminin coating JF - Clinical hemorheology and microcirculation : blood flow and vessels N2 - Bioengineered cell substrates are a highly promising tool to govern the differentiation of stem cells in vitro and to modulate the cellular behavior in vivo. While this technology works fine for adult stem cells, the cultivation of human induced pluripotent stem cells (hiPSCs) is challenging as these cells typically show poor attachment on the bioengineered substrates, which among other effects causes substantial cell death. Thus, very limited types of surfaces have been demonstrated suitable for hiPSC cultures. The multilayer coating approach that renders the surface with diverse chemical compositions, architectures, and functions can be used to improve the adhesion of hiPSCs on the bioengineered substrates. We hypothesized that a multilayer formation based on the attraction of molecules with opposite charges could functionalize the polystyrene (PS) substrates to improve the adhesion of hiPSCs. Polymeric substrates were stepwise coated, first with dopamine to form a polydopamine (PDA) layer, second with polylysine and last with Laminin-521. The multilayer formation resulted in the variation of hydrophilicity and chemical functionality of the surfaces. Hydrophilicity was detected using captive bubble method and the amount of primary and secondary amines on the surface was quantified by fluorescent staining. The PDA layer effectively immobilized the upper layers and thereby improved the attachment of hiPSCs. Cell adhesion was enhanced on the surfaces coated with multilayers, as compared to those without PDA and/or polylysine. Moreover, hiPSCs spread well over this multilayer laminin substrate. These cells maintained their proliferation capacity and differentiation potential. The multilayer coating strategy is a promising attempt for engineering polymer-based substrates for the cultivation of hiPSCs and of interest for expanding the application scope of hiPSCs. KW - Polymeric substrate KW - surface coating KW - induced pluripotent stem cells KW - cell adhesion Y1 - 2019 U6 - https://doi.org/10.3233/CH-189318 SN - 1386-0291 SN - 1875-8622 VL - 70 IS - 4 SP - 531 EP - 542 PB - IOS Press CY - Amsterdam ER - TY - THES A1 - Nie, Yan T1 - Modulating keratinocyte and induced pluripotent stem cell behavior by microenvironment design or temperature control N2 - Under the in vivo condition, a cell is continually interacting with its surrounding microenvironment, which is composed of its neighboring cells and the extracellular matrix (ECM). These components generate and transmit the microenvironmental signals to regulate the fate and function of the target cells. Except the signals from the microenvironment, stimuli from the ambient environment, such as temperature changes, also play an important in modulating the cell behaviors, which are considered as regulators from the macroenvironment. In this regard, recapitulation of these environmental factors to steer cell function will be of crucial importance for therapeutic purposes and tissue regeneration. Although the role of a variety of environmental factors has been evaluated, it is still challenging to identify and provide the appropriate factors, which are required for optimizing the survival of cells and for ensuring effective cell functions. Thus, in vitro recreating the environmental factors that are present in the extracellular environment would help to understand the mechanism of how cells sense and process those environmental signals. In this context, this thesis is aimed to harness these environmental parameters to guide cell responses. Here, human induced pluripotent stem cells (hiPSCs) and human keratinocytes (KTCs), HaCaT cells, were used to investigate the impact of signals from the microenvironment or stimuli from the macroenvironment. Firstly, polydopamine (PDA) or chitosan (CS) modifications were applied to generate different substrate surfaces for hiPSCs and KTCs (Chapter 4 to Chapter 6). Our results showed that the PDA modification was efficient to increase the cell-substrate adhesion and consequently promoted cell spreading. While CS modification was able to decrease the cell-substrate adhesion and enhance the cell-cell interaction, which enabled the morphology shift from monolayered cells to multicellular spheroids. The quantitative result was acquired using the atomic force microscopy (AFM)-based single-cell force spectroscopy. The balance between the cell-substrate and cell-cell adhesion yielded a net force, which determined the preference of the cell to adhere to its neighboring cells or to the substrate. The difference in the adhesive behaviors further affected the cellular function, such as the proliferation and differentiation potential of both hiPSCs and HaCaT cells. Next, the cyclic temperature changes (ΔT) were selected here to study the influence of macroenvironmental stimuli on hiPSCs and KTCs (Chapter 7 and Chapter 8). The macroenvironmental temperature ranging from 10.0 ± 0.1 °C to 37.0 ± 0.1 °C was achieved using a thermal chamber equipped with a temperature controller. This temperature range was selected to explore the responses of hiPSCs to the extreme environments, while a temperature variation between 25.0 ± 0.1 °C and 37.0 ± 0.1 °C was applied to mimic the ambient temperature variations experienced by the skin epithelial KTCs. The ΔT led to cell stiffening in both hiPSCs and HaCaT cells in a cytoskeleton-dependent manner, which was measured by AFM. Specifically, in hiPSCs, the cell stiffening was resulted from the rearrangement of the actin skeleton; in HaCaT cells, was due to the difference of the Keratin (KRT) filaments. Except for inducing cell hardening, ΔT also caused differences in the protein expression profiles in hiPSCs or HaCaT cells, compared to those without ΔT treatment, which might be attributed to the alterations in their cytoskeleton structures. To sum up, the results of the thesis demonstrated how individual factors from the micro-/macro-environment can be harnessed to modulate the behaviors of hiPSCs and HaCaT cells. Engineering the microenvironmental cues using surface modification and exploiting the macroenvironmental stimuli through temperature control were identified as precise and potent approaches to steer hiPSC and HaCaT cell behaviors. The application of AFM served as a non-invasive and real-time monitoring platform to trace the change in cell topography and mechanics induced by the environmental signals, which provide novel insights into the cell-environment interactions. N2 - In vivo interagiert eine Zelle ständig mit ihrer Mikroumgebung, die aus ihren Nachbarzellen und der extrazellulären Matrix (ECM) besteht. Diese Komponenten erzeugen und übertragen die Mikroumgebungssignale, um das Schicksal und die Funktion der Zielzellen zu regulieren. Außer den Signalen aus der Mikroumgebung spielen auch Reize aus der Makroumgebung, wie Temperaturänderungen, eine wichtige Rolle bei der Modulation des Zellverhaltens. In dieser Hinsicht ist es wichtig, diese Umweltfaktoren zur Steuerung der Zellfunktion für therapeutische Zwecke und die Geweberegeneration zu rekapitulieren. Es stellt sich immer noch eine Herausforderung, geeignete Faktoren zu identifizieren und bereitzustellen, die zur Optimierung des Überlebens von Zellen und zur Sicherstellung effektiver Zellfunktionen erforderlich sind. Daher würde die in vitro-Nachbildung der Umweltfaktoren helfen, den Mechanismus zu verstehen, wie Zellen diese Umweltsignale wahrnehmen und verarbeiten. In diesem Zusammenhang zielt diese Dissertation darauf ab, diese externen Parameter zu nutzen, um Zellantworten zu steuern. Hier wurden humaninduzierte pluripotente Stammzellen (hiPSCs) und humane Keratinozyten (KTCs) wie HaCaT-Zellen verwendet, um den Einfluss von Signalen aus der Mikroumgebung oder Stimuli aus der Makroumgebung zu untersuchen. Zunächst wurden Modifikationen mit Polydopamin (PDA) oder Chitosan (CS) angewendet, um unterschiedliche Substratoberflächen für hiPSCs und KTCs zu erzeugen (Kapitel 4 bis Kapitel 6). Unsere Ergebnisse zeigten, dass die PDA-Modifikation die Zell-Substrat-Adhäsion erhöhte und folglich die Zellausbreitung förderte. Während die CS-Modifikation die Zell-Substrat-Adhäsion verringerte und die Zell-Zell-Interaktion verstärkte, verändeite sich die Morphologie von einschichtigen Zellen zu mehrzelligen Sphäroiden. Das quantitative Ergebnis wurde mittels Rasterkraftmikroskopie (AFM)-basierter Einzelzellkraftspektroskopie gewonnen. Das Gleichgewicht zwischen Zell-Substrat und Zell-Zell-Adhäsion ergab eine Nettokraft, die die Präferenz der Zelle bestimmt, an ihren Nachbarzellen oder am Substrat zu haften. Der Unterschied im Adhäsionsverhalten beeinflusste außerdem die Zellfunktion, wie das Proliferations- und Differenzierungspotential von hiPSCs und HaCaT-Zellen. Als nächstes wurden hier zyklische Temperaturänderungen (ΔT) ausgewählt, um den Einfluss von Stimuli aus der Makroumgebung auf hiPSCs und KTCs zu untersuchen (Kapitel 7 und Kapitel 8). Die Makroumgebungstemperatur im Bereich von 10,0 ± 0,1 °C bis 37,0 ± 0,1 °C wurde unter Verwendung einer mit einem Temperaturregler ausgestatteten Wärmekammer erreicht. Dieser Temperaturbereich wurde gewählt, um die Reaktion von hiPSCs auf extreme Umgebungen zu untersuchen, während eine Temperaturvariation zwischen 25,0 ± 0,1 ° C und 37,0 ± 0,1 ° C angewendet wurde, um die Temperaturänderungen nachzuahmen, die die Epithelzellen erfahren. Das ΔT führte zytoskelettabhängig zu einer Zellversteifung sowohl in hiPSCs als auch in HaCaT-Zellen, die mittels AFM gemessen wurde. Insbesondere bei hiPSCs resultierte die Zellversteifung aus der Neuordnung des Aktinskeletts; in HaCaT-Zellen, war auf den Unterschied der Keratin (KRT)-Filamente zurückzuführen. Abgesehen von der festgestellten Erhärtung der Zellen verursachte ΔT auch Unterschiede in den Proteinexpressionsprofilen in hiPSCs oder HaCaT-Zellen im Vergleich zu denen ohne ΔT-Behandlung. Dies könnte auf die Veränderungen in ihren Zytoskelettstrukturen zurückgeführt werden. Zusammenfassend zeigten die Ergebnisse, wie die drei Faktoren (PDA/CS-Modifikation und ΔT) aus der Mikro-/Makroumgebung genutzt werden können, um das Verhalten von hiPSCs und HaCaT-Zellen zu modulieren. Als präzise und wirksame Ansätze zur Steuerung des hiPSC- und HaCaT-Zellen-Verhaltens wurde das Engineering der Mikroumgebungssignale durch Oberflächenmodifikation und die Nutzung der Makroumgebungsreize durch Temperaturkontrolle identifiziert. Die Anwendung von AFM diente als nicht-invasive und Echtzeit-Überwachungsplattform, um die durch die Umweltsignale induzierten Veränderungen der Zelltopographie und -mechanik zu verfolgen, die neue Einblicke in die Zell-Umwelt-Interaktionen liefern. KW - human induced pluripotent stem cells KW - human keratinocytes KW - cell-environment interactions KW - surface modification KW - temperature variations KW - humaninduzierte pluripotente Stammzellen KW - humane Keratinozyten KW - Zell-Umwelt-Interaktionen KW - Oberflächenmodifikation KW - Temperaturänderungen Y1 - 2022 ER - TY - JOUR A1 - Xu, Xun A1 - Nie, Yan A1 - Wang, Weiwei A1 - Ma, Nan A1 - Lendlein, Andreas T1 - Periodic thermomechanical modulation of toll-like receptor expression and distribution in mesenchymal stromal cells JF - MRS communications / a publication of the Materials Research Society N2 - Toll-like receptor (TLR) can trigger an immune response against virus including SARS-CoV-2. TLR expression/distribution is varying in mesenchymal stromal cells (MSCs) depending on their culture environments. Here, to explore the effect of periodic thermomechanical cues on TLRs, thermally controlled shape-memory polymer sheets with programmable actuation capacity were created. The proportion of MSCs expressing SARS-CoV-2-associated TLRs was increased upon stimulation. The TLR4/7 colocalization was promoted and retained in the endoplasmic reticula. The TLR redistribution was driven by myosin-mediated F-actin assembly. These results highlight the potential of boosting the immunity for combating COVID-19 via thermomechanical preconditioning of MSCs. KW - Actuation KW - Antiviral KW - Biomaterial KW - COVID-19 KW - Shape memory Y1 - 2021 U6 - https://doi.org/10.1557/s43579-021-00049-5 SN - 2159-6859 SN - 2159-6867 VL - 11 IS - 4 SP - 425 EP - 431 PB - Springer CY - Berlin ER - TY - JOUR A1 - Xu, Xun A1 - Nie, Yan A1 - Wang, Weiwei A1 - Ullah, Imran A1 - Tung, Wing Tai A1 - Ma, Nan A1 - Lendlein, Andreas T1 - Generation of 2.5D lung bud organoids from human induced pluripotent stem cells JF - Clinical hemorheology and microcirculation : blood flow and vessels N2 - Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70% confluence (SC 70% hom) or a clump seeding group with heterogeneously distributed cells at 90% confluence (CL 90% het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine. KW - lung organoid KW - human induced pluripotent stem cell KW - cell culture Y1 - 2021 U6 - https://doi.org/10.3233/CH-219111 SN - 1386-0291 SN - 1875-8622 VL - 79 IS - 1 SP - 217 EP - 230 PB - IOS Press CY - Amsterdam ER - TY - JOUR A1 - Nie, Yan A1 - Wang, Weiwei A1 - Xu, Xun A1 - Ma, Nan A1 - Lendlein, Andreas T1 - The response of human induced pluripotent stem cells to cyclic temperature changes explored by BIO-AFM JF - MRS advances : a journal of the Materials Research Society (MRS) N2 - Human induced pluripotent stem cells (hiPSCs) are highly sensitive to extrinsic physical and biochemical signals from their extracellular microenvironments. In this study, we analyzed the effect of cyclic temperature changes on hiPSCs behaviors, especially by means of scanning force microscopy (BIO-AFM). The alternation in cellular mechanics, as well as the secretion and pattern of deposition of extracellular matrix (ECM) protein in hiPSCs were evaluated. The arrangement of the actin cytoskeleton changed with the variation of the temperature. The rearranged cytoskeleton architecture led to the subsequent changes in cell mechanics (Young's modulus of hiPSCs). With the exposure to the cyclic cold stimuli, an increase in the average surface roughness (Ra) and roughness mean square (RMS) was detected. This observation might be at least in part due to the upregulated secretion of Laminin alpha 5 during repeated temporary cooling. The expression of pluripotent markers, NANOG and SOX2, was not impaired in hiPSCs, when exposed to the cyclic cold stimuli for 24 h. Our findings provide an insight into the effect of temperature on the hiPSC behaviors, which may contribute to a better understanding of the application of locally controlled therapeutic hypothermia. Y1 - 2021 U6 - https://doi.org/10.1557/s43580-021-00110-4 SN - 2059-8521 VL - 6 IS - 31 SP - 745 EP - 749 PB - Springer CY - Cham ER -