A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in <i>Arabidopsis</i>

Lee, Yuree,Yoon, Taek Han,Lee, Jiyoun,Jeon, So Yeon,Lee, Jae Ho,Lee, Mi Kyoung,Chen, Huize,Yun, Ju,Oh, Se Yun,Wen, Xiaohong,Cho, Hui Kyung,Mang, Hyunggon,Kwak, June M. Elsevier 2018 Cell Vol.173 No.6

<P><B>Summary</B></P> <P>The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in <I>Arabidopsis</I> that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical “brace” to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating <I>de novo</I> specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two neighboring cell types coordinate cellular activities for organ separation </LI> <LI> A honeycomb structure of lignin acts as a “molecular brace” </LI> <LI> The lignin brace spatially restricts cell wall breakdown </LI> <LI> Lignin deposition ensures surface integrity of transdifferentiated epidermal cells </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Lee, Sung-Il,Ko, Youngkyung,Park, Jun-Beom D.A. Spandidos 2017 Experimental and therapeutic medicine Vol.13 No.5

<P>Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10<SUP>5</SUP> (group A) or 8×10<SUP>5</SUP> (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.</P>

Expression of TASK-1 channel in mouse Leydig cells

Min Seok Woo,Eun-Jin Kim,Anjas Happy Prayoga,Yangmi Kim,Dawon Kang 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Leydig cells, crucial for testosterone production, express ion channels like ANO1 that influence hormone secretion. This study investigates the expression and role of the Tandem of P domains in a weak inward rectifying K+ channel-related Acid-Sensitive K+-1 (TASK-1) channel in these cells, exploring its impact on testicular function and steroidogenesis. Methods: TASK-1 expression in Leydig cells was confirmed using immunostaining, while RT-PCR and Western Blot (WB) validated its expression in the TM3 Leydig cell line. The effect of a TASK-1 channel blocker on cell viability was assessed through live/dead staining and MTT assays. Additionally, the blocker’s effect on testosterone secretion was evaluated by measuring testosterone levels. Results: Immunohistochemical analysis revealed a predominant presence of TASK- 1, along with c-Kit and ANO-1, in Leydig cells adjacent to seminiferous tubules and also in Sertoli and spermatogenic cells. Expression levels of TASK-1 mRNA and protein were significantly higher in TM3 Leydig cells compared to TM4 Sertoli cells. In addition, blocking TASK-1 in TM3 cells with ML365 induced cell death but did not affect LHinduced testosterone secretion. Conclusions: These findings suggest that TASK-1 in Leydig cells is crucial for their viability and proliferation, highlighting its potential importance in testicular physiology.

Guided Cell Migration on Microtextured Substrates with Variable Local Density and Anisotropy

Kim, Deok-Ho,Seo, Chang-Ho,Han, Karam,Kwon, Keon Woo,Levchenko, Andre,Suh, Kahp-Yang WILEY-VCH Verlag 2009 Advanced functional materials Vol.19 No.10

<P>This work reports the design of and experimentation with a topographically patterned cell culture substrate of variable local density and anisotropy as a facile and efficient platform to guide the organization and migration of cells in spatially desirable patterns. Using UV-assisted capillary force lithography, an optically transparent microstructured layer of a UV curable poly(urethane acrylate) resin is fabricated and employed as a cell-culture substrate after coating with fibronectin. With variable local pattern density and anisotropy present in a single cell-culture substrate, the differential polarization of cell morphology and movement in a single experiment is quantitatively characterized. It is found that cell shape and velocity are exquisitely sensitive to variation in the local anisotropy of the two-dimensional rectangular lattice arrays, with cell elongation and speed decreasing on symmetric lattice patterns. It is also found that cells could integrate orthogonal spatial cues when determining the direction of cell orientation and movement. Furthermore, cells preferentially migrate toward the topographically denser areas from sparser ones. Consistent with these results, it is demonstrated that systematic variation of local densities of rectangular lattice arrays enable a planar assembly of cells into a specified location. It is envisioned that lithographically defined substrates of variable local density and anisotropy not only provide a new route to tailoring the cell-material interface but could serve as a template for advanced tissue engineering.</P> <B>Graphic Abstract</B> <P>Microtextured substrates with variable local density and anisotropy (see image) are designed to guide the organization and migration of fibroblasts in spatially desirable locations. Cell motility is sensitive to variation in the local density and anisotropy of rectangular lattice, with cell elongation and speed decreasing on symmetric lattice. Also, cells integrate orthogonal spatial cues when determining the direction of their orientation and movement. <img src='wiley_img/1616301X-2009-19-10-ADFM200801174-content.gif' alt='wiley_img/1616301X-2009-19-10-ADFM200801174-content'> </P>

Effective and Intact Cell Detachment from a Clinically Ubiquitous Culture Flask by Combining Ultrasonic Wave Exposure and Diluted Trypsin

Hanako Tauchi,Chikahiro Imashiro,Taiki Kuribara,Genichiro Fujii,Yuta Kurashina,Kiichiro Totani,KenjiroTakemura 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.3

Bioengineering research and applications are supported by cell culture technologies that produce a large number of homogeneous cells. However, trypsin used in the general culture procedure for cell detachment decreases cell activity and culture efficiency. Furthermore, manually conducted culture procedures, especially pipetting after trypsin treatment, can induce inhomogeneous mechanical stress in cells, which may influence cellular functions. Alternate detachment methods using specialized culture devices without trypsin and/or manual pipetting have been reported. However, conventional trypsinization is still widely used. Diluted trypsin increases culture efficiency. Therefore, we developed a cell-detaching method using diluted trypsin and ultrasonic vibration for cell detachment from ubiquitous culture vessels. To demonstrate our concept, we used a T25 flask. Vibration of the culture surface was excited by ultrasonic waves propagated from an ultrasonic transducer placed under the flask. Using the proposed method, cells were completely detached by diluted trypsin, whereas 8.6% of cells remained on the flask with manual pipetting. The viability and proliferation of cells detached by the proposed method were higher than those of cells detached by the conventional method, owing to the low concentration of trypsin. Furthermore, glucose consumption after detachment showed no abnormality, eliminating possible oncogenesis. Two membrane proteins were quantified immediately after detachment and at 24 h of culture, and there were no differences between the detachment methods. Thus, we conclude that our proposed method improves culture efficiency without any adverse effects and ensures homogeneous mechanical stress on cells.

온도감응성 마이크로캐리어를 이용한 세포 계대배양

양희석,김병수,서지혜 한국조직공학과 재생의학회 2009 조직공학과 재생의학 Vol.6 No.13

Microcarrier culture systems are frequently used for large-scale culture of anchorage-dependent mammalian cells. Mass production of a large amount of cells needs serial propagation in microcarrier culture. This requires enzyme treatment which could be a complex step in large-scale cell culture and cause cell damage. Thus, we have developed thermo-sensitive microcarriers by incorporating poly-N-isopropylacrylamide (pNIPAAm) onto Cytodex-3® (pNIPAAm-Cytodex). In this study, we tested the feasibility of serial propagation of Vero cells cultured on pNIPAAm-Cytodex microcarriers in three-dimensional bioreactors. Cells cultured on pNIPAAm-Cytodex were detached from pNIPAAm-Cytodex by a moderate change in temperature without enzyme treatment. This would simplify the complex serial propagation steps, avoid cell damage possibly caused by enzyme treatment, and enable one to easily harvest cells, because cells can be detached from microcarriers without enzyme treatment. Vero cells adhered, spread, and grew successfully on the thermo-sensitive microcarriers similarly to Cytodex microcarriers, and were harvested by simple temperature changes, thereby avoiding the use of proteolytic enzymes and laborious and time-consuming processes. Moreover, Vero cells detached by temperature change reattached and grew on pNIPAAm-Cytodex after scale-up in a manner similar to culture on Cytodex. The pNIPAAm-Cytodex microcarrier culture method would be useful for the mass production of biologicals through large-scale suspension culture of anchorage-dependent mammalian cells. Microcarrier culture systems are frequently used for large-scale culture of anchorage-dependent mammalian cells. Mass production of a large amount of cells needs serial propagation in microcarrier culture. This requires enzyme treatment which could be a complex step in large-scale cell culture and cause cell damage. Thus, we have developed thermo-sensitive microcarriers by incorporating poly-N-isopropylacrylamide (pNIPAAm) onto Cytodex-3® (pNIPAAm-Cytodex). In this study, we tested the feasibility of serial propagation of Vero cells cultured on pNIPAAm-Cytodex microcarriers in three-dimensional bioreactors. Cells cultured on pNIPAAm-Cytodex were detached from pNIPAAm-Cytodex by a moderate change in temperature without enzyme treatment. This would simplify the complex serial propagation steps, avoid cell damage possibly caused by enzyme treatment, and enable one to easily harvest cells, because cells can be detached from microcarriers without enzyme treatment. Vero cells adhered, spread, and grew successfully on the thermo-sensitive microcarriers similarly to Cytodex microcarriers, and were harvested by simple temperature changes, thereby avoiding the use of proteolytic enzymes and laborious and time-consuming processes. Moreover, Vero cells detached by temperature change reattached and grew on pNIPAAm-Cytodex after scale-up in a manner similar to culture on Cytodex. The pNIPAAm-Cytodex microcarrier culture method would be useful for the mass production of biologicals through large-scale suspension culture of anchorage-dependent mammalian cells.

Recent Advances in Cell surface Engineering Focused on Cell Therapy

김종철,태기융 대한화학회 2015 Bulletin of the Korean Chemical Society Vol.36 No.1

Revolutionary progress in stem cell research and the potential of stem cell-based therapeutics promote the translation of cell therapy. However, the therapeutic efficacy of cell therapy is still very low, mainly because of the insufficient homing and survival of the injected cells at the target site. Cell surface engineering is one of the solutions to increase the therapeutic efficacy of cell therapy. Cell surface engineering can modulate the cellular behavior for enhancing targeted delivery, cellular functions, organized structure, and immune evasion by chemical or physical modification of the cell surface. This review summarizes the recent studies in cell surface engineering focused on cell therapy.

Single-Cell RNA Sequencing Shows T-Cell Exhaustion Landscape in the Peripheral Blood of Patients with Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure

( Jia Yao ),( Yaqiu Ji ),( Tian Liu ),( Jinjia Bai ),( Han Wang ),( Ruoyu Yao ),( Juan Wang ),( Xiaoshuang Zhou ) 대한소화기기능성질환·운동학회 2024 Gut and Liver Vol.18 No.3

Background/Aims: The occurrence and development of hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is closely related to the immune pathway. We explored the heterogeneity of peripheral blood T cell subsets and the characteristics of exhausted T lymphocytes, in an attempt to identify potential therapeutic target molecules for immune dysfunction in ACLF patients. Methods: A total of 83,577 T cells from HBV-ACLF patients and healthy controls were screened for heterogeneity by single-cell RNA sequencing. In addition, exhausted T-lymphocyte subsets were screened to analyze their gene expression profiles, and their developmental trajectories were investigated. Subsequently, the expression of exhausted T cells and their capacity in secreting cytokines (interleukin 2, interferon γ, and tumor necrosis factor α) were validated by flow cytometry. Results: A total of eight stable clusters were identified, among which CD4⁺ TIGIT⁺ subset and CD8⁺ LAG-3⁺ subset, with high expression of exhaust genes, were significantly higher in the HBV-ACLF patients than in normal controls. As shown by pseudotime analysis, T cells experienced a transition from naïve T cells to effector T cells and then exhausted T cells. Flow cytometry confirmed that the CD4⁺TIGIT⁺ subset and CD8⁺LAG-3⁺ subset in the peripheral blood of the ACLF patients were significantly higher than those in the healthy controls. Moreover, in vitro cultured CD8⁺LAG-3⁺ T cells were significantly fewer capable of secreting cytokines than CD8⁺LAG-3^- subset. Conclusions: Peripheral blood T cells are heterogeneous in HBV-ACLF. The exhausted T cells markedly increase during the pathogenesis of ACLF, suggesting that T-cell exhaustion is involved in the immune dysfunction of HBV-ACLF patients. (Gut Liver 2024;18:520-530)

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation

Lee, Jae Won,An, Hyoseok,Lee, Kuen Yong Elsevier 2017 Colloids and surfaces. B, Biointerfaces Vol.155 No.-

<P><B>Abstract</B></P> <P>Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. <I>In vitro</I> changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> LRP5 peptide can be successfully conjugated to alginate hydrogel. </LI> <LI> LRP5-alginate gel induces N-cadherin-mediated cell-cell interaction. </LI> <LI> LRP5-alginate gel enhances stem cell aggregation and chondrogenic differentiation. </LI> <LI> Controlling cell-cell interaction is critical in tissue engineering applications. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Strategies for ex vivo Expansion and Cytotoxicity Enhancement of NK Cells Using Mechanical Stimuli

Myeongkwan SONG,Soonjo KWON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Adoptive cell therapy, also known as cellular immunotherapy, has recently attracted attention as a new cancer treatment method. Natural killer (NK) cells with the advantage of allogenic transfer are used as a cell therapy strategy along with Tumor-Infiltrating Lymphocyte (TIL), Engineered T Cell Receptor (TCR), and Chimeric Antigen Receptor (CAR) T Cell. However, for clinical applications, NK cells have problems with low ex vivo expansion and NK cell mediated cytotoxicity. To enhance NK cell expansion and cell-mediated cytotoxicity, we exposed NK cells to two types of mechanical stimuli: vibration and hyper-gravity. Vibration stimulus was calculated by sinusoidal acceleration, and hyper-gravity stimulus was calculated as g-force. CCK-8 assay was performed to determine whether vibration and hyper-gravity stimulus affect cell viability. There was no statistically significant decrease in cell viability at all intensities. We analyzed changes in the expression of cell-mediated cytotoxicity-related genes (granzyme B, perforin, TNF-α, and IFN-γ) and apoptosis related genes (BCL-2, BAX) following exposure of NK cells to two types of mechanical stimuli. In both mechanical stimuli, cytotoxicity enhancement and apoptosis inhibitory effects were observed. In addition, we are observing the changes in cell-mediated cytotoxicity compared to the control group by LDH assay in cancer cells in co-cultures. These findings provide useful insight for improving ex vivo expansion of NK cells and enhancing cell-mediated cytotoxicity.