scholarly journals The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment

2021 ◽  
Vol 22 (6) ◽  
pp. 2978
Author(s):  
Chie-Hong Wang ◽  
Chun-Hao Tsai ◽  
Tsung-Li Lin ◽  
Shih-Ping Liu
Keyword(s):  
Cell Proliferation ◽  
Chondrogenic Differentiation ◽  
Expression Profiles ◽  
Es Cells ◽  
3D Culture ◽  
Ips Cells ◽  
3D Cultures ◽  
Positive Effects ◽  
Cartilaginous Matrix ◽  
Culture Environment

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.

scholarly journals Zebrafish skeletal muscle cell cultures: Monolayer to three-dimensional tissue engineered collagen constructs

2020 ◽  
Author(s):  
K.K Vishnolia ◽  
N.R.W Martin ◽  
D.J Player ◽  
E Spikings ◽  
M.P Lewis
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Morphological Characteristics ◽  
Hypertrophic Growth ◽  
3D Cultures

AbstractZebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models.Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPC’s cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data.Collagen constructs seeded with zebrafish MPC’s were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer.In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

scholarly journals Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e83563 ◽  
Author(s):  
Taiki Hiyama ◽  
Nobuaki Ozeki ◽  
Makio Mogi ◽  
Hideyuki Yamaguchi ◽  
Rie Kawai ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Es Cells ◽  
Ips Cells ◽  
Matrix Metalloproteinase 3 ◽  
Proliferation Response

scholarly journals The circular RNA circSlc7a11 promotes bone cancer pain pathogenesis in rats by modulating LLC-WRC 256 cell proliferation and apoptosis

2021 ◽  
Author(s):  
Han-Wen Chen ◽  
Xiao-Xia Zhang ◽  
Zhu-Ding Peng ◽  
Zu-Min Xing ◽  
Yi-Wen Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cancer Pain ◽  
Expression Profiles ◽  
Bone Cancer ◽  
Circular Rna ◽  
Differentially Expressed ◽  
Bone Cancer Pain ◽  
Circular Rnas ◽  
Altered Expression ◽  
Proliferation And Apoptosis

AbstractTreatment of bone cancer pain (BCP) caused by bone metastasis in advanced cancers remains a challenge in clinical oncology, and the underlying mechanisms of BCP are poorly understood. This study aimed to investigate the pathogenic roles of circular RNAs (circRNAs) in regulating cancer cell proliferation and BCP development. Eight differentially expressed circRNAs in the rat spinal cord were validated by agarose gel electrophoresis and Sanger sequencing. Expression of circRNAs and mRNAs was detected by quantitative RT-PCR. MTS assay and flow cytometry were performed to analyze cell proliferation and apoptosis, respectively. Differentially expressed mRNA profiles were characterized by deep RNA sequencing, hierarchical clustering, and functional categorization. The interactions among circRNAs, microRNAs (miRNAs), and mRNAs were predicted using TargetScan. Additionally, western blot was performed to determine the protein levels of Pax8, Isg15, and Cxcl10. Multiple circRNAs were differentially expressed in the spinal cords of BCP model rats; of these, circSlc7a11 showed the greatest increase in expression. The overexpression of circSlc7a11 significantly promoted cell proliferation and repressed apoptosis of LLC-WRC 256 and UMR-106 cells, whereas circSlc7a11 silencing produced the opposite effects. Altered expression of circSlc7a11 also induced substantial changes in the mRNA expression profiles of LLC-WRC 256 cells; these changes were linked to multiple apoptotic processes and signaling pathways, such as the chemokine signaling pathway, and formed a complex circRNA/miRNA/mRNA network. Additionally, Pax8, Isg15, and Cxc110 protein level in LLC-WRC 256 cells was consistent with the mRNA results. The circRNA circSlc7a11 regulates rat BCP development by modulating LLC-WRC 256 cell proliferation and apoptosis through multiple-signaling mechanisms.

scholarly journals Type II Collagen-Conjugated Mesenchymal Stem Cells Micromass for Articular Tissue Targeting

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 880
Author(s):  
Shamsul Bin Sulaiman ◽  
Shiplu Roy Chowdhury ◽  
Mohd Fauzi Bin Mh Busra ◽  
Rizal Bin Abdul Rani ◽  
Nor Hamdan Bin Mohamad Yahaya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Targeted Delivery ◽  
Expression Profiles ◽  
3D Culture ◽  
Type Ii Collagen ◽  
Cartilage Defects ◽  
Target Tissue ◽  
Type Ii ◽  
Antibody Conjugation

The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis.

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury

2021 ◽  
pp. 039139882098680
Author(s):  
Xuefeng Zhang ◽  
Nan Wang ◽  
Yuhua Huang ◽  
Yan Li ◽  
Gang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Therapeutic Potential ◽  
Expression Profiles ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
3D Culture ◽  
Placental Mesenchymal Stem Cells ◽  
2D And 3D

Background: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). Methods: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. Results: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. Conclusion: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

scholarly journals YAP Regulates the Expression ofHoxa1andHoxc13in Mouse and Human Oral and Skin Epithelial Tissues

2015 ◽  
Vol 35 (8) ◽  
pp. 1449-1461 ◽  
Author(s):  
Ming Liu ◽  
Shuangyun Zhao ◽  
Qingjie Lin ◽  
Xiu-Ping Wang
Keyword(s):  
Cell Proliferation ◽  
Expression Profiles ◽  
Tumor Development ◽  
Gene Expression Profiles ◽  
Tooth Germ ◽  
Conditional Knockout ◽  
Hippo Signaling ◽  
Embryonic Mouse ◽  
Downstream Targets ◽  
Epithelial Tissues

Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues.Yapdeficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5)Yapconditional knockout andYAPtransgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR andin situhybridization. We found that YAP regulates the expression ofHoxa1andHoxc13in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested thatHoxa1andHoxc13are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulateHoxa1andHoxc13expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans.

scholarly journals Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

scholarly journals Overexpression of KIF2A is Suppressed by miR-206 and Associated with Poor Prognosis in Ovarian Cancer

2018 ◽  
Vol 50 (3) ◽  
pp. 810-822 ◽  
Author(s):  
Nan Sheng ◽  
Yun-Zhao Xu ◽  
Qing-Hua Xi ◽  
Hai-Yan Jiang ◽  
Chen-Yi Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Proliferation ◽  
Cancer Cell ◽  
Poor Prognosis ◽  
Ovarian Cancer Cell ◽  
Expression Profiles ◽  
Annexin V ◽  
Prognostic Indicator ◽  
Luciferase Reporter ◽  
Migration And Invasion

Background/Aims: This study aimed to investigate the expression and prognostic value of kinesin family member 2A (KIF2A) and the suppression effects of microRNA-206 (miR-206) on KIF2A in ovarian cancer. Methods: Ovarian cancer tissues from patients and ovarian cancer cell lines (A2780 and SKOV3) were used in this study. miR-206 mimics and control were transiently transfected into cells. RT-qPCR was performed to detect KIF2A mRNA and miR-206 expression levels, Western blot was performed to detect KIF2A protein levels, Dual-Luciferase Reporter Assay was used to examine the inhibition effects of miR-206 on KIF2A mRNA, immunohistochemical staining was used to examine the expression of KIF2A in tissue sections. CCK-8, transwell and Annexin-V-FITC/Propidium Iodide staining with flow cytometry were used to detect the cell proliferation, migration/invasion, and apoptosis respectively. Results: Our study explored the expression profiles of KIF2A and miR-206 in the patients with ovarian cancer. We found that overexpression of KIF2A was associated with a poor prognosis in ovarian cancer. We also found that KIF2A mRNA contains two target sites for miR-206 binding and confirmed that miR-206 directly suppresses KIF2A; inhibits ovarian cancer cell proliferation, migration, and invasion; and induces apoptosis. Conclusion: The results suggest KIF2A could serve a valuable prognostic indicator in ovarian cancer and provide a rationale for treatment of ovarian cancer by targeting KIF2A via miR-206.

scholarly journals SP1-Mediated Upregulation of Long Noncoding RNA ZFAS1 Involved in Non-syndromic Cleft Lip and Palate via Inactivating WNT/β-Catenin Signaling Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Shiyu Chen ◽  
Zhonglin Jia ◽  
Ming Cai ◽  
Mujie Ye ◽  
Dandan Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Noncoding Rna ◽  
Chondrogenic Differentiation ◽  
Cleft Lip ◽  
Cleft Lip And Palate ◽  
Tissue Sample ◽  
Human Umbilical Cord

Non-syndromic cleft lip and palate (NSCLP) is one of the most common congenital malformations with multifactorial etiology. Although long non-coding RNAs (lncRNAs) have been implicated in the development of lip and palate, their roles in NSCLP are not fully elucidated. This study aimed to investigate how dysregulated lncRNAs contribute to NSCLP. Using lncRNA sequencing, bioinformatics analysis, and clinical tissue sample detection, we identified that lncRNA ZFAS1 was significantly upregulated in NSCLP. The upregulation of ZFAS1 mediated by SP1 transcription factor (SP1) inhibited expression levels of Wnt family member 4 (WNT4) through the binding with CCCTC-binding factor (CTCF), subsequently inactivating the WNT/β-catenin signaling pathway, which has been reported to play a significant role on the development of lip and palate. Moreover, in vitro, the overexpression of ZFAS1 inhibited cell proliferation and migration in human oral keratinocytes and human umbilical cord mesenchymal stem cells (HUC-MSCs) and also repressed chondrogenic differentiation of HUC-MSCs. In vivo, ZFAS1 suppressed cell proliferation and numbers of chondrocyte in the zebrafish ethmoid plate. In summary, these results indicated that ZFAS1 may be involved in NSCLP by affecting cell proliferation, migration, and chondrogenic differentiation through inactivating the WNT/β-catenin signaling pathway.

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