scholarly journals Μελέτη έκφρασης γονιδίων αρχέγονων βλαστικών κυττάρων σε τροφοβλαστικά κύτταρα

2016 ◽  
Author(s):  
Ελένη Κατσιάνη
Keyword(s):  
Alcian Blue ◽  
Alizarin Red S ◽  
Cresyl Violet ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Hla Dr

ΕισαγωγήΠαραδοσιακά τα μεσεγχυματικά βλαστικά κύτταρα (MSCs) μπορούν να προκύψουν από πληθώρα ανθρώπινων ιστών. Πρόσφατα, MSCs απομονώθηκαν από εξωεμβρυϊκές πηγές όπως το ομφαλικό αίμα και η γέλη του Wharton, οι αμνιακές μεμβράνες, το αμνιακό υγρό και ο πλακούντας. ΜSCs προερχόμενα από πλακουντιακές χοριακές λάχνες πρώτου τριμήνου φαίνεται να ομοιάζουν, βιολογικά, στα εμβρυϊκά βλαστικά κύτταρα (ESCs), λόγω του πρώιμου σταδίου ανάπτυξης του πλακούντα.Στην παρούσα μελέτη, προσπαθήσαμε να διαλευκάνουμε τα κοινά χαρακτηριστικά μεταξύ των MSCs προερχόμενα από πρώτου τριμήνου χοριακές λάχνες και ESC. Με βάση τα δεδομένα προηγούμενων μελετών, πραγματοποιήθηκαν παρατεταμένες χρονικά καλλιέργειες των MSCs με σκοπό να αξιολογηθούν in vitro, πως διαμορφώνονται το δυναμικό διαφοροποίησης, τα μοριακά χαρακτηριστικά και γενικότερα οι δείκτες πολυδυναμοκότητας καθόλη τη περίοδο καλλιέργειας.Υλικά και ΜέθοδοιMSCs ελήφθησαν από 30 δείγματα χοριακών λαχνών και καλλιεργήθηκαν για να εκτιμηθεί η ικανότητα πολλαπλασιασμού τους και τα μορφολογικά χαρακτηριστικά. Κυτταρομετρία ροής χρησιμοποιήθηκε για να προσδιοριστεί η έκφραση των αντιγόνων επιφανείας των MSCs. Τα MSCs διαφοροποιήθηκαν σε λιποκύτταρα, οστεοβλάστες, χονδροκύτταρα και νευρικά κύτταρα. Μέσω RT-PCR ανάλυθηκε η έκφραση των γονιδίων-δεικτών των ESC και των γονιδίων δεικτών της διαφοροποίησης.ΑποτελέσματαΤα χοριακά λαχνικά κύτταρα, αναπτύσσονται εύκολα, είναι φαινοτυπικά σταθερά με χαρακτηριστική ινοβλαστοειδή μορφολογία. MSCs ανακαλλιεργήθηκαν για 20 φορές κατά τη διάρκεια περιόδου 120 ημερών. Ρυθμός πολλαπλασιασμού των MSCs παρέμενε σταθερός στα πρώιμα στάδια (3η-12η ανακαλλιέργεια) και παρουσίασε πτώση στα μεταγενέστερα στάδια (13η-20η ανακαλλιέργεια). Καθόλη τη διάρκεια της παρατεταμένης χρονικά καλλιέργειας ο καρυότυπος των κυττάρων δεν τροποποιηθήκε.Η ανάλυση κυτταρομετρίας ροής έδειξε ότι τα κύτταρα χοριακών λαχνών ήταν θετικά για CD90, CD73, CD105, CD29, CD44, HLA ABC αντιγόνα και αρνητικά για CD14, CD34, AC133, και HLA DR αντιγόνα, για ολόκληρη την περίοδο καλλιέργειας.Τα από χοριακές λάχνες προερχόμενα MSCs διαφοροποιήθηκαν επιτυχώς σε λιποκύτταρα, οστεοβλάστες, χονδροκύτταρα αλλά και σε νευρικά κύτταρα. Διαφοροποίηση επιβεβαιώθηκε με χρώσεις Oil Red Ο, Alizarin Red S, Alcian Blue και Cresyl Violet αντίστοιχα και έκφραση των γονιδίων PPARγ2, SPP1, Sox9 και Νεστίνης αντίστοιχα.Η έκφραση ESCs γονιδίων-δεικτών POU5F1 (Οct-4) και Nanog παρατηρήθηκε σε πρώιμα στάδια (4-12 περάσματα), ενώ τα τελευταία στάδια (14-20 περάσματα) δεν παρουσίασαν ανιχνεύσιμα επίπεδα έκφρασης. ZFP42 και Sox2 έκφραση δεν παρατηρήθηκε. Επιπλέον, MSCs βρέθηκαν να εκφράζουν το γονίδιο GATA4 αλλά όχι το NES (νεστίνη) σε μη διαφοροποιημένα κύτταρα.ΣυμπεράσματαΤα από χοριακές λάχνες προερχόμενα MSCs έχουν πολυδύναμες ιδιότητες μεταξύ εμβρυϊκών και ενήλικων βλαστικών κύτταρα. Στην παρούσα μελέτη με τη χρήση ενός ευρύτερου φάσματος δεικτών, διαπιστώθηκε ότι MSC από χοριακές λάχνες εμφανίζουν υψηλό ρυθμό πολλαπλασιασμού, διατηρώντας αναλλοίωτο το χρωμοσωμικό τους προφίλ, και την αυτό-ανανεωτική ικανότητα τους, μοιράζονται κοινή έκφραση αντιγόνων επιφάνειας με τα ενήλικα MSCs και εκφράζουν ορισμένα γονίδια μάρτυρες των εμβρυϊκών βλαστικών κυττάρων. Αυτά τα χαρακτηριστικά θέτουν τις χοριακές λάχνες ως μια ελκυστική πηγή MSC για τις ανάγκες της αναγεννητικής ιατρικής.

Human Umbilical Cord Mesenchymal Stem Cells Induce into Chondrocytes in 3D Cultured System

2013 ◽  
Vol 749 ◽  
pp. 198-205
Author(s):  
Li Yu ◽  
Jing Liu ◽  
Chao Xu ◽  
Er Mei Luo ◽  
Ming Qiao Tang
Keyword(s):  
Real Time ◽  
Alcian Blue ◽  
Culture System ◽  
Type Ii Collagen ◽  
Human Umbilical Cord ◽  
Type Ii ◽  
Pellet Culture ◽  
Hla Dr

Objective: To investigate a better method of inducing hUC-MSCs into chondrocytes in different culture system in vitro. Method: hUC-MSCs were isolated and cultured by tissue block culture, and the cells surface antigens were identified by flow cytometry, hUC-MSCs were cultured with chondrogenic media and stained with Alcian Blue. The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method. Expressions of glycosaminoglycan (GAG), type II collagen and Sox-9 were assayed by real-time fluorescence quantitative PCR. Results: The cultured hUC-MSCs phenotype was CD105+/CD29+/CD44+/ CD31-/CD34-/ CD40-/CD45-/HLA-DR-. hUC-MSCs weakly expressed chondrocyte marker, which strongly expressed GAG and type II collagen after chondrogenic induction, and the cells were incubated in pellet culture with higher expression. Real-time PCR results demonstrated that chondrogenic induction cells were expressed GAG, type II collagen and Sox-9, and the cells were incubated in pellet culture with higher expression. Conclusion: hUC-MSCs incubated in pellet culture is more conducive to differentiate into chondrocytes than those cultured in monolayer culture system.

scholarly journals The Role and Mechanism ofα-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Tianlei Chen ◽  
Huijuan Mao ◽  
Cheng Chen ◽  
Lin Wu ◽  
Ningning Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Signaling Pathway ◽  
Vascular Smooth Muscle Cells ◽  
Calcium Content ◽  
Alizarin Red S ◽  
Alizarin Red ◽  
Calcium Deposits

Objective. To investigate the role and possible mechanism ofα-Klotho in the calcification and the osteogenic transition of cultured VSMCs.Methods. VSMCs were culturedin vitroand divided into 5 groups, each using a different medium: (1) control; (2)β-GP; (3)β-GP + Klotho; (4)β-GP + LiCl; (5)β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 andβ-catenin levels were estimated by western blotting, and the level ofα-SMA was determined by using immunofluorescence at day 12.Results.β-GP induced an increase in the expression of BMP2, Runx2, andβ-catenin. The calcium content increased, and the expression ofα-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, andβ-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.Conclusions. Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced byβ-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.

scholarly journals Alizarin Red S-Alcian Blue Staining for Regenerated tail of Common House Gecko (Hemidactylus frenatus)

2018 ◽  
Vol 7 (2) ◽  
pp. 57-59
Author(s):  
Rakhmiyati Rakhmiyati ◽  
Muhammad Ja’far Luthfi
Keyword(s):  
Alcian Blue ◽  
Histochemical Staining ◽  
Alizarin Red S ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Tail Regeneration ◽  
Alizarin Red ◽  
Regenerate Tail ◽  
Hemidactylus Frenatus ◽  
Common House

Common House Gecko (Hemidactylus frenatus) is one of reptiles that have ability to autotomy their tails. Tail autotomy is a mechanism to protect it self from predators. After the tail broke, there will be wound healing on the tail which is then followed by a tail regeneration event. Original tail and regenerate tail is very different morphologically and anatomically. The original tail is composed of bones while the tail of the regenerate is composed of cartilage. Histochemical staining using Alizarin Red-S Alcian Blue was done to differentiate bone and cartilage. This method will stained bones red while the cartilage will stained blue.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

Double staining technique for rat foetus skeletons in teratological studies

1979 ◽  
Vol 13 (4) ◽  
pp. 309-310 ◽  
Author(s):  
J. Whitaker ◽  
Kathleen M. Dix
Keyword(s):  
Alcian Blue ◽  
Staining Technique ◽  
Alizarin Red S ◽  
Double Staining ◽  
Alizarin Red

Cartilage and bone were differentiated using alcian blue and alizarin red S respectively. Anomalies of both cartilaginous and bony parts of the skeleton could be examined.

Role of Osteogenesis of MC3T3-E1 Induced by Demineralized Tooth Block with Composite Acids: An In Vitro Study

2020 ◽  
Vol 10 (8) ◽  
pp. 1085-1093
Author(s):  
Liu-Zhu Jin ◽  
Xiao-Qian Gu ◽  
Jing-Jing Li ◽  
Yuan Ma ◽  
Lu Cai ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Attachment ◽  
Alizarin Red S ◽  
Permanent Teeth ◽  
Alizarin Red ◽  
Superior Result ◽  
Significant Difference ◽  
Group A ◽  
Demineralized Dentin

More and more research had focused on the osteogenesis of demineralized dentin in clinic, especially when the first application of deminerized dentin in 2008. The study tried to compare the osteogenetic ability of the demineralized dentin block, which were processed two different regents by VacuaSonic system. The extracted human permanent teeth were demineralized by two different methods. Then the MC3T3-E1 cells were invited to culture on the surface of these demineralized dentin blocks (DDB). The cell attachment, proliferation and differentiation were tested. Adhesion of MC3T3-E1 on DDM was observed using scanning electron microscopy and confocal test, the Alizarin Red S, ALP activity, and the protein of BMP-2/-7 and OCN were employed to confirm the level of cell differentiation. The P value was set at 0.05. The microfilaments established a good contact and formed a network in Group A. The Group A had more full cytoskeleton and actin stretched more obviously than Group C, the number of cells on three scaffolds were difference (p < 0 05). The MTT results showed no cytotoxicity in all experiment groups, and Group C had a significant difference in cell proliferation than other groups (p < 0 05) except for day 1. While when related to the cell differentiation, Group A showed a similar result with Group C, but in Alizarin Red S, Group A had a superior result (p < 0 05). The tooth dentin scaffold processed with composite acids in Group A presents the superiority in osteoconduction and preferable osteogenesis ability, which could be an alternative method to process the tooth scaffold.

scholarly journals A Novel Fragment Derived from Laminin-411 Facilitates Proliferation and Differentiation of Odontoblast-Like Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Jia Tang ◽  
Takashi Saito
Keyword(s):  
Proliferative Activity ◽  
Mrna Level ◽  
Cell Counting ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Alp Activity ◽  
Proliferation And Differentiation ◽  
Post Hoc ◽  
Laminin 411

The aim for the present study was to evaluate the in vitro effects of iMatrix-411 in odontoblast-like cells. To that end, iMatrix-411 was coated to both nontissue culture treated- (Non-PS) and tissue culture treated-polystyrene (TCPS) multiwells. MDPC-23 cells were seeded into noncoated (control) or coated wells. Optimal coating density and cell proliferation were assessed by cell counting kit-8 (CCK-8) at day two, day three, and day five. Osteo/odontogenic differentiation was evaluated by real-time RT-PCR and alkaline phosphatase (ALP) activity at days seven and eight, respectively. Calcific deposition of cells was visualized by alizarin red staining. Data were analyzed with post hoc Tukey HSD test (p<0.05). Optimal coating density for iMatrix-411 was 8 μg/cm2. Exposure of MDPC-23 cells to iMatrix-411 in either non-PS or TCPS significantly enhanced proliferative activity. iMatrix-411 elevated ALP activity in both types of culture plates. iMatrix-411 significantly increased the mRNA level of OCN, BSP, OPN, ALP, and DMP-1. Meanwhile, it enhanced the expression of several integrin subunits: ITGA1, ITGA5, ITGAV, ITGB1, and ITGB5. Finally, iMatrix-411 also accelerated the mineralization at day eight in Non-PS. The results indicated iMatrix-411 stimulates proliferation and favours differentiation of odontoblast-like cells.

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