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Effect of the 3D Microenvironment on the Gene Expression of

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Melva Suarez Muñoz

on 9 April 2014

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Transcript of Effect of the 3D Microenvironment on the Gene Expression of

Thank you for your attention!
Effect of the 3D Microenvironment
on the Gene Expression
of Mouse Embryonic Fibroblasts
when Cultured in RAD16-I

Autor: Melva Suarez Muñoz
Academic Advisor: Dr. Christian Klein
External Supervisor: Dr. Carlos E. Semino
Introduction
Materials and Methods
Results and Discussion
Conclusions
Three main approaches to tissue engineering:
To use isolated cells or cells substitutes as cellular replacement parts.
To use acellular materials capable of inducing tissue regeneration.
To use a combination of cells and materials (typically in the form of scaffolds).
Basic principles of Tissue Engineering
Aspects of Self-Assembly
Peptide RAD16-I:
Synthetic matrix of repeated units of hydrophilic and hydrophobic aminoacids  AcN-RADARADARADARADA-CONH2 (R = Arginine, A = Alanine, D = Aspartic acid).
Responsible of the self-assembling process, forming a nanofiber network (pore size: 50-200 nm). Truly 3-dimensional (3D) environment.
Main advantages:
Stable at room temperature
Ability to form a hydrogel by increasing the ionic strength
Chondrogenesis
Schematic representation of the steps involved in chondrogenesis (Pizette & Niswander, 2000).
Multistep process
1st step: aggregation of mesenchymal cells into prechondrogenic condensations.
Expression of markers specific for the chondrogenic lineage.
Differentiation of the prechondrogenic condensed mesenchyma into small immature chondrocytes.
A change in cell shape, gene expression, and increased deposition of cartilage matrix characterize this event.
Endochondral Ossification
This process involves
Terminal differentiation of chondrocytes to the hypertrophic phenotype,
cartilage matrix calcification,
vascular invasion and
ossification.

Takes place in the cartilage templates, at the primary site of ossification. The next step is marked by two events:
Apoptosis of hypertrophic chondrocytes,
Blood vessels invasion bringing in differentiated osteoblasts and osteoclasts from the bone collar, which remove mineralized cartilage matrix and replace it with bone.

Steps involved in endochondral ossification (Ray Boot-Handford).
Tissue Engineering
1. To study the effect of the microenvironment on the expression of chondrogenic markers by mouse embryonic fibroblasts, when cultured in the self-assembling peptide RAD16-I.

2. To study the influence of the cross-talk between human umbilical vein endothelial cells with mouse embryonic fibroblasts, on the gene expression of the latter, to determine osteogenic differentiation via endochondral ossification, when co-cultured in self-assembling peptide RAD16-I.
Objectives
3D Cell Culture Using RAD16-I
Cell encapsulation protocol with self-assembling peptide RAD16-I
Chondrogenic Commitment
- Mouse embryonic fibroblasts (MEFs), when cultured in RAD16-I at a specific concentration (0.07%), can suffer chondrogenic differentiation, supported specifically by the expression of chondrogenic markers such as Sox9 and BMP4, as well as by the expression of extracellular matrix (ECM) proteins type I and II collagens, and the lack of expression of type X collagen.

- Another evidence that suggest the chondrogenic commitment by MEFs cultured in 0.07% RAD16-I, is that when comparing those results with the results obtained by MEFs cultured in a higher concentration of RAD16-I (0.25%), these last were not capable of neither contract the cell matrix nor reproduce the same results, which suggests that matrix stiffness with the consequence of cell-cell contact is an important feature to obtain chondrogenic differentiation.

Osteogenic Commitment
- Unlike MEFs cultured alone, when co-cultured by both methods with human umbilical vein endothelial cells (HUVECs) showed a different commitment, demonstrated by the expression of type X collagen.

- Additionally, other factors expressed by the mentioned co-cultures were Runx2 and VEGF-A, which can indicate hypertrophy and possible formation of new blood vessels, necessary for the endochondral ossification to occur.

- Finally, the observation of black precipitates in the Von Kossa staining indicates the presence of a mineralization process of the cell constructs induced by the direct interaction between the two cell types.

- In sum, those results may indicate an effective cross-talk between MEFs and HUVECs, which can be studied more in detail and used in future research projects.
Co-culture of MEFs and HUVECs
Co-culture of MEFs and HUVECs by loading a HUVECs suspension over a MEFs culture in 0.07% RAD16-I
Co-culture of MEFs and HUVECs by encapsulating them together in 0.07% RAD16-I
Molecular Biology Techniques
RT-PCR
Western Blot
The RNA was extracted from the samples using peqGold total RNA kit.
cDNA was synthesized using QuantiTect reverse transcription kit.
The cDNA obtained was then analyzed by RT-PCR:
50 ng of cDNA sample,
dNTPs at a final concentration of 200 M
primers at a final concentration of 0.3 M
Deep Vent, 0.2 units or Taq polymerase 1 unit
Amplification reaction:
Stage 1: 5 min at 95°C,
Stage 2: 40 cycles of 30 s at 95°C, 15 s at T°m (depending on the primers used) and 20 s at 72°,
Stage 3: 1 min at 72°C.
The gene used as a housekeeping was the ribosomal subunit 18S.
(previous step) Quantification step using BCA Protein Assay kit.

According to the size of the proteins, acrylamide gel was prepared (7 or 12%).

The samples were prepared and loaded into the acrylamide gel.

Marker: Precision Plus Protein Standards dual color.
Run of the gel
Transference and Detection of the Proteins
Detection and Analysis
Acrylamide gel and Sample preparation
Performed in electrophoresis buffer.

Applying 150V during 1 h (depending on the size of the proteins and the concentration of the gel).
After the run, the proteins were transferred to a PVDF membrane in transfer buffer, applying 40V for 2 h.
The membrane was incubated at RT for 2 h in blocking buffer.
Then incubated with the primary Ab for 1 h, washed with PBST and incubated with the secondary Ab IgG-HRP.
Finally revealed with SuperSignal WestPico Chemiluminescent substrate, for detection of HRP and analyzed using ImageQuant LAS 4000 mini.

To normalize the results obtained, actin was used as a standard protein.
Stainings
DAPI and Phalloidin
For the observation of the cell nuclei and cytoskeletons.
Cell Viability
To qualitatively determine proportion of live and dead cells.
Toluidine Blue
To determine chondrogenic phenotype.
Von Kossa
To determine the mineralization of cell cultures, which indicates osteogenic differentiation.
Immunofluorescence
To detect the presence of a protein in the whole cell construct.
Labeling of HUVECs
For better identification of HUVECs when co-cultured with MEFs.
Chondrogenic Differentiation Model
Osteogenic Differentiation Model
Encapsulation at Different Peptide Scaffold Concentrations
1 day
5 days
14 days
0.07% RAD16-I
0.25% RAD16-I
DAPI-Phall on 3D cultures
Live-dead staining (21 days)
Toluidine Blue Staining
0.07% RAD16-I
0.25% RAD16-I
Analysis of the Gene Expression of MEFs Cultured in RAD 16-I
Stages involved in the differentiation of MEFs into chondrocytes and the factors and extracellular matrix proteins mainly involved on each stage (adapted from Vinatier et al. 2009).
Immunoassay, BMP4 (10 days of culture).
0.25% RAD16-I
0.07% RAD16-I
0.07%
0.07%
0.25%
0.25%
0.25%
0.25%
0.07%
0.07%
Coll I
0.25%
0.07%
Coll I
Coll II
Coll II
0.07%
0.25%
0.07%
0.25%
Collagen I expression. PCR
0.07%
0.25%
0.07%
0.07%
0.25%
ECM Remodeling During Endochondral Ossification
Cross-talk model between HUVECs and MEFs
Von Kossa Staining
HUVECs loaded over MEFs
Co-culture MEFs + HUVECs (21 days)
1 day
5 days
10 days
HUVECs Fluorescently-Labeled
HUVECs loaded over MEFs cultures at day 1 of culture, and fixed 7 days before
Analysis of the Gene Expression of Co-Cultures of MEFs and HUVECs in RAD16-I
Ortega et al. 2004
Coll I
Coll I
Coll II
Coll II
Immunoassay, PECAM. HUVECs and MEFs (10 days)
HUVECs in EBM
HUVECs + MEFs in DMEM
HUVECs + MEFs in DMEM + EBM
HUVECs + MEFs in EBM
BMP4 Expression. WB and PCR
Noggin Expression. WB and PCR
Sox9 Expression. WB
Coll I and Coll II Expression. WB
Runx2 Expression. WB and PCR
Collagen X Expression. WB and PCR
Runx2 Expression. WB
Co-culture MEFs + HUVECs
HUVECs loaded over MEFs
Co-culture MEFs + HUVECs
HUVECs loaded over MEFs
Collagen X Expression. WB
Coll I and Coll II Expression. WB
Coll I
RT-PCR Results
18S
Runx2
Coll I
Coll X
VEGF-A
Co-cultures MEFs + HUVECs
HUVECs loaded over MEFs
Full transcript