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In vitro generation of chondrocyte-osteoblast hybrid cells through differentiation of ATDC5 cells
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In vitro generation of chondrocyte-osteoblast hybrid cells through differentiation of ATDC5 cells
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Adrian Wang 1
5516738666
In Vitro Generation of Chondrocyte-Osteoblast Hybrid Cells
Through Differentiation of ATDC5 Cells
Adrian Wang
Master of Science (Biochemistry and Molecular Biology)
University of Southern California Graduate School
August 2018
Principal Investigator: Dr. Francesca Mariani, PhD
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Table of Contents
Abstract…………………………………………............................................................................3
Background………………………………………………………………………………………..4
Introduction………………………………………………………………………………………..6
Methods………………………………………………………………………………………......10
Figures…………………………………………………………………………………………....15
Figure 1: Protocol and Methods………………………………………………………………………………………….15
Figure 2: Alcian Blue and Alizarin Red Staining Indicates Presence of Both Cartilage and Biomineralization …….....17
Figure 2a: 1-2 Week Differentiation Cycle…………………………………………………………………...17
Figure 2b: 6 Week Differentiation Cycle; Single Reagent……………………………………………………18
Figure 2c: 6 Week Differentiation Cycle; Multiple Reagents………………………………………………...19
Figure 2d: Undifferentiated ATDC5 Cells……………………………………………………………………20
Figure 3: Chondrogenic and Osteogenic Markers are Present in Cells exposed to Differentiation Media ……………..21
Results……………………………………………………………………………………………22
Discussion………………………………………………………………………………………..27
Further Directions………………………………………………………………………………..38
Acknowledgements………………………………………………………………………………42
References………………………………………………………………………………………..43
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Abstract
Murine-derived ATDC5 cells were exposed to different media which have been shown to
induce differentiation into both chondrocytes and osteoblasts from ATDC5 cells. The exposure
time to the differentiation media varied between 2 to 6 weeks, and the treatments were used to
produce cell cultures which exhibit characteristics of both chondrocytes and osteoblasts. The
goal of this project was to generate and optimize a protocol which could be used to efficiently
create hybrid chondrocyte-osteoblast cells which would later be used in regenerative bone
research.
The cells were treated with reagents which are known to have effects on the chondrocyte
and osteoblast differentiation pathways. The three pathways chosen for investigation were the
Hedgehog (Hh) pathway, TGF-β pathway, and the Notch pathway. To study the effect of
stimulating these pathways on osteoblast formation, I treated ATDC5 cells using three separate
reagents in varying quantities, smoothened agonist (SAG), Bone Morphogenetic Protein 2
(BMP2), and GÖ6983, mixed into bone differentiation media.
Successful differentiation was tested using both RT-PCR and by extracellular matrix
staining using Alcian blue and Alizarin red for cartilage matrix and bone matrix respectively.
Gene expression was expected of certain osteogenic markers, such as osteocalcin, was thought to
be increased in cells that had successfully differentiated into osteoblast-type cells. Similarly,
expression of chondrogenic markers such as collagen, type X, was predicted to be expressed in
chondrocyte-type cells. Hybrid cells were expected to express both these markers at least in early
differentiation stages. Subsequent treatment of ATDC5 cells with chondrogenic and osteogenic
media seemed to result in cells that share extracellular and genetic characteristics with
chondrocytes and osteoblasts, but whether individual cells have become hybrid is still unknown.
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Background
Currently, when treating patients who have experienced substantial bone loss or bone
damage, the primary method is a replacement of missing or fractured bone by either a graft or
prosthetic implant. The organic grafts that are used can either be autografts—bone taken from
elsewhere in the patient’s own body—or an allograft—sterilized bone taken from a cadaver.
Autografts are used because they can often decrease the chance of complications and rejection of
the transplant, and can significantly decrease hospitalization time,198 days compared to 416 days
(Flierl et al, 2013) but they still carry the risk of post-operational problems. 12.4% of autograft
recipients still experience infection, compared to 26.3% of allograft patients (Flierl et al, 2013).
Alternatively, synthetic bone grafts can also carry their own degree of risk, depending on the
type of graft used. Certain synthetics may contribute to tissue inflammation and may even
require revision surgery (Friesenbichler et al, 2014)
Regardless of the type of graft being used, surgery inherently carries with it some degree
of risk, and a replacement surgery is no exception to the fact. During hip arthroplasty,
approximately 20% of patients experience problems, such as postoperative delirium and even
heart failure, which can dramatically increase time in the hospital or mortality rates (Carpintero
et al, 2014). The surgical risks of replacement surgeries can dramatically increase for patients in
poor health, such as smokers, who experience deep infections after hip replacement surgery with
3 to 4 times more likelihood than non-smokers (Teng et al, 2015)
To avoid the side effects associated with replacement surgery, one prospective method of
treating bone injuries would be the stimulation of regenerative pathways in the bone. Humans
have two methods by which bone is produced during initial skeletal development: endochondral
ossification, where a cartilage matrix is first generated by chondrocytes, onto which bone is
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produced by mineralization of the matrix, and intramembranous ossification, where bone is
generated directly by mesenchymal cells. During bone fracture repair in mice, both pathways are
active, but endochondral ossification is required to bridge the fracture gap (Hu et al, 2017).
Neither pathway is active at high levels in adult humans, even at fracture sites, and in about 10%
of all cases, bone fractures will not properly heal at all (Einhorn and Gerstenfeld, 2015).
Certain animals, such as salamanders and zebrafish, have exceptionally potent
regenerative properties. Mature zebrafish can regenerate their spinal columns (Goldshmit et al,
2012), retina (Fausett and Goldman, 2006), and heart after injury (Jopling et al, 2010), with the
aid of precursor-type cells in each respective area. Jawbone regeneration in zebrafish is similarly
facilitated with the assistance of hybrid-type repair cells (Paul et al, 2016). More closely related
to humans are mice, which exhibit the ability to regenerate the cartilage matrix and establish
biomineralization after rib resection (Srour et al. 2015), and can be used as a model organism to
study bone fracture repair pathways in humans (Holstein et al, 2011).
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Introduction
ATDC5 cells are a pre-chondrocytic cell line, derived from mouse teratocarcinoma
AT805 cells. These cells are a common model for studying differentiation into chondrocytes in
vitro, because they exhibit a process analogous to chondrocyte differentiation (Wang & Yao,
2013). By studying the differentiation of ATDC5 cells into chondrocytes, the goal of the project
was to generate a protocol by which chondrocytes may begin to exhibit characteristics of
osteoblast differentiation. Under the proper growth conditions, ATDC5 cells can be stimulated
into either osteogenesis or chondrogenesis. There is evidence that stimulation of the
BMP2/TGFβ signaling pathways can lead to phosphorylation of Smad1/5 proteins, which in turn
stimulates the process of bone formation in ATDC5 cells (Matsumoto et al, 2012). As a result, it
may be possible to stimulate the differentiation of ATDC5 cells into a hybrid state.
This project focused on stimulating hybrid cell differentiation by emulating and
modifying the process of endochondral ossification. Endochondral ossification in vivo occurs
first when chondrocytes begin to release extracellular matrix proteins, such as collagen type II,
and a mesh of proteoglycans, hyaluronic acid, and chondroitin sulfate (Gao et al, 2014). After an
initial matrix of cartilage has been formed, the cartilage cells become hypertrophic, secreting
collagen type X and matrix metalloprotease 13 and angiogenic growth factors VEGF and CTGF
(Ortega, Behonick, & Werb, 2004).
Hypertrophic chondrocytes stimulate initial calcification by production of hydroxyapatite
(Mackie et al, 2007) from the subchondral layer (Andreassen et al, 2012), before beginning to
degrade to allow for the influx of osteoprogenitor cells. Osteoprogenitor cells differentiate into
mature osteoblasts within the developing bone (Maes et al, 2010), occupying the space in the
cartilage matrix left behind by the dying chondrocytes (Mackie et al, 2007). These osteoblasts
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then initiate final biomineralization of the extracellular matrix by continued secretion of
hydroxyapatite crystals (Boonrungsiman et al, 2012), forming the rudimentary scaffold on which
organic components in the bone can be produced, such as collagen type I. The final structure of
bone tissue is a combined matrix of organic and inorganic components.
At different stages of chondrocyte differentiation, separate markers of the extracellular
collagen protein family are produced, with collagen type II being produced in early stage
chondrocytes and decreasing progressively as chondrocytes become hypertrophic (Hamada et al,
2013). Conversely, in vitro collagen type I expression levels increase as chondrocytes become
more hypertrophic, and play a role in facilitating bone formation (Hamada et al, 2013). The
transition to bone is associated with an increase in the expression of biomineralization proteins,
such as the osteoblast marker osteocalcin, which stimulates osteoid mineralization in osteoblasts
(Wheater et al, 2013), or osteopontin, which inhibits excess biomineralization (Kalmar et al,
2012). Both proteins are viable markers for osteogenesis in this system because they are found
bound to hydroxyapatite structures, and are therefore only present in biomineralized systems
(Wheater et al, 2013), (Kalmar et al, 2012).
The generation of chondrocytes and the differentiation into osteoblasts relies on the
activity of separate distinct characterized pathways. Of focus for this project were the Hedgehog
pathway (Hh), the Notch Pathway, and the BMP2 pathways. All three pathways regulate the
transcription of genes in many cell types throughout the body, and influence the generation of
osteoblasts and differentiation into chondrocytes. The activity of each pathway has already been
characterized in osteoblast and chondrocyte formation, and are therefore ideal for study in the
formation of hybrid cells.
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The primary pathway of interest for this project was the Hedgehog pathway. Indian
Hedgehog is one of the three hedgehog signaling pathways commonly found in mammalian
systems, and is necessary for the proper progression of bone formation. Ihh signaling has been
found to play a key role in stimulating osteoblast differentiation in mice (Hu et al, 2004). More
so, Ihh signaling plays a role in positively regulating chondrocyte proliferation, and is expressed
in both pre-hypertrophic and early hypertrophic chondrocytes (Long et al, 2004), and acts to
push proliferating chondrocytes into hypertrophy (St-Jacques, Hammerschmidt, & McMahon,
1999). Furthermore, after differentiation, Ihh is functional in controlling both bone formation and
reabsorption in mature osteoblasts (Mak et al, 2008).
Ihh has also been found to be important in hybrid osteochondral cell formation in
zebrafish jaw regeneration (Paul et al, 2016), the effect of which is likely amplified by Ihh
signaling directed towards the surrounding perichondral layer (Long et al, 2004). To stimulate
the hedgehog signaling pathway, a smoothened agonist reagent, SAG, was chosen for use. SAG
functions by targeting the Smoothened coreceptor, and stimulates the Hedgehog signaling
pathway (Lewis and Krieg, 2013) by increasing transcription of Gli1, the downstream mediator
of all Hedgehog signaling pathways (Bragina et al, 2010). Because of its direct link to hybrid cell
formation, the Hh pathway was prioritized over the other pathways, with regulation of other
pathways being secondary or additional to Hh regulation.
The secondary pathways being studied in this experiment were the BMP2 pathway and
the Notch pathway. BMP2, short for Bone Morphogenetic Protein 2, is a member of a family of
proteins which play a key role in regulating development of various organ systems in the body
(Wang et al, 2014). Of the BMP family, BMP2 is an essential protein in chondrocyte
differentiation, with BMP2 knockout mice showing “severe chondrodysplasia” (Shu et al, 2011).
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BMP2 also acts to stimulate osteogenesis by stimulating Runx2 activity, both transcriptionally
and post-transcriptionally (Shu et al, 2011), which itself plays an important role in regulating the
generation of both mature chondrocytes and osteoblasts. Runx2 is necessary for the
differentiation of young chondrocytes into a hypertrophic state (Chen et al, 2014), and stimulates
the differentiation of precursor cells into young osteoblasts (Komori, 2010).
The final pathway of interest is the Notch signaling pathway, which acts by modifying
transcription factors in the nucleus once activated. Notch signaling is activated by binding of the
Notch receptor to the ligand Delta (Berndt et al, 2017), which, after binding, stimulates cleavage
of the NICD domain, allowing it to travel to the nucleus (Hori, Sen, & Artavanis-Tsakonas,
2013). During endochondral ossification, the Notch pathway acts by stimulating the production
of Sox9 (Kohn et al, 2015), which in turn is necessary for both chondrocyte proliferation and
differentiation into hypertrophy (Akiyama et al. 2004). With respect to osteoblast differentiation,
increases in Notch signaling can suppress differentiation into mature osteoblasts, maintaining
cells in an osteoprogenitor state (Hilton et al, 2008).
To regulate the Notch Pathway, I have chosen to use the general PKC inhibitor molecule,
GÖ6983 as a Notch pathway activator. GÖ6983 acts as a competitive inhibitor for PKC,
occupying the same binding site as ATP and preventing kinase activity (Wu-Zhang and Newton,
2013). While GÖ6983 has a more substantial inhibitory effect on conventional and novel
isozymes of PKC, it is also effective against atypical isozymes such as PKCζ and PKCι (Wu-
Zhang and Newton, 2013). PKCι has been found as an inhibitor of Notch signaling, through
phosphorylation of the Numb protein (Mah et al, 2015). Numb, when phosphorylated, is
localized to the cell membrane (Smith et al, 2007), where it can act to inhibit the activation of the
Notch pathway through various mechanisms (Andersson et al, 2011).
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Methods
Differentiation and Growth Media: ATDC5 growth media was created in bulk using 500
mL DMEM/F-12 (Corning 10-090-CV), 25 mL of HI-FBS, and 5 mL of 100U/mL Penicillin and
172uM Streptomycin mixture (Corning 30-002-CI), for a total of 555 mL of growth media.
Chondrogenic differentiation media consisted of the ATDC5 media, as well as 12 uL of 0.1mM
dexamethasone, 13.7uL of 0.5M ascorbic acid, 6 uL of 20ug/mL TGFβ, and 120uL of ITS+
Premix per every 12 mL of growth media. The reagents used are commonly used in chondrocyte
micromass differentiation experiments (Enochson, Brittberg, & Lindahl, 2012), with Penicillin
and Streptomycin being supplied in the original ATDC5 growth media. The basic osteoblast
differentiation media used 220 mL aMEM (Corning 15-012-CV), 25mL of HI-FBS, 2.5mL of
Penicillin/Streptomycin, and 2.5 mL of 1x L-glutamine. Immediately prior to feeding the cells,
dexamethasone, ascorbic acid, and β-glycerophosphate were added to the osteoblast
differentiation media.
Ascorbic acid was used because of its ability to stimulate chondrogenesis by stimulating
ERK signaling and synthesis of the collagenous extracellular matrix in ATDC5 cells (Temu et al,
2010). Dexamethasone is a glucocorticoid analogue that inhibits Wnt/β-catenin signaling (Naito,
Ohashi, & Takahashi, 2015), simultaneously decreasing proliferation and differentiation of
chondrocytes (Miyazaki et al, 2000) in ITS+ and TGFβ induced ATDC5 cells. TGFβ, or
transforming growth factor beta 1, is a key factor in initiating differentiation into pre-
hypertrophic chondrocytes (Kraan et al, 2009), largely through the chondrogenic factor Sox9
(Furumatsu et al, 2009). TGFβ also plays a role in repressing both hypertrophy and apoptosis of
chondrocytes (Kraan et al, 2009). ITS Premix solution consists of insulin, transferrin, and
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selenium, and is a nutritional supplement which increases chondrogenic differentiation of cells in
vitro compared to cells grown without it (Liu et al, 2014)
In osteoprogenitor cells, ascorbic acid also stimulates the differentiation into osteoblasts
by activation of osteoblast marker Runx2 (Hadir et al, 2014). Ascorbic acid does so by increasing
Col1a1 secretion into the extracellular matrix, at which point, interactions with the extracellular
matrix increase Runx2 activity (Langenbach and Handschel, 2013) Dexamethasone, like ascorbic
acid, stimulates the activation of Runx2 and also regulates phosphorylation of Runx2 by MKP-1
(Langenbach and Handschel, 2013). β-glycerophosphate is necessary for in vitro osteogenesis
because it supplies phosphate for both formation of hydroxyapatite crystals and for kinase related
phosphorylation (Langenbach and Handschel, 2013).
Generation of ATDC5 Cells: ATDC5 cells were obtained frozen in a solution containing
4 parts HI-FBS (Omega Scientific FB-12) and 1 part DMSO, in equal volume with ATDC5
growth serum for a total of 1 mL The cells were thawed at 37
o
C in a water bath and resuspended
with an additional 1 mL of ATDC5 media. The resuspension was then transferred to a 15 mL
conical centrifuge tube, where an additional 5 mL of ATDC5 media were added dropwise, before
centrifugation at 1000 rpm for 5 minutes. The supernatant was then suctioned off, and the
resulting cell pellet was resuspended in 5 mL of ATDC5 media, and then transferred to a gelatin-
coated 10cm plate, where another 5mL of ATDC5 media was added. After plating, the cells were
placed in an incubator at 37
o
C and 5.0% CO2 to grow overnight. After the initial plating, the cells
were fed with 10mL of ATDC5 media and placed back into the incubator each day for 5-7 days,
until the cells reached above 80% confluency.
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Micromass cell generation: After 80% confluency, ATDC5 cells were trypsinized and
counted. The cells were resuspended in 1 mL of ATDC5 media, and a 10 μL drop of the
suspension, containing approximately 100000 cells, was added to each well in two copies of
three different 12-well plates, in the center of the well, and allowed to adhere for 2 hours in the
incubator. After the 2 hours were over, 1 mL of ATDC5 media was added to each well and the
plates were placed back in the incubator over to grow overnight. The protocols for generating
micromass cultures established in Greco et al was employed for this experiment, with
modifications to the cell count per well.
Chondrocyte and osteoblast differentiation: Micromass cell cultures were cleaned and
exposed to fresh cell media every other day, for a maximum of 2 weeks to induce chondrocyte
differentiation. After the chondrocyte differentiation cycle, the cells were exposed to an
osteoblast induction media for a minimum of 1 week (plate 1), and up to a maximum of 4 weeks
(plates 2 and 3). Cells in plates 2 and 3 were exposed to chondrogenic differentiation media for 2
weeks, prior to transitioning to osteogenic media, and cells in plate 1 were exposed to
chondrogenic media for 1 week prior to osteogenesis. In each plate, wells may either be exposed
to the basic osteoblast media, an osteoblast media containing smoothened agonist (SAG), Bone
Morphogenetic Protein-2 (BMP-2), GӦ6983, or a combination of SAG and the other reagents.
SAG was added at a concentration of 1 μM, BMP2 and 10 ng/μL for low concentration media,
20 ng/μL for high concentration media, and GÖ6983 was added at 5μM for low concentration
media and 10μM for high concentration media
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RNA Isolation: RNA was extracted at the end of the differentiation cycle from the cells
using the Invitrogen PureLink RNA Mini Kit from ThermoFisher Scientific. Cells were first
trypsinized and the total cell count of 3 wells was used to isolate the RNA, and the final
extracted RNA was collected in 40 μL of RNAse-free water per 3 wells of cells.
cDNA Synthesis: Up to 13 μL of RNA solution, at a maximum of 1 μg of RNA, was
mixed with a solution consisting of 2 μL of a random hexamer, 1 μL of 10mM dNTPs, and
DEPC-water was added until the final solution was 16 μL. The solution was heated at 65
o
C for 5
minutes, before being treated with 1 μL of M-MulV RT enzyme and 1 μL of RNAse inhibitor
enzyme in an extra 2 μL of 10X RT Buffer, and placed at 42
o
C for an hour. Finally, the solution
was set at 90
o
C for 10 minutes.
RT-PCR: 1.5 μL of the newly synthesized cDNA was mixed with 1.5 μL each of the
forward and reverse primers in question, as well as 12 μL of ThermoFisher Scientific 2X Master
Mix and 7.5 μL of DEPC-water. The primers used tested for collagen, type II, alpha 1 (Col2a1),
collagen, type X, alpha 1 (Col10a1), bone γ-carboxyglutamate protein 3 (Osteocalcin), and
secreted phosphoprotein 1 (Osteopontin), with GAPDH also used as a housekeeping gene
marker. The PCR was run at 35 cycles for all genes—except for GAPDH, which was run at 28
cycles—and at 55
o
C. The DNA was then examined on a 5% agarose-EtBr gel, at 120V for 20 to
25 minutes. RT-PCR was examined and compared over two trials.
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Cell Staining Protocol: The remaining 3 plates were fixed with 0.4 μL of 4% PFA per
well for 15 minutes, before being stained with 1 mL of either Alcian Blue or Alizarin Red.
Alcian Blue was created at a concentration of 20mg 8GX in 100 mL of solvent, 97 mL of diH2O
and 3mL of glacial acetic acid. Alizarin Red was created at a concentration of 6 mg in 100 mL of
diH2O, and brought to pH 4.1-4.3 with 10% ammonium hydroxide. The cells were covered and
allowed to incubate overnight at room temperature at a slow rotation. The cells were then washed
with PBS, before a final mL of PBS was added to maintain moisture and prevent damage. If the
cells were stained by both reagents, then they were stained first with Alcian Blue overnight,
before being washed and subsequently stained with Alizarin Red for a second night. The cells
were then imaged at 4X and 20X magnification. Images were edited in Photoshop to improve
photo clarity and contrast. Cell stains were examined and compared over two trials.
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Figures
Figure 1: Protocol and Methods
B) Timeline of Cell Differentiation
A) Cells Were Distributed into 3 Separate Plates
ATDC5
Cell
Growth
2 Week Cartilage
Differentiation (Plate 1)
4 Week Bone Differentiation (Plates 2 and 3)
5 Days 19 Days 6 Weeks
1 Week Cartilage
Plate 1: 1-2 Week
Differentiation
2 Weeks Cartilage
1 Week Cartilage + 1
Week Bone
1 Week Cartilage + 1
Week Bone + SAG
Bone
Plate 2: 6 Week
Differentiation; Single
Reagents
SAG
BMP2
Gö6983
SAG+Gö6983
Low Concentration
SAG+BMP2
High Concentration
SAG+BMP2
Low Concentration
SAG+Gö6983
High Concentration
Plate 3: 6 Week
Differentiation; Multiple
Reagents
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Gene Forward Reverse Band
Length
GAPDH 5’-ACC ACA GTC CAT GCC
ATC AC-3’
5’-TCC ACC ACC CTG TTG CTG
TA-3’
452bp
Col2a1 5’-AGA ACA TCA CCT ACC
ACT GTA AGA ACA-3’
5’-TGA CGG TCT TGC CCC ACT
T-3’
189bp
Col10a1 5’-CCT TTC TGC TGC TAA
TGT TCT TGA-3’
5’-ATG CCT TGT TCT CCT CTT
ACT GGA-3’
167bp
OCN 5’-GGC CCT GAG TCT GAC
AAA GC-3’
5’-GCT CGT CAC AAG CAG GGT
TAA-3’
151bp
OPN 5’-CCC ATC TCA GAA GCA
GAA TCT CC-3’
5’-TTC ATC CGA GTC CAC AGA
ATC C-3’
189bp
C) PCR Primers
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Figure 2: Alcian Blue and Alizarin Red Staining Indicates Presence of Both Cartilage and
Biomineralization
20X
Alizarin Red
4X
Alizarin Red
4X
Alcian Blue
20X
Alcian Blue
A) Plate 1: 1-2 Week Differentiation Cycle
1 Week
Cartilage
2 Week
Cartilage
Bone Bone + SAG
Figure 2A. ATDC5 cells stained with Alcian Blue and Alizarin Red after a
maximum of 2 weeks in differentiation media. Cells were either grown for 1
week in chondrogenic media, 2 weeks in chondrogenic media, 1 week in
chondrogenic media followed by 1 week in control osteogenic media, or 1
week in chondrogenic media followed by 1 week in SAG-modified
osteogenic media, prior to staining
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20X
Alizarin Red
4X
Alizarin Red
B) Plate 2: 6 Week Differentiation Cycle; Single Reagent
4X
Alcian Blue
20X
Alcian Blue
Bone SAG BMP2
GÖ6983
Figure 2B. ATDC5 cells stained with Alcian Blue and Alizarin Red after 6
weeks in differentiation media. All cells were differentiated for 2 weeks in
chondrogenic media, before being transitioned to either control or modified
osteogenic media containing a single reagent (SAG, BMP2, or GÖ6983) for 4
weeks.
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20X
Alizarin Red
4X
Alizarin Red
Gö6983 Low
Concentration
Gö6983 High
Concentration
BMP2 Low
Concentration
BMP2 High
Concentration
C) Plate 3: 6 Week Differentiation Cycle; Multiple Reagents
4X
Alcian Blue
20X
Alcian Blue
Figure 2C. ATDC5 cells stained with Alcian Blue and Alizarin Red after 6
weeks in differentiation media. All cells were differentiated for 2 weeks in
chondrogenic media, before being transitioned osteogenic media containing
either BMP2 or Gö6983 for 4 weeks. Each well had an additional 20uL of
1uM SAG added to the osteogenic media during each feeding cycle.
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Alcian Blue, 4X
Alizarin Red, 4X
Undifferentiated ATDC5 Stains
D) Untreated ATDC5 Cells Show no Indication of Chondrogenesis or Osteogenesis
E)
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Figure 3: Chondrogenic and Osteogenic Markers are Present in Cells exposed to Differentiation
Media
A) 2 Week Differentiation Cycle
Col10
Col2
OCN
OPN
GAPDH
B) 6 Week Differentiation Cycle
Col10
Col2
OCN
OPN
GAPDH
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Results
The following results are based on two complete runs of the treatment protocol detailed
above in Methods, and without repeated trials, it is not possible to determine whether the results
are representative. The results are described starting with analysis of the Alizarin Red and Alcian
Blue staining, followed by RT-PCR results detailing chondrocyte and osteoblast gene
expression. Finally, the results are concluded with a comparison of the RT-PCR and staining
results.
Treatment of Differentiated ATDC5 Cells with Osteogenic Media can Induce
Biomineralization: The cells across all groups were treated in chondrogenic media, 1 week for
plate 1, 2 weeks for plates 2 and 3, before being treated with osteogenic media, 1 week for plate
1, 4 weeks for plates 2 and 3. The expression of both osteopontin (secreted phosphoprotein 1)
and osteocalcin (bone γ-carboxyglutamate protein 3) can be evidenced in the cells that have been
cultured in osteogenic media over the 6-week period, but is less evident in the cells that were
only exposed for a short period of time. The evidence of biomineralization is more apparent
when viewing the cell stains though (Fig. 2), as Alizarin red staining was generally absent in the
2-week cycle plate across both trials. Interestingly, osteopontin and osteocalcin were shown to
be expressed in many of the cells that had had a shorter differentiation cycle (Fig. 3A), including
in cells that had not been exposed to any osteogenic differentiation at all. The evidence of
biomineralization is more apparent when viewing the cell stains in Fig. 2, as Alizarin red staining
was generally absent in the 2-week cycle plate across both trials.
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Alcian Blue Indicates Cartilage Presence In all Cell Cultures: Across the different plates,
staining by Alcian blue has shown no remarkable differences between treatment groups. The
staining patterns are a strong indicator that chondrogenesis has occurred (Fig. 2), even by 1-week
since the beginning of treatment (Fig. 2A). This is in comparison to the Alcian Blue stain of
undifferentiated ATDC5 cells, which show no indications that any chondrogenesis has occurred
when not exposed to the differentiation media (Fig. 2D). Therefore, it is safe to assume that
exposure to chondrogenic media for at least 1 week is sufficient for chondrogenesis to begin.
Furthermore, across all treatment groups that also indicated biomineralization, the presence of
Alcian Blue has not changed (Fig. 2B,2C), implying that subsequent differentiation media can
lead to a cell culture containing both chondrocytes and osteoblasts, or at least cells that can
produce both a cartilage-like matrix and a mineralized matrix.
Biomineralization has Occurred in Most Cell Populations, but the Patterns of Which Vary
by Treatment Type: Cells that were exposed to osteogenic differentiation media for the 4-week
cycle—those found on plates 2 and 3—showed indications of biomineralization (Fig. 2B,2C), as
evidenced by the prominent red stain, while cells that were only exposed to osteogenic media for
a maximum of 1 week showed no markers of biomineralization (Fig. 2A). It is important to note
though that the patterns of biomineralization vary greatly between plate 2 and plate 3. Treatment
groups that were not exposed to multiple reagents seem to show a more dispersed pattern of
biomineralization, where small clusters of cells are stained all throughout the well (Fig. 2B). On
the other hand, cells that were treated with SAG as well as other reagents, BMP2 and GÖ6983,
have substantially larger clusters of cells, with much more space in between clusters (Fig. 2C).
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The reason behind this is still unknown, but it may be related to the presence of SAG in the
differentiation media.
Variation of Treatment Conditions Does Not Appear to Affect Col2a1 Expression
Levels: Col2a1, commonly found expressed in pre-hypertrophic chondrocytes, is found to be
expressed across almost all treatment groups except the 2-week chondrocyte treatment (Fig. 3A).
The presence of Col2a1 indicates that chondrogenesis has begun to occur in the ATDC5 cells,
even when only grown for 1 week. It is interesting to note that the 2-week chondrogenic
treatment is missing Col2a1 expression, since the cells in plates 2 and 3 are all grown using cells
that had been previously exposed to the 2-week treatment, and they all show evidence of Col2a1
(Fig. 3B). It should be noted that the expression level across all treatment groups appears to be
reasonably low based entirely on band intensity (Fig. 3), so the absence in the 2-week
differentiation cycle may just be indicative of low expression levels that were unable to be
detected under the given PCR conditions.
Col10a1, a Marker for Hypertrophy, is Expressed Across All Treatment Groups: During
endochondral ossification, the eventual fate of chondrocytes is generally to become hypertrophic
and undergo apoptosis to allow for osteogenesis. Col10a1 is generally expressed by hypertrophic
chondrocytes but not pre-hypertrophic chondrocytes, and is found to be expressed across all the
given treatment groups (Fig. 3). On plate 1, cells that were switched to osteogenic differentiation
after a single week though do not seem to express as high levels of Col10a1 when compared to
the other cells (Fig. 3A). This is in comparison to both cells that were maintained in
chondrogenic media (Fig. 3A), and cells that had been switched to osteogenesis after 2-weeks in
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chondrogenesis (Fig. 3B). Of interest is that the cells, even after not being exposed to
chondrogenic media for weeks, are still showing indications of chondrocyte hypertrophy,
implying that the treatment may be maintaining some of the cells in a hypertrophic state.
Osteocalcin Levels are Unexpectedly Elevated in Chondrogenic Treatment Groups:
Osteocalcin as a protein is found in an unstable state when not bound to the crystalline structures
developed during biomineralization. As a result, it can be used as a marker of biomineralization
and osteogenesis. But in all cells in the experiment, osteocalcin expression was not only present
(Fig. 3), but expression levels were reasonably comparable between most treatment groups. It
was not expected that osteocalcin would be present at such high levels in cells that were only
exposed to chondrogenic media, but it is expressed in both the 1-week and 2-week treatment
groups (Fig. 3A). While it is possible that the initial biomineralization during chondrocyte
hypertrophy may allow for osteocalcin to be stabilized, it seems unlikely to be the case.
Expression Patterns of Osteopontin are Difficult to Characterize: The other osteogenic
marker for this experiment, osteopontin, is not consistently expressed across the different
treatment groups. While it is expected that expression of osteopontin would be low in the 1-week
treatment plate (Fig. 3A), osteopontin was also not expressed across various treatment groups in
the 2- and 6-week treatment groups. It is not expressed in the treatment group grown using only
added GÖ6983 (Fig. 3B), which may be attributed to the Notch signaling suppressing maturation
into osteoblasts. Interestingly, expression is also absent in two of the SAG treatment groups,
GÖ6983 at a low concentration and BMP2 at a high concentration, but not the other SAG
treatment groups (Fig. 3B).
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The Presence of Col10a1 is a Stronger Indicator for Cartilage Staining that Col2a1:
Across the treatment groups, hypertrophy marker Col10a1 is more strongly expressed than
Col2a1—an indicator of pre-hypertrophic chondrocytes (Fig. 3) Similarly, Alcian Blue has
successfully stained for chondrocytes and cartilage in all cell cultures (Fig. 2), even those that are
showing very little or no presence of Col2a1, such as in plate 2, SAG and BMP2 treatment
groups (Fig. 2B, 3B). Together, the presence of Col10a1 and Alcian Blue staining indicates that
the ATDC5 cells have successfully undergone chondrogenesis, and Col10a1 also hints that the
cells may have entered hypertrophy during this experiment.
The Expression Patterns of Osteogenic Biomarkers Do Not Appear to be Entirely
Correlated with the Biomineralization Patterns: Osteocalcin and osteopontin, the two biomarkers
of osteogenesis that I primarily focused on, do not seem to completely match the pattern of
biomineralization that is found in the Alizarin Red stains. The presence of osteocalcin in plate 1
(Fig. 3A), would suggest that there should be biomineralization, but there is no evidence that
there is any (Fig. 2A). Similarly, the absence of osteopontin in the SAG/GÖ6983 low
concentration and SAG/BMP2 high treatment groups (Fig. 3B), should suggest that there is
limited osteogenesis in those cell treatments, and as a result, minimal biomineralization, but the
opposite appears to be true (Fig. 2B, 2C).
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Discussion:
Control Treatment Groups Appear to Stimulate Differentiation of ATDC5 Cells: In both
the chondrocyte differentiation treatment groups, and the osteoblast differentiation treatment
groups, the cells show indications of differentiation into the expected cell type. In all conditions,
Alcian Blue stains indicate the presence of chondrocytes (Fig. 2), which implies that the cells,
even after only a single week in chondrogenic media, have begun to differentiate into
chondrocytes. It also appears that the expression levels of cells that are exposed to chondrogenic
media for a single week differ than those of the cells treated for 2 weeks (Fig. 3A). It is
surprising to see that the 2-week differentiation cycle is showing decreased expression levels of
Col2a1 comparatively (Fig. 3A), because one would expect that Col2a1 would be present since it
is expressed in much of plates 2 and 3 (Fig. 3B), albeit at low levels of expression. It may be
possible though that Col2a1 expression is still present, but just undetectable.
Cells that are transitioned into osteogenic media also show indications of osteogenesis
when grown for the entire 6-week cycle, exhibiting osteogenic markers osteocalcin and
osteopontin (Fig. 3B). Alternatively, the cells that had been grown for only a single week after
transitioning from chondrogenic media do not show the same expression patterns, only
exhibiting osteocalcin, but not osteopontin (Fig. 3A). This matches the pattern shown in the 2
separate Alcian Red stains, comparing short-term growth (Fig. 2A) and long-term growth (Fig.
2B). In cells only induced for 1-week in osteogenic media, there is no apparent evidence of
biomineralization occurring (Fig. 2A), but there is evidence that it has occurred after the 6-week
cycle (Fig. 2B)
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Instability of the Chondrogenic Micromass may have Affected the Differentiation of the
ATDC5 Cells: The cells were originally plated as micromass cultures, after the initial ATDC5
growth phase, following the protocol set out in Greco et al, 2011. It becomes apparent, when
looking at the images of the Alcian Blue and Alizarin Red stains (Fig. 2), that the cells seemed to
have spread from their initial micromass, which may be affecting their ability to differentiate
properly into chondrocytes. The cells that have managed to differentiate into chondrocytes
appear to be forming clusters of cells (Fig. 2), which may indicate that better cohesiveness of the
micromass may have induced a greater level of chondrogenesis in the wells.
Another problem that arose was that, due to the spread of cells from the initial
micromass, the cells had the chance to become highly confluent. Because the cells were grown
for 6 weeks in the wells, it is possible that the cells, given the opportunity to grow, became too
confluent and started to initiate apoptosis. Cells that have become too confluent show signs of
cell death, because of increased cell density (Song, Caplan, & Dennis, 2009), which may explain
the large regions within the wells in which no cells are visible. During the staining protocol, it
seemed as if a large portion of the cells had lifted off, which may indicate that the cells had been
dying during differentiation.
Cells That are Exposed to SAG Seem to Show Slightly More Clustered Osteogenesis
than Cells Only Treated with Bone Media: Examining the early expression patterns of the SAG
treatment group, contrary to cells exposed to only bone media, the SAG cells do show some,
although limited, expression levels of osteopontin as well as osteocalcin (Fig. 3A). This implies
that the cells treated with SAG may be beginning to initialize osteogenesis earlier than the cells
without SAG. After 6 weeks, the gene expression patterns appear to be comparable to those of
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the bone media treatment group, which also does imply that osteogenesis is occurring at the time
of analysis (Fig. 3B). Unlike the 6-week bone media treatment group, the SAG treatment group
staining results do appear to be slightly more clustered, with a noticeably large region of cells
becoming stained red (Fig. 2B), although the results are not quite as prominent as those in plate 3
(Fig. 2C). In terms of chondrogenesis, the SAG treatment does not appear to have any noticeable
difference from the control conditions in terms of staining (Fig. 2). Furthermore, the expression
of chondrocyte markers Col2a1 and Col10a1 in SAG cells is similar to that of bone cells (Fig.
3), indicating that, like the control groups, chondrogenesis has occurred.
Differentiation with the Addition of BMP2 Does Not Appear to be Substantially Distinct
from the Control Osteogenic Media: BMP2, when mixed with osteogenic differentiation media,
shows very similar results to those found in cells grown only with the osteogenic media.
Comparing the RT-PCR results, BMP2 treated cells exhibit decreased levels of Col2a1 (Fig. 3B)
than the control condition, but this may be due to the cells being pushed more towards
hypertrophy in BMP2 cells. The Alcian Blue stain does not appear to show any difference when
compared to the control condition though, indicating that chondrogenesis had occurred during
the experiment (Fig. 2B). In the same way, there is no evidence of changes in osteogenesis
between the BMP2 treated cells and the control osteogenic cells with respect to
biomineralization (Fig. 2B)
GÖ6983 Inhibits the Ability of ATDC5 Cells to Fully Mature as Osteoblasts: Cells that
were treated with GÖ6983 show a definite reduction in osteogenic characteristics when
compared to the control cells. While there is no change in chondrogenic markers (Fig. 3B) or
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Alcian Blue staining patterns (Fig. 2B), the cells treated with GÖ6983 do not express osteopontin
(Fig. 3B), a common osteogenic biomarker. Furthermore, the GÖ6983 treated cells were the only
cells grown for the 6-week period which show a complete absence of biomineralization (Fig.
2B). This implies that GÖ6983, which acts as an activator of the Notch pathway, may be limiting
the ability of ATDC5 cells to mature into osteoblasts, similar to its inhibitory effect on
osteoprogenitor cells (Hilton et al, 2008).
SAG, When Acting in Tandem with Other Reagents, has a Noticeable Effect on the
Levels of Biomineralization in Differentiated ATDC5 Cells: Across all the treatment groups
presented in plate 3, the cells show a substantially different pattern biomineralization than the
cells that have been treated with only a single reagent (Fig. 2B,2C). Namely, the regions in
which Alizarin Red has successfully stained the cells are much larger than in the cells that were
only treated with a single reagent. Interestingly, while there seems to be significantly larger
regions of biomineralization, there also appears to be larger gaps in between regions of
biomineralization in the cells on plate 3 (Fig. 2C) than in the cells on plate 2 (Fig. 2B). This
result seems to fit well with the biomineralization seen in SAG treated cells, which appears to be
more clustered than that of cells grown without SAG (Fig. 2B).
It should be noted that the cells in plate 3 do not have consistent expression patterns of
osteogenic genes (Fig. 3B). More specifically, 2 of the 4 treatment groups exposed to multiple
reagents do not show any indication of osteopontin at all (Fig. 3B). While it may be expected
that a treatment containing GÖ6983 may have limited osteogenesis, as is found in the cells only
grown with GÖ6983, indicated by the lack of osteopontin (Fig. 3B, 3B), the cells that are grown
with both SAG and BMP2 also show a surprising absence of osteopontin (Fig. 3B). This acts in
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contrast to the very prominent levels of biomineralization in both the SAG/GÖ6983 low
concentration cells and the SAG/BMP2 high concentration cells (Fig. 2B). It may be therefore
viable to test the cells at varying concentrations of GÖ6983 and BMP2 to determine whether
there is a reason for this result.
The Presence of BMP2 may have a Similar Effect on Hybrid Cell Generation as SAG:
BMP2 has been well characterized as a necessary component in the generation of chondrocytes
and achieving chondrocyte hypertrophy (Kobayashi et al, 2005). Therefore, if the formation of
hybrid cells is reliant on chondrocyte hypertrophy, it is highly possible that BMP2 is necessary
for the generation of hybrid cells. When comparing the results between SAG treated cells and
BMP2 treated cells, there are no substantial differences in both extracellular markers and in gene
expression. Both treatment groups show evidence of biomineralization after 6 weeks, and
therefore, so I cannot make any conclusion on whether BMP2 or SAG contributes more to the
biomineralization patterns seen on plate 3.
The State of Chondrocytes Necessary for Hybrid Cell Formation is Still Uncharacterized:
Chondrocytes can exist in multiple stages of their differentiation pathway—pre-hypertrophic and
hypertrophic. Hypertrophic chondrocytes, characterized for example by the presence of Col 10 in
vitro, are classically part of the endochondral ossification pathway, and as a result, I have
theorized that hypertrophy may be necessary in the formation of hybrid cells. While it appears
that in my results, I have detectable levels of Col 10 across all the different treatment groups
(Fig. 3), indicating that my cells have likely achieved hypertrophy, it is not enough to say that
hypertrophy is required for my differentiation pathway.
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I have not yet verified hypertrophy in my cell cultures, and as a result, it is entirely
possible that the formation of hybrid cells may replicate the formation of osteoblasts during
endochondral ossification. Conversely, it is possible that the cells need to be pre-hypertrophic to
reach a sufficiently hybrid state, in order to still retain markers more indicative of chondrocytes
rather than entirely becoming osteoblast-like. Therefore, while hypertrophy may be important in
the generation of hybrid cells, it is not guaranteed that the chondrocytes are becoming
hypertrophic before differentiating.
Osteocalcin is Limited as an Osteogenic Marker Specifically in ATDC5 Cells: At the
onset of the experiment, osteocalcin was chosen as a biomarker for osteoblasts because of its role
in osteogenesis. Unfortunately, osteocalcin appeared to be present in all the RT-PCR results (Fig.
3), which makes it appear to be less viable as an osteogenic marker, since it was also present in
cells that were not exposed to any osteogenic differentiation media. While one possibility is that
osteocalcin is present due to the ability of hypertrophic chondrocytes to produce part of the
crystalline biomatrix, it would not be expected to be present in cells that are not showing any
visible forms of biomineralization. It is more probable that the apparent expression of
osteocalcin is due to the usage of ATDC5 cells, which show increased osteocalcin expression
during chondrogenic differentiation because of the MAP kinase pathway (Han et al, 2016). As a
result, the potential of osteocalcin as a biomarker for osteogenesis in ATDC5 cells is severely
limited, because it is increasingly expressed in the control chondrogenic conditions.
Osteogenesis may be Occurring from Initially Undifferentiated ATDC5 Cells, rather than
from Chondrocytes: Despite having evidence that cells treated with osteogenic media begin
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biomineralization after 6 weeks, I have yet to test whether the differentiation into chondrocytes
was necessary for biomineralization to occur. The common pathways of osteogenesis are
endochondral ossification and intramembranous ossification, which rely on cartilage formation
and mesenchymal stem cells respectively. While ATDC5 cells are neither chondrogenic nor
mesenchymal stem cells, they do show pre-chondrogenic characteristics, and it would have been
beneficial for my project to test whether direct differentiation into osteoblasts can occur from
ATDC5 cells, bypassing the differentiation into chondrocytes.
The Presence of Osteopontin is not a Guarantee for Osteogenesis: Despite endochondral
ossification being a well-established pathway for the formation of bone, it is not a certainty that
the ATDC5 cells are being differentiated into first chondrocytes and then osteoblasts.
Osteopontin is known to be “involved in multiple steps of tumor biology, including cell
proliferation, survival, angiogenesis, chemoresistance, stem-like properties, tumor invasion, and
metastasis” (Wei, Wong, Kwok, 2017). As a result, it is difficult to distinguish the emerging cells
as osteoblasts without further considering the morphology or other biological properties of these
resultant cells, tests for which have yet to be done in this experiment.
The conclusion is further complicated by the presence of osteopontin in the cartilage cells
that have only been induced for 1 week (Fig. 3A). If osteopontin were a marker indicating
biomineralization, and as a result, indicating the presence of osteoblasts in the cell, then one
would expect to evidence of biomineralization in the 1 week grown chondrocyte culture.
Furthermore, the fact that the marker was present in 1-week chondrocytes means that the
presence of osteopontin in longer grown cells (Fig. 3B) is no longer a guarantee of osteogenesis,
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and may in fact be from the osteopontin-expressing chondrocytes which were differentiated into
biomineralizing cells.
Furthermore, on the opposite end, certain treatment groups that show biomineralization
show a definite lack of osteopontin expression (Fig. 3B). Neither cells that are exposed to SAG
and GÖ6983 at a low concentration nor SAG and BMP2 at a high concentration show any visible
expression levels of osteopontin, despite there being biomineralization in both treatment groups
(Fig. 2C). It is possible that this is a result of biomineralization occurring at an earlier time point
than at 6-weeks, with the expression levels of osteopontin decreasing after biomineralization has
occurred. Unfortunately, this conclusion seems to lack validity because of the comparable band
intensity of osteocalcin compared to other cells in other treatment groups (Fig. 2C), although, as
mentioned above, osteocalcin presence is not the strongest indicator for the state of the
differentiating ATDC5 cells. Another possibility though is that the cells have become more like
hypertrophic chondrocytes, which still produce a mineralized matrix, rather than differentiating
fully into osteoblasts.
Cells Grown for 6 Weeks More Closely Resemble the Cells Differentiated for a Shorter
Period: It is well established that biomineralization will often not occur within 1 to 2 weeks of
the beginning of differentiation, and as a result there is no indications of such presented in
Alizarin Red (Fig. 2A). Despite this, the treatment group of only 1 week of cartilage induction
seems to show markers, such as osteopontin and osteocalcin, that are indicative of eventual
osteoblast formation, or biomineralization at the very least (Fig. 3A). Conversely, the cells that
were differentiated for the entire 2-week cycle do not show the same markers, showing a lack of
both osteopontin and collagen type II (Fig. 3A), which is particularly interesting. The cells in
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plates 2 and 3 in general seem to have more similar expression patterns to the 1-week
differentiated cells, exhibiting both osteopontin—except for some of the cells grown with
GÖ6983—and collagen type II (Fig. 3B). It may be possible that the treatment with osteogenic
media forced the chondrocytes into a less hypertrophic state, but due to a lack of evidence of
hypertrophy, this cannot effectively be concluded.
Expression of Hybrid Cell Markers May Need to be Tested at Different Time Points:
Currently, in Zebrafish models, hybrid cells have been located which exhibit markers of both
chondrocytes and osteoblasts. Of note is that the different expression markers have appeared at
different time points post resection (Paul et al, 2016), with osteoblast markers Col1a1a and
Runx2 for example appearing by 6 days post resection, but Col10a1, which marks for
hypertrophy, not appearing until approximately 10 days post resection. Not only does expression
of the different markers occur progressively, it is important to note that, in the in vivo model,
there was evidence of some osteogenic markers appearing much earlier than the chondrogenic
markers. This acts opposite to my project, which has been attempting to synthesize hybrid cells
by first initiating chondrogenesis, followed by osteogenesis. This observation means that, even if
hybrid cells were produced in vitro by the protocol I had followed, it would not guarantee similar
hybrid cell functionality.
To validate any type of protocol I may be following, it would be beneficial to monitor the
progression of cell markers over different time points. Furthermore, I may need to reevaluate
whether the cells should be first differentiated into chondrocytes, because it appears that that
may not be the pathway from which hybrid cells are produced in vivo. There is a potential that
the osteogenesis pathway may occur prior to the chondrogenesis pathway in hybrid cell
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formation, which illustrates a further need to test the osteogenic differentiation of untreated
ATDC5 cells.
Osteoblasts have Limited Mitotic Activity, which may Negate the Effects of Chondrocyte
Induced Hybrid Cells: Osteoblasts have been found to be antiproliferative, because of increased
osteoblast-specific runx2 expression (Galindo et al, 2005). The process of endochondral
ossification, which is commonly found in fracture repair, requires that bone length be increased
by the rapid proliferation of chondrocytes via interstitial proliferation. The differentiation
therefore of chondrocytes into hybrid cells, which should express characteristics of osteoblasts,
will likely decrease the mitotic activity of the cell culture. As a result, the production of in vitro
hybrid cells may not be very applicable in the long term for injury repair, or at the very least,
further research must be done to done to ensure that there are sufficient cells during treatment to
assist in repair.. While the limited proliferation of cells would reduce the risk of tumorigenesis, a
lack of proliferation may simultaneously render the use of the cells as a treatment as ineffective.
Depending on the function of the hybrid cells in bone repair—whether they act along the
entire length of the regeneration site, or they act as an initial starting colony for production of
other hybrid or regenerative cells—it is possible that the induction of hybrid cells may need to
happen in vivo. In vivo, the hybrid cells are primarily responsible for the biomineralization
process, but these cells are largely produced from the periosteum, which is largely known for
producing osteoprogenitor cells (Paul et al, 2016). Therefore, it may be more viable to study
whether the hybrid cells can be produced from osteoprogenitor cells, rather than from
chondroprogenitor cells such as ATDC5 cells.
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Murine Chondrocyte-Osteoblast Hybrid Cells are not Currently Well Characterized or
Defined: One of the problems surrounding this project is a lack of information of whether hybrid
cells can be reasonably generated in a mouse cell line. So far, hybrid cells have been in the
jawline of zebrafish, which exhibit innate highly regenerative properties. But because they have
not yet been well characterized in mice in vivo, it is entirely possible that ATDC5 cells, which
are derived from mice teratocarcinomas, may lack the ability to differentiate into the type of
regenerative hybrid cell which exhibits the properties we are interested in. Instead, if they can
differentiate into a hybrid state, they may instead proceed into an intermediate form, that does
not exhibit the same regenerative properties as found in Zebrafish. Until they are better defined, I
am currently unable to conclude whether the cells that I have produced would even showcase
similar properties to an in vivo produced hybrid mouse cell, and by extension, whether they
would have the same beneficial efficacy as an in vivo mouse hybrid cell.
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Further Directions
Assays to Determine whether Cell Cultures are Mixed or Hybrid are Necessary Before
any Further Experimentation: Based on only the results present, it is currently impossible to
conclude whether the cell cultures which are showing indications of both chondrogenesis and
osteogenesis are a mixed culture of both chondrocytes and osteoblasts, or are true hybrid cells.
To address this issue, individual cells would need to be tested for the presence of both osteogenic
markers and chondrogenic markers simultaneously. The cells which have progressed into the
biomineralization stage are still showing indications of being chondrocyte like, in the shape of
Col2a1 and Col10a1 signaling still being present (Fig. 3B). It may be the case though that there
are still cells which are still chondrocytes, which becomes apparent when looking at the presence
of Alcian blue staining after 6 weeks (Fig. 2B.3C).
A colocalization assay, by using a secondary antibody stain marking for two separate
proteins—one osteogenic, one chondrogenic—would be a strong indicator of whether individual
cells are exhibiting characteristics of both cells. If there is little overlap in the fluorescent readout
of the two antibodies, then it can be assumed that the cells are not differentiating into a hybrid
state. If there is an overlap of the antibody fluorescent signal, then one of two conclusions can be
made: either the cells are exhibiting hybrid qualities, or that some of the cells within a cluster of
chondrocytes are differentiating into osteoblasts and beginning to biomineralize.
To test this second possibility, one method may be to use multiple sequences of FACS to
separate the cells. The cells would be first sorted by whether they show specific osteogenic
markers, and followed by another sorting step to separate the cells which also show
chondrogenic markers. In theory, if none of the cells are hybrid, and therefore do not show any
indication of both osteogenic and chondrogenic markers, then there should be no cells after both
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sorts. This would give insight as to whether the cells are hybrid or are a mixed colony existing
within the same location.
Quantification of Both Gene Expression and Cell Count Would Allow for Statistical
Analysis: As of now, this project has relied entirely on qualitative results and observations,
which results in a very vague understanding of the underlying nuances of the experiment. To
better test whether any of the treatment groups had a statistically significant impact on osteoblast
or hybrid cell differentiation, I would need to test for quantitative data. There are two primary
aspects which I may be able to analyze quantitatively: the relative gene expression of hybrid cell
biomarkers such as osteogenic marker Col1a1a and chondrogenic marker Col10a1, and the
relative cell count for those that exhibit hybrid cell biomarkers.
To test for gene expression, I would use a quantitative RT-PCR assay for specific
biomarkers, normalized to the gene expression levels of a housekeeping gene such as GAPDH.
Doing so would allow me to determine whether any of the treatments substantially changed gene
expression compared to the control conditions. Measuring cell count on the other hand may give
some insight as to whether differentiation into biomineralizing cells is occurring more
frequently—either because differentiation is occurring earlier or at a faster rate—in different
osteogenic treatment groups. This could be achieved through FACS, sorting the cells by the
presence of osteogenic markers, and normalized as a percentage of the total cell count per well.
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Reorganization of the Differentiation Protocol May Generate Results Closer to that of the
in vivo Model: The current protocol that I have been using for this experiment follows a protocol
which serves to copy endochondral ossification. While this is acceptable for the formation of
biomineralizing osteoblasts, it may be counterproductive to produce hybrid cells. The zebrafish
model of hybrid cells seems to indicate the presence of certain osteogenic markers prior to the
production of chondrogenic markers, which should not be the case under endochondral
ossification methods. As a result, it may be productive to study whether a hybrid-like state could
be induced by first stimulating partial osteogenic differentiation, and then transitioning to a
chondrogenic differentiation condition.
As it is also important to understand the role that hypertrophy has in the generation of
hybrid cells, it would be useful to test the formation of hybrid cells from cells which are limited
from becoming hypertrophic. One method to do so would be to generate ATDC5 cells which
may be Col10a1
-/-
, or using an inducible Cre system or miRNA to knockdown expression of
Col10a1 in the ATDC5 cells, or to use a compound such as Pterosin B (Yahara, 2016). Since
Col10a1 has been found to play a role in induction of hypertrophy (Dong et al, 2006), and
eventual removal of chondrocytes and generation of osteoblasts (Yahara. 2016), a knockout line
could be used to prevent the transition into chondrocytes. Using the same protocol on the
Col10a1 impaired cells, we could test the importance of hypertrophy on hybrid cell formation.
Reorganization of Reagents May Help Determine Whether the Patterns of
Biomineralization Can be Attributed to SAG: To test whether BMP2 has a comparable effect as
SAG on osteogenesis, it would be beneficial to create a treatment group that combines BMP2
and GÖ6983, because I have already tested the effects of SAG combined with GÖ6983. If a
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combined treatment of BMP2 and GÖ6983 could stimulate biomineralization like SAG and
GÖ6983, then it would help provide evidence that stimulating the hedgehog pathway may not be
necessary for sufficient biomineralization. It appears that combining treatments does stimulate a
different biomineralization pattern than treatment with only a single reagent, but it cannot be
concluded that it is inherently because of SAG as the second reagent that we see these
biomineralization patterns.
Hybrid Cell Culture Application to Injured Bones May Aid in Regeneration: The
eventual goal of the project is to create a working protocol for generating hybrid cells which can
be used help in skeletal regeneration. This implies that the hybrid cells that are currently being
grown need to have some positive effect on the rate of regeneration in injured mice for the
project to be successful. It is therefore important to test the hybrid cells, grown in vitro, in an in
vitro model to determine whether there is any impact on the bone repair pathways in mice.
In the original study, the hybrid cells facilitated the process of jawbone repair of a
zebrafish by producing their own biomineralized extracellular matrix, despite being more
chondrocyte-like (Paul et al, 2016). It is therefore hypothesized that the dish grown hybrid cells
should have similar effects and function, providing their own degree of biomineralization to help
with the progression of bone repair. Because it can already be seen that the cells that have been
generated through this project are exhibiting biomineralization when grown in a well (Fig.
2B,2C), it is very possible that these same cells, when implanted into an injury site, will begin to
generate a biomineralized matrix and facilitate bone repair.
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Acknowledgements
I would like to acknowledge the members of my laboratory, especially my principal
investigator, Dr. Francesca Mariani, as well as fellow researchers Neel Hedge and Zeferino
Reyna, and our laboratory technician Ashlie Muñoz, for helping me to create and conduct my
experiment over the last 2 years. Without their help, I would not have had the tools or the
experience to start any of my own project. I would also like to acknowledge Dr. Amy Merrill and
Dr. Jian Xu for sitting on my committee and giving me suggestions as to how to improve the
work that I had been doing so far. Finally, I would like to give a special acknowledgement to Dr.
Merrill for lending me my initial batch of frozen ATDC5 cells, without which I would not have
been able to do any of my research for my thesis project.
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References
Akiyama, Haruhiko et al. “Interactions between Sox9 and Β-Catenin Control Chondrocyte
Differentiation.” Genes & Development 18.9 (2004): 1072–1087. PMC. Web. 25 May
2018.
Andersson, Emma R., et al. “Notch Signaling: Simplicity in Design, Versatility in Function.”
Development, vol. 138, no. 17, 2011, pp. 3593–3612., doi:10.1242/dev.063610.
Andreassen, Kim Vietz, et al. “Investigation of Chondrocyte Hypertrophy and Cartilage
Calcification in a Full-Depth Articular Cartilage Explants Model.” SpringerLink,
Springer, Dordrecht, 28 Mar. 2012, link.springer.com/article/10.1007/s00296-012-2368-
6.
Berndt, Nicole et al. “Ubiquitylation-Independent Activation of Notch Signalling by Delta.”
Ed.KVijayRaghavan. eLife 6 (2017): e27346. PMC. Web. 24 May 2018.
Boonrungsiman, Suwimon, et al. “The Role of Intracellular Calcium Phosphate in Osteoblast-
Mediated Bone Apatite Formation.” PNAS, National Academy of Sciences, 28 Aug.
2012, www.pnas.org/content/109/35/14170.long.
Bragina, Olga, et al. “Smoothened Agonist Augments Proliferation and Survival of Neural
Cells.” Neuroscience Letters, vol. 482, no. 2, 2010, pp. 81–85.,
doi:10.1016/j.neulet.2010.06.068.
Carpintero, Pedro et al. “Complications of Hip Fractures: A Review.” World Journal of
Orthopedics 5.4 (2014): 402–411. PMC. Web. 22 May 2018.
Chen, Haiyan et al. “Runx2 Regulates Endochondral Ossification through Control of
Chondrocyte Proliferation and Differentiation.” Journal of bone and mineral research :
the official journal of the American Society for Bone and Mineral Research 29.12 (2014):
2653–2665. PMC. Web. 24 May 2018.
Dong, Yu-Feng, et al. “Wnt Induction of Chondrocyte Hypertrophy through the Runx2
Transcription Factor.” Journal of Cellular Physiology, vol. 208, no. 1, 2006, pp. 77–86.,
doi:10.1002/jcp.20656
Einhorn, Thomas A., and Gerstenfeld, Louis C. “Fracture Healing: Mechanisms and
Interventions.” Nature reviews. Rheumatology 11.1 (2015): 45–54. PMC. Web. 22 May
2018.
Enochson, Lars, Mats Brittberg, and Anders Lindahl. “Optimization of a Chondrogenic Medium
Through the Use of Factorial Design of Experiments.” BioResearch Open Access 1.6
(2012): 306–313. PMC. Web. 25 May 2018.
Adrian Wang 44
5516738666
Fausett, Blake V., and Daniel Goldman. “A Role for α1 Tubulin-Expressing Müller Glia in
Regeneration of the Injured Zebrafish Retina.” Journal of Neuroscience, Society for
Neuroscience, 7 June 2006, www.jneurosci.org/content/26/23/6303.
Flierl, Michael A et al. “Outcomes and Complication Rates of Different Bone Grafting
Modalities in Long Bone Fracture Nonunions: A Retrospective Cohort Study in 182
Patients.” Journal of Orthopaedic Surgery and Research 8 (2013): 33. PMC. Web. 22
May 2018.
Friesenbichler, Joerg et al. “Adverse Reactions of Artificial Bone Graft Substitutes: Lessons
Learned From Using Tricalcium Phosphate geneX
®
.” Clinical Orthopaedics and Related
Research 472.3 (2014): 976–982. PMC. Web. 22 May 2018.
Furumatsu, Takayuki, et al. “Smad3 Activates the Sox9-Dependent Transcription on
Chromatin.” The International Journal of Biochemistry & Cell Biology, vol. 41, no. 5,
2009, pp. 1198–1204., doi:10.1016/j.biocel.2008.10.032.
Galindo, Mario et al. “The Bone-Specific Expression of Runx2 Oscillates during the Cell Cycle
to Support a G1-Related Antiproliferative Function in Osteoblasts.” The Journal of
biological chemistry 280.21 (2005): 20274–20285. PMC. Web. 18 May 2018.
Gao, Yue, et al. “The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes.”
BioMed Research International, Hindawi Publishing Corporation, 18 May 2014,
www.ncbi.nlm.nih.gov/pmc/articles/PMC4052144/.
Goldshmit, Yona, et al. “Fgf-Dependent Glial Cell Bridges Facilitate Spinal Cord Regeneration
in Zebrafish.” Journal of Neuroscience, Society for Neuroscience, 30 May 2012,
www.jneurosci.org/content/32/22/7477.
Greco, K.V. et al. “High Density Micromass Cultures of a Human Chondrocyte Cell Line: A
Reliable Assay System to Reveal the Modulatory Functions of Pharmacological Agents.”
Biochemical pharmacology 82.12 (2011): 1919–1929. PMC. Web. 26 May 2018.
Hadzir, Siti Norhaiza, et al. “Ascorbic Acid Induces Osteoblast Differentiation of Human
Suspension Mononuclear Cells.” Cytotherapy, vol. 16, no. 5, 2014, pp. 674–682.,
doi:10.1016/j.jcyt.2013.07.013.
Hamada, Takashi et al. “Surface Markers and Gene Expression to
Characterize the Differentiation of Monolayer Expanded Human Articular
Chondrocytes.” Nagoya Journal of Medical Science 75.1-2 (2013): 101–111. Print.
Han, Yingchao et al. “Leptin Induces Osteocalcin Expression in ATDC5 Cells through
Activation of the MAPK-ERK1/2 Signaling Pathway.” Oncotarget 7.39 (2016): 64021–
64029. PMC. Web. 11 June 2018.
Adrian Wang 45
5516738666
Hilton, Matthew J. et al. “Notch Signaling Maintains Bone Marrow Mesenchymal Progenitors by
Suppressing Osteoblast Differentiation.” Nature medicine 14.3 (2008): 306–314. PMC.
Web. 25 May 2018.
Holstein, Joerg H., et al. “Mouse Models for the Study of Fracture Healing and Bone
Regeneration.” Osteoporosis Research, 26 Apr. 2011, pp. 175–191., doi:10.1007/978-0-
85729-293-3_14.
Hori, Kazuya, Anindya Sen, and Spyros Artavanis-Tsakonas. “Notch Signaling at a Glance.”
Journal of Cell Science 126.10 (2013): 2135–2140. PMC. Web. 24 May 2018.
Hu, Diane P. et al. “Cartilage to Bone Transformation during Fracture Healing Is Coordinated by
the Invading Vasculature and Induction of the Core Pluripotency Genes.” Development
(Cambridge, England) 144.2 (2017): 221–234. PMC. Web. 22 May 2018.
Hu, Hongliang, et al. “Sequential Roles of Hedgehog and Wnt Signaling in Osteoblast
Development.” Development, vol. 132, no. 1, 2004, pp. 49–60., doi:10.1242/dev.01564.
Jopling, Chris, et al. “Zebrafish Heart Regeneration Occurs by Cardiomyocyte Dedifferentiation
and Proliferation.” Nature, U.S. National Library of Medicine, 25 Mar. 2010,
www.ncbi.nlm.nih.gov/pmc/articles/PMC2846535/.
Kalmar, Lajos, et al. “Structural Disorder in Proteins Brings Order to Crystal Growth in
Biomineralization.” Bone, vol. 51, no. 3, 2012, pp. 528–534.,
doi:10.1016/j.bone.2012.05.009.
Kobayashi, Tatsuya, et al. “BMP Signaling Stimulates Cellular Differentiation at Multiple
Steps during Cartilage Development.” PNAS, National Academy of Sciences, 13 Dec.
2005,
Kohn, Anat, et al. “Notch Signaling Controls Chondrocyte Hypertrophy via Indirect Regulation
of Sox9.” Bone Research, vol. 3, no. 1, 2015, doi:10.1038/boneres.2015.21.
Komori, Toshihisa. “Regulation of Osteoblast and Odontoblast Differentiation by RUNX2.”
Journal of Oral Biosciences, vol. 52, no. 1, 2010, pp. 22–25.,
doi:10.2330/joralbiosci.52.22.
Kraan, P.m. Van Der, et al. “TGF-Beta Signaling in Chondrocyte Terminal Differentiation and
Osteoarthritis.” Osteoarthritis and Cartilage, vol. 17, no. 12, 2009, pp. 1539–1545.,
doi:10.1016/j.joca.2009.06.008.
Langenbach, Fabian, and Jörg Handschel. “Effects of Dexamethasone, Ascorbic Acid and Β-
Glycerophosphate on the Osteogenic Differentiation of Stem Cells in Vitro.” Stem Cell
Research & Therapy 4.5 (2013): 117. PMC. Web. 26 May 2018.
Adrian Wang 46
5516738666
Lewis, Cristy, and Paul A. Krieg. “Reagents for Developmental Regulation of Hedgehog
Signaling.” Methods, vol. 66, no. 3, 2014, pp. 390–397.,
doi:10.1016/j.ymeth.2013.08.022.
Long, Fanxin, et al. “Ihh Signaling Is Directly Required for the Osteoblast Lineage in the
Endochondral Skeleton.” Development, vol. 131, no. 6, 2004, pp. 1309–1318.,
doi:10.1242/dev.01006.
Liu, Xia et al. “Role of Insulin-Transferrin-Selenium in Auricular Chondrocyte Proliferation and
Engineered Cartilage Formation in Vitro.” International Journal of Molecular Sciences
15.1 (2014): 1525–1537. PMC. Web. 26 May 2018.
Maes, Christa et al. “Osteoblast Precursors, but Not Mature Osteoblasts, Move into Developing
and Fractured Bones along with Invading Blood Vessels.” Developmental cell 19.2
(2010): 329–344. PMC. Web. 23 May 2018.
Mackie, E J, et al. “Endochondral Ossification: How Cartilage Is Converted into Bone in the
Developing Skeleton.” The International Journal of Biochemistry & Cell Biology., U.S.
National Library of Medicine, 29 Jan. 2007, www.ncbi.nlm.nih.gov/pubmed/17659995.
Mah, In Kyoung et al. “Atypical PKC-Iota Controls Stem Cell Expansion via Regulation of the
Notch Pathway.” Stem Cell Reports 5.5 (2015): 866–880. PMC. Web. 6 June 2018.
Mak, Kinglun Kingston, et al. “Hedgehog Signaling in Mature Osteoblasts Regulates Bone
Formation and Resorption by Controlling PTHrP and RANKL Expression.” Bone, vol.
42, 2008, doi:10.1016/j.bone.2007.12.032.
Matsumoto, Goichi, et al. “Bone Regeneration by Polyhedral Microcrystals from Silkworm
Virus.” Scientific Reports, vol. 2, no. 1, 6 Dec. 2012, doi:10.1038/srep00935.
Miyazaki, Y., et al. “Dexamethasone Inhibition of TGFβ-Induced Cell Growth and Type II
Collagen MRNA Expression through ERK-Integrated AP-1 Activity in Cultured Rat
Articular Chondrocytes.” Osteoarthritis and Cartilage, vol. 8, no. 5, 2000, pp. 378–385.,
doi:10.1053/joca.1999.0313.
Naito, Masako, et al. “Dexamethasone Inhibits Chondrocyte Differentiation by Suppression of
Wnt/β-Catenin Signaling in the Chondrogenic Cell Line ATDC5.” Histochemistry and
Cell Biology, vol. 144, no. 3, 2015, pp. 261–272., doi:10.1007/s00418-015-1334-2.
Ortega, Nathalie, Behonick, Danielle J., and Werb, Zena. “Matrix Remodeling during
Endochondral Ossification.” Trends in cell biology 14.2 (2004): 86–93. PMC. Web. 23
May 2018.
Adrian Wang 47
5516738666
Paul, Sandeep et al. “Ihha Induces Hybrid Cartilage-Bone Cells during Zebrafish
Jawbone Regeneration.” Development (Cambridge, England) 143.12 (2016): 2066–2076.
PMC. Web. 30 Jan. 2018.
Shu, Bing et al. “BMP2, but Not BMP4, Is Crucial for Chondrocyte Proliferation and Maturation
during Endochondral Bone Development.” Journal of Cell Science 124.20 (2011): 3428–
3440. PMC. Web. 24 May 2018.
Smith, Christian A et al. “aPKC-Mediated Phosphorylation Regulates Asymmetric Membrane
Localization of the Cell Fate Determinant Numb.” The EMBO Journal 26.2 (2007): 468–
480. PMC. Web. 25 May 2018.
Song, In-Hwan, et al. “Dexamethasone Inhibition of Confluence-Induced Apoptosis in Human
Mesenchymal Stem Cells.” Journal of Orthopaedic Research, vol. 27, no. 2, 2009, pp.
216–221., doi:10.1002/jor.20726.
Srour, M. K. et al. (2015), Natural Large-Scale Regeneration of Rib Cartilage in a Mouse Model.
J Bone Miner Res, 30: 297–308. doi:10.1002/jbmr.2326
St-Jacques, Benoit, Matthias Hammerschmidt, and Andrew P. McMahon. “Indian Hedgehog
Signaling Regulates Proliferation and Differentiation of Chondrocytes and Is Essential
for Bone Formation.” Genes & Development 13.16 (1999): 2072–2086. Print.
Temu, Tecla M. et al. “The Mechanism of Ascorbic Acid-Induced Differentiation of ATDC5
Chondrogenic Cells.” American Journal of Physiology - Endocrinology and Metabolism
299.2 (2010): E325–E334. PMC. Web. 25 May 2018.
Teng, Songsong et al. “Smoking and Risk of Prosthesis-Related Complications after
Total Hip Arthroplasty: A Meta-Analysis of Cohort Studies.” Ed. Masaru Katoh. PLoS
ONE 10.4 (2015): e0125294. PMC. Web. 22 May 2018.
Wang, Richard N. et al. “Bone Morphogenetic Protein (BMP) Signaling in Development and
Human Diseases.” Genes & diseases 1.1 (2014): 87–105. PMC. Web. 24 May 2018.
Wei, Ran, Janet Pik Ching Wong, and Hang Fai Kwok. “Osteopontin -- a Promising Biomarker
for Cancer Therapy.” Journal of Cancer 8.12 (2017): 2173–2183. PMC. Web. 15 May
2018.
Wheater, Gillian et al. “The Clinical Utility of Bone Marker Measurements in Osteoporosis.”
Journal of Translational Medicine 11 (2013): 201. PMC. Web. 23 May 2018.
Wu-Zhang, Alyssa X., and Alexandra C. Newton. “Protein Kinase C Pharmacology: Refining
the Toolbox.” The Biochemical journal 452.2 (2013): 195–209. PMC. Web. 25 May
2018.
Adrian Wang 48
5516738666
Yahara, Yasuhito et al. “Pterosin B Prevents Chondrocyte Hypertrophy and Osteoarthritis in
Mice by Inhibiting Sik3.” Nature Communications 7 (2016): 10959. PMC. Web. 17 May
2018
Yao, Y. and Wang, Y. (2013), ATDC5: An excellent in vitro model cell line for skeletal
development. J. Cell. Biochem., 114: 1223–1229. doi:10.1002/jcb.24467
Zanotti, Stefano, and Ernesto Canalis. “Notch and the Skeleton .” Molecular and Cellular
Biology 30.4 (2010): 886–896. PMC. Web. 15 May 2018.
Abstract (if available)
Abstract
Murine-derived ATDC5 cells were exposed to different media which have been shown to induce differentiation into both chondrocytes and osteoblasts from ATDC5 cells. The exposure time to the differentiation media varied between 2 to 6 weeks, and the treatments were used to produce cell cultures which exhibit characteristics of both chondrocytes and osteoblasts. The goal of this project was to generate and optimize a protocol which could be used to efficiently create hybrid chondrocyte-osteoblast cells which would later be used in regenerative bone research. ❧ The cells were treated with reagents which are known to have effects on the chondrocyte and osteoblast differentiation pathways. The three pathways chosen for investigation were the Hedgehog (Hh) pathway, TGF-β pathway, and the Notch pathway. To study the effect of stimulating these pathways on osteoblast formation, I treated ATDC5 cells using three separate reagents in varying quantities, smoothened agonist (SAG), Bone Morphogenetic Protein 2 (BMP2), and GÖ6983, mixed into bone differentiation media. ❧ Successful differentiation was tested using both RT-PCR and by extracellular matrix staining using Alcian blue and Alizarin red for cartilage matrix and bone matrix respectively. Gene expression was expected of certain osteogenic markers, such as osteocalcin, was thought to be increased in cells that had successfully differentiated into osteoblast-type cells. Similarly, expression of chondrogenic markers such as collagen, type X, was predicted to be expressed in chondrocyte-type cells. Hybrid cells were expected to express both these markers at least in early differentiation stages. Subsequent treatment of ATDC5 cells with chondrogenic and osteogenic media seemed to result in cells that share extracellular and genetic characteristics with chondrocytes and osteoblasts, but whether individual cells have become hybrid is still unknown.
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Wang, Adrian Cheng
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In vitro generation of chondrocyte-osteoblast hybrid cells through differentiation of ATDC5 cells
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Biochemistry and Molecular Biology
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