Gene Expression Patterns of Bone under normal mechanical load versus LSJL

Each day I go through studies to put into old posts and most of them are not that helpful but I found one that's full of one and insight's into the legendary microfracture theory sprouted by Sky and others.  Search for (*NEW*) to find it.  The study has a lot of great diagrams and the full study is available.  It's a rich informative study so it would be beneficial for other people to look into it.  Ultimately, you can see that the load required to induce a microfracture may be too much and if kicking with ankle weights induced the microfractures such as in this study you would notice them due to the massive appositional bone formation(thus if you did grow taller with such a method you'd have lots of bumps on your bones--chicks dig scars but do they dig microfractures?).  However, it may be possible for tapping with a sufficient load enough times may be enough to induce such a fracture.  I have no idea what that load is though.

CH Turner was one of the leading researchers behind Lateral Synovial Joint Loading before he passed away and he was the researcher who was most directly interested in bone(Hiroki Yokota for example was more interested in mechanotransduction).

CH Turner's studies are still coming out.  Here's one about the gene expression of bone on mechanical loading.  It will be interested to compare that to gene expression under LSJL.<-Read that study, it's important!

Gene expression patterns in bone following mechanical loading.

"The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day{so axial loading rather than our lateral loading, note that the epiphysis still gets loading under axial loading as there's no way to avoid it}, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1[The C-Fos and c-Jun complex]-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Chemokine-related genes, were upregulated early but downregulated later in the time course; solute carrier genes{these help with chondrocyte hypertrophy}, were both upregulated and downregulated; and muscle-related genes,  were primarily downregulated{This is interesting, as many anabolic pathways are shared between muscle and bone; this gives weight to the theory that muscle and bone compete for resources}."

The Peak Load used was 13N. "Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz"  24 hours between loading.  Genes upregulated in supplementary material were taken from all time points from 4 hours to 32 days.  Mice were 20 weeks old.

"Bone responds in an anabolic manner to physiologic dynamic loading. For example, the midshaft humerus in the throwing arm of baseball pitchers and catchers showed enhanced bone mass, structure, estimated strength, and resistance to torsion compared with the nonthrowing control arm.{bone length was measured but the comparative length was not presented, something to ask Stuart J. Warden} In contrast, bone mineral density (BMD) in astronauts decreased 1.0% and 1.5% in the spine and hip, respectively, per month of spaceflight{This is not that much considering astronauts are reported to gain 3 inches in space, this shows you the potential of your intervertebral discs in terms of height gain}"

"Mechanical loading uses pathways currently being investigated for new drug development, such as low density lipoprotein receptor–related protein 5 (LRP5) and sclerostin"<-supplements to look into

"New osteoblasts appear on the bone surface 24 to 48 hours after initiating mechanical loading, and bone formation is observed within 96 hours of loading. Bone formation increases between 5 and 12 days after starting loading, but after 6 weeks of loading, bone formation returns to baseline levels."

This is for osteoblasts not chondrocytes which is what we are mainly looking for with LSJL but  it may take 12 days after starting LSJL to notice new osteoblast bone deposition(never mind that LSJL requires a chondrocyte phase beforehand).  A month is not enough time to measure results.

The shaft of the bone was used so stem cell genes should be detected.  So if any mesenchymal chondrogenesis occurred it should show up.

Looking at what was upregulated in axial Loading and Lateral Loading share many of the same pathways like TGF-Beta and WNT/B-Catenin in addition to many ECM related proteins.

Some genes involved in LSJL that were not involved in axial loading hyaluronan synthase(involved in hyaluronic acid).  No MMP3 in axial loading in contrast to lateral loading(MMP-3 is stimulatory to chondrocytes). 

And of course no induction of chondrogenic differentiation of stem cells in axial loading(that's more due to hydrostatic pressure though than genes).  Though it should be represented by genes which I think it is given the upregulating of ECM genes.  Chondrogenic differentiation produces ECM but ECM doesn't always indicate chondrogenic differentiation.

BMP-2 and TGF-Beta were produced by axial loading.  Both of which can induce chondrogenic differentiation.  Axial Loading + LIPUS may be enough to gain height.

Interleukin 1 receptor-like 1 was expressed by both axial and LSJL.  Stat3 was expressed which induces Lin28B expression.  It also downregulated FGF23 which may be involved in growth plate reactivation.

Upregulated Genes of Note(see supplementary material):
Acan(upregulated in LSJL)
ADAMTS1(up)
Adh7(up)
Angptl2(up)
Apcs(down)
Apln(up)
Arg1(up)
Bambi
Bgn(up)
BMP2(up)
c1qtnf5(up)
c3ar1(up)
Capn6(up)
Car8(up)
ccl2(up)
ccl7(up)
cd14(up)
cd276(up)
CCND1
Cdh15(down)
Cgref1(up)
Chgb(down)
Cit(down)
Cntn1(up)
Fn1
Follistatin
Cntn1(up in LSJL)
Col2A1 alpha1(up in LSJL)
Col3A1 alpha1(up in LSJL)
Col16A1(up)
crabp2(up)
creb3l1(up)
cspg4(up)
cthrc1(up)
cxcl1(up)
dbx1(down)
dlg4(up)
dnm1(up)
ENPP3(down)
fkbp10(up)
Gfpt2(up)
ggcx(up)
glrb(up)
GPR180(down in LSJL)
grin2d(up)
Hapln1(up in LSJL)
Hdlbp(down)
Hif1alpha
Hnf4a(down in LSJL)
HTRA1(up)
Id2(down in LSJL)
il1rl1(up)
IRS-1(down in LSJL)
Junb(also upregulated in LSJL)
Kcnn2(up)
Lepre1(up)
Leptin
lmna(up)
lox(up)
Lrat(up)
Mall(up)
Metrnl(down)
MMP2(up in LSJL)
MMP9
MMP14(up in LSJL)
NDRG4(up)
Neurod2(down)
Ninj1(down)
Nkx2.5
Nos3
Nr4a2(up)
Pacsin1(down)
Pcdhb2(up)
pcsk6(up)
pdgfc(up)
pdpn(up)
prrx1(up)
prss35(up)
PTHR1
PTGS2(up in LSJL)
PTN(up in LSJL)
RPL36al(down)
S100A4(up)
Scn1a(up)
Sct(down)
Serpina3n(up)
Serpine1(up)
Sept5(down)
Slc1a4(up)
Slc6a2(up)
Slc6a15(up)
Slco2a1(up)
Smad9(up)
Smpd3(down)
SOCS2(Anti-height gene)
SOCS3(upregulated in LSJL)
Sp7
Stat3
Syndecan 4(also upregulated in LSJL)
TGFbp1
TGFbp3
tnfrsf12a(up)
TIMP1(up in LSJL)
Vcan(up in LSJL)
VDR
Zfp36(upregulated in LSJL)

Axial loading upregulated a few chondrogenic genes like Acan and COL2A1 but nowhere near the amount of Collagens upregulated by LSJL which also upregulated Col9.  Also key, is that Sox9 is not upregulated in axial loading whereas it is in LSJL.

Downregulated genes of note:
Acacb(down)
Acsl6(down)
Anxa3(down in LSJL)
Arl6ip1(down)
Asb2(down)
Asph(up)
BMPR1B(up in LSJL)
Btla(up)
ccnb1(down in LSJL)
ccr1(up)
dpp4(down)
Egr1(up in LSJL)
Fgr(down)
Fnbp1(down)
GADD45A
Galc(down)
Gas6
Ghitm(down)
GHR
IGFBP6(up in LSJL)
Kynu(up)
Leptin Receptor
Mkrn1(down)
Mrps18b(up)
Myl1(up)
Nexn(down)
Ntn1(up)
Pcsk1(up)
Pdlim3(up)
Pkia(up)
Plag1
Ppp1r3c(up)
Prkaa2(down)
Prkg2(up in LSJL)
Pygl(down)
Rsad2(down as Pcaf)
Sdpr(down)
Sla(down)
Slc16a1(down)
Slc25a30(down)
Sost
Srpkg3(down)
TGFBR3
Tnnt3(down)
Trim55(down)
Tsc22d3(down)
Ucp2(down)
Vav1(down)
Vcam1(down in LSJL)

The differential expression of Egr and BMPR1B between Axial Loading and Lateral Loading could be key to LSJL's ability to induce chondrogenesis.

The gene expression data for LSJL was taken 1 hour after the last loading and in this study genes were taken 4 hours after the first loading.  So we can compare these early response genes to see how they compare to LSJL.

Upregulated:
Fosl1
Junb(up in LSJL)
Anxa2
S100A4(up)
S100A10
CCBP2
CCL2(up)
CCL7(up)
CXCL1(up)
CXCL13
IL1RL1(up)
IL1RL2
Osm
Osmr
Socs3(up)
Stat3
Tnfrsf12a(up)
Adamts1(up)
ECM1
Serpina3n(up)
Serpine1(up)
Tfpi2
CCND2
Clic1
Gpr1
KCNE4
Lep
Syndecan4 (up)

Regulatory mechanisms in bone following mechanical loading.

"The right forelimb [of rodents] was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnas were sampled at 11 time points, from 4 hours to 32 days after beginning loading."

Mice were 20 weeks old.

"The peak load achieved during loading was 13 N"

Stat5B was upregulated 4 hours following loading. Stat5b is downregulated in LSJL

The expression of COL1 differs greatly with LSJL.  At 14 days there was almost no COL1 expression whereas at 14 days there was still extremely high COL1 levels with Axial Loading.

"The CREB-related transcription factors are important for bone formation, specifically ATF4, which is required for collagen synthesis by mature osteoblasts. The CREB motif was predicted to be positive at 2d, 4d, 6d, and 8d. The transcription factors that bind to the CREB motif include cAMP responsive element binding protein 1 (CREB1), cAMP responsive element modulator (CREM), activating transcription factor 1 (ATF1), ATF2, ATF3, ATF4, and ATF7. The CREB motif was present in the promoter of an important matrix gene, fibronectin 1 (Fn1), and in genes that promote collagen construction and cross-linking, including Lox, prolyl 4- hydroxylase beta polypeptide (P4hb), and procollagen C-endopeptidase enhancer (Pcolce)"

"at 32d, the system was less responsive to loading and had shifted from bone forming to baseline bone maintenance"<-maybe every 32 days take a break from LSJL?

Collagen 1 alpha 1 did not begin to rise until two days after loading.  The LSJL study took gene expression at 49 days after first loading.  In the axial loading Col1A was upregulated 3-fold seven days after loading whereas with LSJL it was upregulated only 2 fold.

Alternative Splicing in Bone Following Mechanical Loading

Alternative splicing means that gene expression was altered in mRNA.  The whole bone was ground.

"Compressive load was applied as an oscillating Haversine waveform for 360 cycles at a frequency of 2 Hz using a Bose ElectroForce 3200 Series electromechanical actuator"<-Peak Load was 13N.  Axial loading was used.

"Rats were subjected to loading sessions every day, with 24 hours between sessions."  Rats were 20 weeks old.  Gene expression data was taken up to 4 hours to 32 days.

The greatest alteration of gene expression occurred at 16 days or about 2 weeks.  Maybe this is when conditioning effect starts to inhibit gene expression?

According to this Study Sox9 mRNA was not altered at any time point.  Col2a1and Acan mRNA were altered.  Tgfbeta1 and Tgfbeta2 expression was altered.  The key stature genes HMGA2 and Lin28b were altered in LSJL but not here.

Altered genes of note:

Akt1

Akt2

BMPr1a

BMPr1b{up in LSJL}

BMP2{up in LSJL}

BMP4

CNP

CREB3l1

Esrra

Esrrb

Esr2{up in LSJL}

FGF2{up in LSJL}
FGF4

FGF21

FGFR1{up}

FGFR3

GH1{down in LSJL}

GHR

GHRHR

GPC3

HMGA1

ID2{down}

ID4

IGF1

IGF1R

IGF2R

IGF2bp1

NPR1

NPR2

NPR3

PLAG1

PRKG2{up}
RARA

Runx3

Shh

SHOX2

Smad1{down}

Smad2

Smad3

Smad4

Smad5

Smad7(inhibits BMP signaling, Smad6 which inhibits TGF-Beta signaling is not altered)

Smad9

Sox10

Sox11

Syn3

Twist1

Wnt4

Wnt5a

Here's the Partek GSEA Analysis to compare to LSJL for chondrogenic related genes, Bold means the p-value < 0.05, no fold changes were given and no fold cutoff is used:

Chondroblast Differentiation(6.77) 100%:
RARA
FGF4
FGF2
Cyr61

Chondrocyte Differentiation(5.16) 45%:
Col2a1
Creb3l2
MAPK14
Col11a2
TGFB1
Mef2d
FGFR1
OSR1
FGF9

Cartilage Condensation(8.32) 58.32%:
THRA
COL2A1
Tgfb2
Uncx
Ctgf
Bmpr1b
Acan

Chondrocyte Development(1.80) 33.33%<-this could be a key between LSJL and axial loading which has an enrichment score of 3.80.

Cartilage Development(12.70) 44.2%

Cartilge Development Involved in Endochondral Bone Morphogenesis(2.10) 42.8%

Growth Plate Cartilage Development(0.27) 14.29%

Endochondral Ossification(1.66) 29.41%

Systemic effects of ulna loading in male rats during functional adaptation.

"The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague-Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue- and sham-loaded animals, blood plasma concentrations of TNF-α, RANKL, OPG, and TRAP5b were determined. Loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated{LSJL increased length in bones not loaded}. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue-loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue-loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. "

"The periosteum is the skeletal tissue with the greatest density of sensory nerve fibers, which are arranged in a dense netlike meshwork that is optimized for detection of mechanical distortion. Nerve branches or single neurons enter the bone cortex, often in association with the microvasculature, and connect individual bone cells to the central nervous system via unmyelinated sensory neurons."

"In the load and block + load groups, loading was performed for 1500 cycles at 4 Hz, with an initial peak strain of −3,750 µɛ (−18 N entered into materials testing machine, −16.8 N applied to ulna). In the fatigue and block + fatigue groups, cyclic loading was performed at 4 Hz. Loading was initiated at −16 N, and the load applied to the ulna was increased incrementally until fatigue was initiated. Loading then was terminated when 40% loss of stiffness was attained."

"TRAP5b is expressed on both immature and mature osteoclasts; plasma TRAP5b concentrations are proportional to osteoclast number."

(*NEW*)

Healing of non-displaced fractures produced by fatigue loading of the mouse ulna.

"Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage."

Monotonic loading: "Both forelimbs of five mice were loaded by a displacement ramp (0.5 mm/sec) to complete, displaced fracture in order to determine monotonic mechanical properties. Mice were euthanized immediately after loading. Ultimate force (mean ± SD) was 4.32 ± 0.21 N, and stiffness was 3.98 ± 0.26 N/mm."

Fatigue loading:  "Both forelimbs of 14 mice were cyclically loaded at peak compressive forces (F) ranging from 2.1 to 3.5 N (50 to 80% of average ultimate force) until complete fracture. "

Force loading, to partial non-displaced fracture: "Right forelimbs of 80 mice were cyclically loaded at peak forces ranging from 3.75 to 4.10 N (70–75% of ultimate force) while displacement was monitored. Loading was terminated when peak displacement increased by 0.75 mm relative to the peak displacement at cycle 10."

The key here is to see if any chondrogenic genes were upregulated in partial, non -displaced fractures(so like a microcrack).  Col2a1 was highly upregulated at day 7.  BMP2[1.1-1.9 fold] and FGF2[1.1-1.8 fold] were moderately upregulated.  FGF2 was more highly upregulated in LSJL than here whereas BMP2 was more highly upregulated here than LSJL.  Hif1a the chondrogenically related transcription factor was more significantly upregulated peaking at 3.0 at day 3 and increasing before and decreasing after.

It should be noted that LSJL gene expression was done by microarray whereas this study was done with RT-PCR with the exception of BMP2 which was also done by PCR.

Here's what a bone microfracture-microcrack looks like:

M stands for marrow. CB stands for cortical bone.  WB stands for woven bone.

"Longitudinal sections of fatigue-loaded ulnae (H&E) show that the fracture occurred as a non-displaced, oblique crack through the medial cortex (arrows). On day 1 after loading, a clot is seen on both ends of the crack. On day 3, the periosteum is expanded and filled with cellular, fibrovascular tissue; nascent woven bone is seen sub-periosteally. On days 7 and 11 there is abundant woven bone on the medial periosteum. In approximately one-half of specimens the callus contained no cartilage (not shown), but in the others there was cartilage (*) in the center of the woven bone."

"Cyclic loading of the rat forelimb (~18 N peak force) to 85% of fracture displacement resulted in a non-displaced fracture localized to the medial cortex of the ulna and an associated loss of ulnar strength and stiffness of 55 and 80%, respectively. Because the fracture in the rat ulna was partial and non-displaced, and because the repair process involved negligible cartilage formation, we referred to this as a “stress fracture”, consistent with descriptions by others"<-now we've considered that that 0.5N in LSJL is equivalent to 100N on a 200lbs human which is already a challenge.  Imagine the challenge of generating 18N peak force.  It's possible that less force may be needed if the force is applied cyclically over a long period of time as long as that force causes residual damage in the bone.

"Cartilage was often observed at 7 and 11 day timepoints and appeared only on the medial surface, corresponding to the periosteal fracture location. By comparison, in studies of complete fracture in mice cartilage is seen on both sides of the bone as well as between the fractured ends"

The fracture occurred on the medial side and although the majority of the activity is on the medial side there is some enhanced activity on the lateral side giving weight to the possibility of gradually lengthening the bone through microfracture(as the unfractured side does seem to adapt).  Although the force required to induce a sufficient microfracture may be too large to be induced under normal physiological circumstances(and you would notice a bone adaptation as large as that depicted).  That does not preclude the possibility that rapid loading that is large enough to induce residual damage to bone is enough to induce such a microfracture as well.  Something like tapping.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

"We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months. There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months. We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls. In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias, due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%). Non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice."

Unfortunately, no chondrogenic genes were studied.

Global gene expression analysis in the bones reveals involvement of several novel genes and pathways in mediating an anabolic response of mechanical loading in mice

"We applied mechanical loads[4-point bending] to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls"

"4 days of loading"

"Twenty-four hours after last stimulation"<-whereas LSJL was 1 hour after last stimulation.

"Ten-week-old C57BL/6J female mice"

Complete list of supplementary gene comparison to LSJL to be done.  Gene comparison of just genes on main paper and spot comparisons done below.
Genes upregulated in bone to four point bending also upregulated by LSJL:
Ptn
Ogn{down}
Itm2a
Lepre1
Col6a3
Col14a1
Col18a1
Matn2
Lox
Gas1
Timp1
Acta2
Ppfibp1{down}
Fer1l3
Spon2
Wnt2
Lmna
Sgk
Odz3
Anxa8
Chl1
Adamts4
Tcf12{down}
Col4a2
Junb
Tnc{down}
Bgn
Egfr
MMP2
BSP

Downregulated:
Mkrn1

Height Growth Protein Map

 Key: 
- downregulate
+ upregulate
-> transcription regulation
+/- means the result is dose or experiment dependent


Okay here's a draft of the genetic regulation involved height growth.  We want to focus mainly on the genes that promote chondrogenesis although eventually we'll go through genes that maximize height.  These up- and down- regulations may not happen all the time but showed up in at least one study and may be dose and/or environment dependent.


Key factors in initiating height growth seem to be inhibiting the NFKappaB pathway and Runx2.   Although this study here shows no effect of Runx2 on bone length. Sox9 and the interplay between TGFBeta1 and BMP2 seem key to initiating a new growth plate.  Finding the TGF and BMP Smad interplay will be key in developing therapies to grow taller as will PTH/IHH interplay.


Posting this now to get feedback on genes/interactions I'm missing.  Eventually I'll draw a graphic representing the interaction.  Also, take note of Harpogoside and Acteoside which inhibit NFKappaB activity.  Two supplements that I looked at earlier that take on additional relevance now due to the importance of NFKappaB on height growth.


Also, tomorrow I'll be posting an LSJL finger update.




Sox9 -> Col2a1(intron 1, intron 6)


IL1:
- Sox9
- chondrocyte proliferation
+MMPs


TNFalpha:
-Sox9


FGFs:
+Sox9
-Ihh


PKA+Sox9
S181+Sox9


TGFBeta:
+Sox9
+Aggrecan
+Col2A1
+BMP2
+Smad2/3P
+F-spondin
-cartilage tissue growth(encourages homeostasis)
+Fibronectin


BMPs:
+Ihh
-PTHrP


BMP2:
+Sox9
+Aggrecan
+Col2A1
+Smad1/5/8P
+Beta-Catenin
-TGFBeta1


BMP7:
+cartilage tissue growth
+GAGs


LIPUS:
+BMP2
+FGF7
+TGFBetaR1
+EGFR1
+VEGF
+PI3K
-GSK3Beta


IGF-1:
-RhoA
+cartilage tissue growth
+GAGs
+Twist1


Noggin:
-BMP
-Height


Gremlin:
-BMP


Chordin:
-BMP


Bezafibrate:
+BMP2
+RUNX2


RhoA:
-chondrogenesis
-chondrocyte maturation
+PI3K


Rac1:
-chondrogenesis
+chondrocyte maturation
+alkaline phosphatase
+adhesion
+Col2A1
+Aggrecan
-GAGs
+Sox9


Mechanical Loading:
+TGFBeta1
+IGF-1


LSJL:
+/-MMP3(depends on load)
-MMP1
-MMP8
-MMP13
+hyaluranon synthase 1
+TIMP1
+GAGs
+Interleukin 1 receptor-like 1
-TNF
+TGF-Beta1
+TGF-BetaR1
+Follistatin
+ActivinR1
-EIF2alpha phosphorylation
+CITED2
+Actin filament remodeling


Icariin:
+BMP2
+BMP4
+Cpfalpha1
-COX2
-PGE


PEMF:
+proteoglycans
+ECM
+Col2A1
+/-ALP
+MMP2
+Sox9
+MT1-MMP
+proteoglycan sulfation
-Collagen Type X


FGF:
-Ihh


FGF2:
-cellular senescence


FGFR3:
-height


SUMF1:
+Activate sulfates that catalyze desulfation of the GAGs moiety of proteoglycans in the intracellular and extracellular space. 
+ECM
+height
-FGF


CHOP:
+apoptosis
+IL1
+IL6


AKT1:
-caspase3
+BclXL
+chondrocyte survival


Salubrinal:
+EIF2alpha phosphorylation
+ATF3
+ATF4
+Chop
-Cell death


Mechanical Loading:
Alteration in cortical actin dynamics resulting in adaptation to load.


MT1-MMP:
+cartilage canals


Endoglin:
+Smad 1/5P
-Smad 2P


Smad2/3P(ALK5):
+Type II Collagen


Smad 1/5/8P(ALK1):
+Type X Collagen
+MMP13
-Height


ESL1:
+Height
-TGFBeta1


Cnidium:
+bFGF
+BMP-2
+IGF-1


RA:
+F-Spondin


Beta-Catenin:
-Chondrogenesis
-Sox9


F-Spondin:
-Height
+MMP-13
+ALP


Extracellular P(i):
+ALP
+RUNX2
+MMP-13


Smurf1:
-BMPs
-Height


Smad6:
-Height
-Smad1/5/8P
-BMPs


Smad7:
-TGF-Beta1
-BMP2


PTHrP:
+Sox9
+chondrocyte hypertrophy


IHH:
+chondrocyte proliferation
-Nkx3.2


FGFR1:
-Chondrogenesis


FGFR3:
+STAT1
-Height
-Ihh


STAT1:
-CCND1


CCND1:
-Runx2


FGF18:
-chondrocyte proliferation


Dynamic Compression(60min):
+COL2A1


MAPK(in chondrocytes):
+accelerates endochondral ossification


Microgravity:
+Sox9
+Aggrecan
+Type II Collagen


Ecdysterone:
+chondrogenesis


Twist1:
-Sox9


NfKappaB:
-Sox9


Runx2:
-Sox9


PTHrP:
+NLK3.2


Nkx3.2:
+Sox9
+Col2A1


Silicon:
+ALP
-Height


TSP3:
+Height


TSP5:
+Height


Col9:
+Height


Insulin:
-IGF1R
-IR


PGE2:
+IL6
+Type II Collagen
+MMP13
-Type X Collagen
-BMPs


Ascorbic Acid:
+ERK
+helps synthesize collagen matrix


Hydrostatic Pressure:
+Mitotic cell rounding into chondrogenesis


CNP:
+Height
+cGMP


CCN2:
+/- Height


Estrogen:
+/- Height


Proepithelin:
-NFKappaB
+Height


Harpogoside:
-MMP1
-MMP3
-MMP13
-NFKappaB


Acteoside:
-NFKappaB

What's the typical gene expression of bone marrow?

Since all the LSJL studies have been done on rats, it would be informative to know how genes are expressed in typical rat bone marrow.  And for humans it would be helpful to know if there's any chondroinhibitory expression in typical bone marrow.

Gene and protein expression analysis of mesenchymal stem cells derived from rat adipose tissue and bone marrow.

"In secretory protein analysis, ASC secreted significantly larger amounts of growth factor and inflammatory cytokines, such as vascular endothelial growth factor, hepatocyte growth factor and interleukin 6, whereas BM-MSC secreted significantly larger amounts of stromal-derived factor-1α."

"Adult Lewis rats were used"

Genes upregulated in MSCs versus ASCs also upregulated in LSJL:
Wisp2
Slco2a1
Col11a1
Cdh13
Crabp2

Downregulated:
Cxcl1{up}
IL6{up}
Rasd1{up}
Nox1{up}
Cxcl5
Ccl2{up}

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets.

Genes can be expressed differently in different cell lines.  Chondrogenic related genes that were higher in non-rapidly dividing cells than radiply dividing cell lines: ADAMTS5, VCAM1{downregulated by LSJL}, and FGF2{upregulated by LSJL}.  So perhaps slower dividing cells may aid in inducing bone marrow chondrogenesis.  However in rapidly dividing cells the following were expressed more: HMGA2{which is upregulated in LSJL}.  

Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow. found no specific chondrogenic-related genes between bone marrow stem cells and other stem cell types.

One adult male and female(18 and 19) were used.

Gene expression profiling suggests a pathological role of human bone marrow-derived mesenchymal stem cells in aging-related skeletal diseases. states that the following genes may upregulate with age: HEXA, HEXB, CTSK, SULF1{up in LSJL}, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29.  Bone Marrow donors were 36-74 and only a few did not have osteoarthritis.

Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes. found that TGF-Beta1 expression increased in bone marrow adipocytes with age however numerous pro-adipogenic genes were increased as well.

Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.

"we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin{up in LSJL}, alpha-ATPase, mitochondrial superoxide dismutase and cyclophilin A. All these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism and response to stress processes that take place in the early steps of chondrogenesis."

"makes use of MSCs derived from the bone marrow of femoral condyles"

"The average age of the patients was 70 years (range 65-74 years)."

"64 of the identified proteins were found to be quantitatively altered at day 14 of chondrogenesis when compared the initial steps (day 2). Some of these proteins were recently characterized by 2D-DIGE as modulated in a chondrogenesis model of umbilical cord stroma cells. These include some metabolism related proteins (ENOA, NNMT, PGAM1 or TPIS), chaperones (GRP78, PRDX6 and SODM), annexins (ANXA2 and ANXA6) and vimentin."

"annexins-2, -5 and -6 were found increased in a murine embryonic mesenchymal cell line"

"GELS is induced by integrin signaling, and [is upregulated] in BMSCs undergoing BMP2-induced

chondrogenesis"

"the intermediate filament VIME [is] a positive regulator of adult hBMSC chondrogenesis"

"SODM is a major intracellular antioxidant protein [that is reduced in osteoarthritic chondrocytes].  The detected increase after chondrogenic induction in a chondrogenesis model of BMSCs induced by TGF-β1 exposure, and in [a' chondrogenesis model of umbilical cord stroma cells, might be a mark of increased oxidative stress in the micromasses."

"While ROS levels have been shown to regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes in a mice model, a recent work showed its increase during the chondrogenic differentiation of ATDC5 cells and primary chondrocytes derived from mouse embryos, and demonstrated that ROS generated by NADPH oxidases 2 and 4 are essential for survival and differentiation in the early stage of chondrogenesis"

Gene expression increased during 14 days of chondrogenic differentiation versus day 2 that were also up-(or down) regulated by LSJL:

PTRF

COL6A2(both up- and down-)

COL6A3

LMNA

Down:

None

Appropriate reference gene selection for real-time PCR data normalization during rat mesenchymal stem cell differentiation.

"The expression stability of six widely used HKGs[House Keeping Genes] including Actb, Btub, Hprt, B2m, Gusb and Tfrc was investigated during rat MSC differentiation into osteocytes, adipocytes and chondrocytes lineages using geNorm and NormFinder software. RT-PCR data analyzed by geNorm revealed the different sets of suitable reference genes for each cell type. NormFinder also showed similar results. Analysis of the combined data of MSCs with each differentiated cell type revealed the considerable shift in expression of some reference genes during differentiation; for example Gusb and B2m were among the least stable genes in MSCs but the most stable in chondrocytes. Normalization of specific genes for each lineage by different reference genes showed considerable difference in their expression fold change."

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers.

"The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. We have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states."

Col2a1 and Acan were upregulated in CH Turner's axial loading study but no Sox9.

Proteins identified in the metaphysis that were upregulated in LSJL(Bolded genes are also identified in diaphysis):
Col1a1
Col2a1

Detailed comparison of LSJL genes to these genes to be completed.

Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation.

"TGF-beta coordinates the increase of alpha-actin and the decrease of gelsolin to promote MSC differentiation"

"TGF-β induces chondrogenic differentiation of MSCs in the presence of dexamethasone or three-dimensional cell aggregates"

"Our results indicate a decrease of HSP27 phosphorylation at Ser-82 after TGF-β stimulation. TGF-β also decreased T-plastin (fimbrin) expression in MSCs"

"T-plastin is normally found in epithelial and mesenchymal cells and is an actin-bundling protein regulating microvilli actin filaments"

Endogenous bone morphogenetic proteins in human bone marrow-derived multipotent mesenchymal stromal cells.

"upon prolonged in vitro culture, MSCs tend to undergo spontaneous osteogenic differentiation. Here, we address the possible role of endogenous osteogenic bone morphogenetic proteins (BMPs) in in situ osteoblastic differentiation of human MSCs. Human MSCs consistently express biologically active BMP-2, BMP-4 and BMP-6 in addition to all BMP-activated receptors, which are functional as shown by the induction of alkaline phosphatase (ALP) activity and up-regulation of osteogenic genes (ALP, BSP1, collagen I and Runx2) following BMP-2 exposure. Since glycosaminoglycans (GAGs) have been implicated in the modulation of the osteogenic bioactivity of BMPs, we reduced sulphated cell surface GAGs by NaClO(3) treatment and found significantly reduced osteogenic gene expression and ALP activity, suggesting that this was partly due to the reduced biological activity of endogenous BMPs. Antagonising osteogenic BMP activity led to a significant reduction in the ALP activity and down-regulation of the transcription factor Runx2 associated with osteogenic development. Blocking BMP receptor type I kinase function with dorsomorphin demonstrated that endogenous osteogenesis was independent of Smad activation but was dependent on phosphatidylinositol 3-kinase (PI-3K). Inclusion of the PI-3K kinase inhibitor Ly294002 significantly reduced osteogenic gene expression and ALP activity. Spontaneous mineralisation was also abrogated following PI-3K inhibition. Thus, endogenous BMPs could contribute to spontaneous osteogenesis through Smad-independent PI-3K-dependent signalling."

"MSCs showed spontaneous mineralisation after 3 weeks of culture in the presence of additional ascorbic acid and glycerophosphate"

"BMP-2, -4 and -6 but not BMP-3, -7 or -9 were consistently expressed in MSCs and showed a tendency to increase with time in culture"

"MSCs treated with the BMP antagonist showed significantly reduced ALP activity as well as reduced expression of the key osteogenic marker gene Runx2."

BM-MSCs had greater expression of CD90, CD73, and CD166.

BMP-4 and BMP-6 increased linearly until 6 days of culture whereas BMP-2 peaked at day 4.

Analysis of Differentiation Potentials and Gene Expression Profiles of Mesenchymal Stem Cells Derived from Periodontal Ligament and Wharton's Jelly of the Umbilical Cord.

Supplementary Material

"the osteo-/dentinogenic, adipogenic and chondrogenic differentiation potentials of PDLSCs[tooth ligamental stem cells] were more powerful than those of WJCMSCs{So we can learn which genes encourage chondrogenic differentiation}. Microarray analysis discovered that 903 genes were significantly down-regulated and 726 genes up-regulated in WJCMSCs compared with PDLSCs. Based on the microarray data, we found that several genes may be associated with MSCs characteristics. Further bioinformatic analysis identified that TGF-β and WNT signaling pathways, and several genes, including STAT5B{down in LSJL} and ITGA4{down in LSJL}, may play key roles in MSCs. Our results indicate that the differentiation potentials of WJCMSCs are far less than those of PDLSCs, and that unmodified WJCMSCs may not be good seeding cells for periodontal tissue regeneration. Our results also help to elucidate the differentiation mechanisms in MSCs and to find the key factors to prompt WJCMSC-mediated periodontal tissue regeneration."

"cell surface marker analysis of [PLDSCs and WJCMSCs] showed they were positive for CD73, CD90 and CD105. These cells were negative for CD45, CD34, CD19, CD11b and HLA-DR"

"WJCMSCs showed enhanced proliferation ability compared with PDLSCs"<-which is consistent with the tendency for differentiation and proliferation to be inversely related.

Sox9 and Col2 was more strongly induced in PDLSCs than WJCMSCs after being placed in chondrogenic medium for two weeks.

"The chondrogenic potentials were decreased in WJCMSCs compared with PDLSCs after chondrogenic induction; SOX2 and NANOG were highly expressed in PDLSCs compared with WJCMSCs"

Genes upregulated in PDLSCs versus WJCMSCs also upregulated in LSJL:

S100A4
Hhip
Vgll3
Cdh13
Col6a1
IRS1
Zfp36
Crabp2
Igfbp6
Col14a1
Nr4a2
Htra1
Steap2
Nov
Itgbl1
Acan
Adamts1
Epha5
Steap1
Osr1{down}
Grem2

-2fold cutoff detailed analysis TBD

Genes downregulated in PDLSCs versus WJCMscs also downregulated in LSJL:

Hoxd10{up}

ITGA4

STAT5B

TMEM154

SPON2{up}

DPP4

ANXA3

GFPT2{up}

Ifne1{up}

Pdlim3{up}

Dsc2{up}

P4ha3{up}

Cxcl1{up}

PTGS2{up}

LAMC2{up}

PLEK2{up}

ARSI{up}
Plekha2
Il33{up}
Col3a1{up}
Vcam1
Cxcl5
Matn2{up}
Pdpn{up}
Tgfbr1
Pgm2l1
F11r
Col11a1{up}
Matn3{up}
Cyp2s1
C3ar1{up}
Cnn1{up}
Zfpm2
Kdr{up}
Hbegf{up}
D5na5{up}
Col4a1{up}
Cadm1
Cth{up}
Thbs2{up}
Prss35{up}
Slc6a15{up}
SULF1{up}
Ddx26b{up}
IL6{up}
Rhbdf2
Baiap2l1{up}
Fry
Col4a2{up}
Csnk1d
Atp2b1
Anxa8

-2 fold cutoff

"STAT5B is a transcription factor that is triggered by various cell ligands and different growth hormones; it mediates signal transduction, and STAT5A/B activation is required for self-renewal and quiescence of stem cells"

The Impact of Cell Source, Culture Methodology, Culture Location, and Individual Donors on Gene Expression Profiles of Bone Marrow-Derived and Adipose-Derived Stromal Cells.

"MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements."

"genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs."

The youngest donor for MSCs was 29.

Pro-chondrogenic genes expressed in BM-MSCs at a higher fold than Adipose Tissue MSCs:
COMP
COL10A1
TGFBR1
IGF2
HAS1
IGF1
Wnt5b
Lum
Wnt5a

The key height gene SHOX2 is expressed at higher levels in adipose tissue stem cells than bone marrow MSCs but that gene is not chondroinductive.

Pro-chondrogenic genes expressed higher in BONE Marrow MSCs cultured in fetal calf serum versus human platelet lysate:

ACAN
IGF2
COMP
FOS
DCN
SULF1
FOSB
SMAD9
COL11A1

No strikingly pro-chondrogenic genes were noted in the reverse condititions.

A mesenchymal stromal cell gene signature for donor age.

"early passage hMSCs were isolated from bone marrow of 61 donors, with ages varying from 17-84, and clinical parameters, in vitro characteristics and microarray analysis were assessed."  Follistatin was the common marker gene for aging.  Follistatin expression levels increase with age.

HMGA2 expression also increases with age.

Identification of a common gene expression signature associated with immature clonal mesenchymal cell populations derived from bone marrow and dental tissues.

"human dental pulp stem cells (DPSCs) do not form bone and hematopoietic supportive stroma in vivo, but rather exhibit the capacity to regenerate an ectopic dentin-pulp-like microenvironment following transplantation into immunocompromised mice. Similarly, when transplanted under the same conditions, human periodontal ligament stem cells (PDLSCs) are capable of regenerating calcified structures of cementum impregnated with periodontal ligament-like filaments (Sharpe's fibers) that sprout from the surrounding collagen-rich fibrous tissue"

"Human bone marrow aspirates were obtained from the posterior iliac crest of normal adult volunteers (20–35 years old)"

"Within this gene data set, the transcription factors, E2F2, PTTG1, TWIST-1, and transcriptional cofactor, LDB2, were found to be commonly up-regulated in long-lived, multipotent cells derived from all 3 tissues."

"E2F1, E2F2, and E2F3 activate hTERT in normal somatic cells suggesting that activation of E2F2 in high proliferative MSC clones may maintain expression levels of hTERT and, hence telomerase activity, previously shown to greatly prolong the lifespan of culture-expanded BMSC"

Geometric cues for directing the differentiation of mesenchymal stem cells.

"cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature-and with each occupying the same area-display different adipogenesis and osteogenesis profiles. geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. vontractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling."

"The degree of cell spreading permitted by the culture density or micro-island size led to a higher degree of cytoskeletal tension and differential expression of the small GTPase RhoA and its downstream effector Rho-associated protein kinase (ROCK). Round cells promoted adipogenesis while cells with high spreading preferred an osteoblast fate."

"cells assemble stress fibers along edges that overlap regions of substrate that are nonadhesive"

"On average, cells in star shapes show larger focal adhesions and stress fibers than cells in flower shapes."

Based on tests with inhibitors, F-actin and ROCK seem to be the mechanism in which shape affects differentiation.

In basal media

Both flower and star shapes in basal media upregulated the pro-osteogenic Runx2 and the pro chondrogenic Sox9 and neutral TGF-Beta1.  Sox9 was upregulated higher in Flower shapes than star shapes but still less than Runx2(which was lower in Flower than star shape).

Although in mixed osteogenic/adipogenic medium, flower shape downregulated Sox9.  This was the only condition where Sox9 was downregulated.

"[There's] an increasingly contractile cytoskeleton in cells as they moved from a flower to a pentagon and finally to a star shape "

Transcriptional comparisons between equine articular repair tissue, neonatal cartilage, cultured chondrocytes and mesenchymal stromal cells.

"culture-expanded de-differentiated chondrocytes and primary bone marrow stromal cells at a pre-transplantation time-point were compared along with neonatal cartilage to repair tissue."

"Expression patterns and gene ontology analyses indicated that while the repair cells were more chondrogenic than bone marrow stromal cells and de-differentiated cultured chondrocytes, steady-state levels of transcripts encoding cartilage biomarkers were substantially lower than the amounts found in neonatal articular cartilage."

"Repair tissue that forms in full-thickness articular cartilage lesions is often described as fibrocartilage or hyaline-like cartilage. The fibrocartilage typically contains more type I collagen than type II collagen, and is deficient in proteoglycans relative to normal articular cartilage"

"repair tissue fails to fully anchor or attach to the surrounding articular cartilage."

"while cells of articular repair tissue may also be of mesenchymal origin, they do not successfully recapitulate differentiation to the chondrogenic phenotype of normal articular chondrocytes during the healing process"

Genes upregulated in MSCs versus repair cartilage also up in LSJL:
COL1A1
Col5a2
Sulf1
TIMP1
Acta2
Tgfbr1{down}
Sox9

Genes downregulated:

COL2A1{up}

Col6a1{up}

Col6a2{up and down}

Col6a3{up}

Col9a1{up}

Col9a3{up}

Acan{up}

Hapln1{up}

Tnc

MMP3{up}

Sox9{up}

BMP2{up}

FGF2{up}

Fgfr1{up}

IGFBP6{up}

Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis.

"isolated single stromal cells simultaneously express transcripts associated with osteoblast, fibroblast, muscle, and adipocyte differentiation. Furthermore, isolated single stromal cells simultaneously express transcripts characteristic of epithelial cells, endothelial cells, and neural/glial cells. Isolated single stromal cells also express transcripts for CD45, CD19, CD10, CD79a, and representative proto-oncogenes and transcription factors, which are typically associated with normal and neoplastic hematopoietic cells. These findings suggest that the nonhematopoietic mesenchymal cells and the hematopoietic B-lymphocytes have a common progenitor. This is consistent with the idea that progenitor cells express genes that are characteristic of the multiple lineage paths that such cells may be capable of adopting.  Bone marrow stromal cells are relatively homogeneous and show a phenotypic signature of potential multilineage differentiation capacity."

" The marrow samples were obtained from 4 normal healthy adult human subjects and consisted of mixtures of unfractionated stromal cells (collective USCs or cUSCs; 8 samples), Percoll gradient-purified mesenchymal progenitor cells (collective MPCs or cMPCs; 5 samples), and single-cell MPCs (sMPCs; 10 samples)"

"The isolated single stromal cells were selected on the basis of morphology. Wright–Giemsa (or hematoxylin) stained cytospin preparation revealed characteristically large nonhematopoietic cells with a relatively irregular nucleus and cytoplasm compartmentalized into ectoplasm and endoplasm"

Chondrocyte-related genes(although they were not characterized as such) detected in stromal cells:

In osteoblast category:

Bhlhb2{also upregulated in LSJL and known as DEC1}

BGN{up}

Cdh11{down, actually an osteoblast related gene}

Vcan{up}

CSPG6

EXT1

c-fos{up}

Key transcription factors like osteopontin are expressed in stromal cells which explains their propensity to an osteogenic lineage.

In muscle category:

Acta2{up}

In fibroblast:

SDC2{down}

Epithelial:

CD44

Endothelial:

Vcam1{down}

Since this study was done in single cells and chondrocytes are more commonly differentiated in groups it makes sense that there weren't very many chondrogenic genes expressed.

Sox9 is expressed by single cells at a low level.  It's expressed at almost 2-3 fold higher in a group than in single cells.  Col2a1 is expressed at very low levels in both groups and single cells.  Aggrecan and Sox6 are not expressed at all.  Sox5 is similarly expressed low levels.

In contrast the more osteogenic gene Beta-Catenin is detected at over 10 fold and much higher in single cells versus groups.

The chondrocyte progenitor gene Cyr61 is expressed at high levels.  FGF2, FGF4, and RARa are not present at all.

Barx2 the gene associated with cartilage condensation is expressed at low levels.  THRA1 is expressed at moderate levels.  TGFB2, PKD1, and ROR2 are expressed at fairly high levels.  Bmpr1b and other cartilage condensation genes were not detected at all.  COL11A1 is expressed at exceptionally high levels.

Gene expression profiling of bone marrow stromal cells from juvenile, adult, aged and osteoporotic rats: With an emphasis on osteoporosis

"[Female Lewis Rats]: geriatric rats (more than 2 years old), osteoporotic rats (7 months old), non-osteoporotic adult rats (7 months old) and juvenile rats (7 weeks old)"

Genes downregulated in aged rats(7 month old) versus normal rats(7 week old) in bone marrow stromal cells also downregulated in LSJL:

IGFBP6{up}

Cyr61{up}

Crabp2{up}

Egr1{up}

Braf

Gsta1

Eef2k

IRS1
Ggcx{up}
Clcn4-2
Pfn2{up}
Sdc2

Genes upregulated:
Ddah1
Olfr78
Edn1
Cnn1
Sstr4{down}

Gene and protein expression profile of naive and osteo-chondrogenically differentiated rat bone marrow-derived mesenchymal progenitor cells.

"this study was carried out for a quantitative RT-PCR based time-course profiling of osteo- and chondrogenesis related gene expression in undifferentiated and differentiated rat adult MPCs. In addition, with an antibody array analysis TIMP-1{up}, MCP-1 and VEGFalpha-164 were detected in the culture supernatant and CINC-2 and beta-NGF in the cell lysate of MPCs according to their differentiation commitment."

""During induction of chondrogenic differentiation, tissue inhibitor of matrix metalloproteases (TIMP-1) became downregulated at day 21 while TIMP-2, MMP-2{up} and -13 remained upregulated until day 21"

"Sox9 and Tbox2 are essential components for the BMP-dependent onset of chondrogenesis"

Genes upregulated during chondrogenic differentiation also up in LSJL:
Sox9
Col1a1
Col2a1
Col10a1
Col16a1
Aggrecan