Thursday, May 10, 2012

The genes involved in growth plate chondrogenesis

Here's an excellent paper that elucidates the genes involve in growth plate chondrogenesis.  It can let us know which genes can be activated in enduce chondrogenesis and if any of these genes are activated by LSJL.

Relating the chondrocyte gene network to growth plate morphology: from genes to phenotype.

"The resting and proliferating chondrocytes secrete structural proteins, such as collagen type II, that form a hyaline cartilage matrix."<-So an upregulation of the type II collagen expression could indicate formation of new growth plates.

"Important local signalling pathways regulating the endochondral development of bones are the parathyroid hormone related peptide (PTHrP), Indian hedgehog (Ihh), bone morphogenic proteins (BMPs), transforming growth factors β (TGFβs), Wnts and Fibroblast growth factors (FGFs). These pathways exert their influence on the growth plate, at least in part, by regulation of the key transcription factors Sox9 and Runx2. The former is crucial for chondrogenesis, whereas the latter is a central regulator in chondrocyte hypertrophy."<-LSJL upregulates TGF-Beta pathway.

So TGF-Beta pathway activates Sox9 and ColII which means that the stimulation of the pathway is highly promising for inducing chondrogenesis.

"Achondroplasia can be simulated by forcing the FGFR3 node to be maximally activated. The FGFR3-ACH mutant does not change chondrocyte behaviour in the resting zone, the increased FGF signalling has little effect here as there is no BMP and wild type chondrocytes do not proliferate in this zone. The more activated ERK pathway might stimulate Runx2 activity, but the activation of the ERK node is insufficient to change Runx2 expression. In the proliferating zone BMP ligands allow for more active Sox9. However, due to dominant FGF signalling Sox9 is less activated in the FGFR3-ACH proliferating chondrocytes[so dwarfism is related to Sox9 activation thus things that stimulate Sox9 like BMP ligands may increase height]. Furthermore, the CCND1 gene, which is associated with an increased proliferation rate, is not expressed (not shown). When perichondrial TGFβ signalling diminishes, the chondrocytes will undergo hypertrophy. The way this hypertrophy is achieved is different than in the case of the normal network, where MEF2C, a transcription factor regulating hypertrophy, will be expressed before Runx2. In the mutant network this sequence is reversed. Additionally, due to the absence of the Smad complex, a lower amount of Ihh secretion is predicted."

"chicken limbs where a stabilized form of -catenin is expressed in chondrocytes showed shorter cartilage elements and ectopic FGF8 expression"<-So destabilizing Beta-Catenin may be a way to increase height.

"many ligands appear to be primarily expressed in the perichondrium (e.g. FGF, certain Wnts, some BMPs), supporting that the growth plate core is partly dependent on these signals."<-The perichondrium is a younger periosteum thus there is evidence that stimulation of the periosteum may help with height growth.

"an increased proliferation and differentiation of chondrocytes was seen after the perichondrium was removed. The researchers also noticed that the cartilage could no longer be divided into distinct zones as
the expression of Ihh became diffuse"<-So the periosteum is vital for growth plate organization but there also seems to be inhibiting factors there.

Other than general activation of TGF-Beta pathways there aren't matches between LSJL gene upregulation and growth plate pathways.  However, that could be due to fact that only bone cells were measured and not chondrocyte cells.  In additional gene expression data provided in the LSJL gene expression study however, Smad5B expression is altered by LSJL and Smad/Dlx 5 plays an important role in the pathway.

The study provides a very important insight and that is that any gene that is mentioned as a continuous source is a very important target for loading regimes and supplements.

What's the significance of Nkx3.2 in this pathway and is there any to increase it?

Nkx3.2 promotes primary chondrogenic differentiation by upregulating col2a1 transcription.

"The Nkx3.2 transcription factor promotes chondrogenesis by forming a positive regulatory loop with a crucial chondrogenictranscription factor, Sox9[so stimulating the Nkx3.2 is a way to increase height by increasing Sox9 levels]. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms.
C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan (GAG) production and expression of type II collagen α1 (Col2a1) mRNA[so upregulating Nkx3.2 may increase height by increasing GAG production and Col2a1 levels], and these effects were evident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expression of Col2a1 mRNA. Dual luciferase reporter assays revealed that WT-Nkx3.2 upregulated Col2a1enhancer activity in a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assays revealed that Nkx3.2 bound to the Col2a1 enhancer element.
Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independent upregulation ofCol2a1 transcription and upregulation of Sox9 mRNA expression under positive feedback system."

"hypoxia (5% oxygen tension) promotes chondrogenesis and glycosaminoglycan (GAG) production and that it suppresses hypertrophy of chondrocytes and osteoblastic differentiation in C3H10T1/2 cell culture. Nkx3.2 (also known as Bapx1), a member of the NK homeobox gene family, is a transcriptional repressor that regulates Runt related transcription factor 2 (Runx2) expression as part of a tight positive regulatory system that includes Sox9; this system is initiated by Sonic hedgehog (Shh)"<-So Runx2 and Shh are also target genes for inducing or enhancing height increase.

There are no known methods of upregulating Nkx3.2 directly but here's a study linking Ihh to Nkx3.2.

Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation.

"The Ihh (Indian Hedgehog) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development.  Nkx3.2 (NK3 homeobox 2) is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in the present study, we investigated whether Nkx3.2, an early-stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. We show that Ihh signalling can induce proteasomal degradation of Nkx3.2[thus inhibiting Ihh signaling may increase height by reducing degradation of Nkx3.2]. In addition, we found that Ihh can suppress levels of Lrp (low-density-lipoprotein-receptor-related protein) (Wnt co-receptor) and Sfrp (secreted frizzled-related protein) (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocytes. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a[thus inhibiting Wnt5a would be another way to reduce height if Wnt5a wasn't important for growth plate column formation, LSJL does affect the Wnt signaling pathway but no mention is made in the LSJL gene expression study of the effect on Wnt5a], which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signalling by deletion of either Ihh or smoothened. Thus these results suggest that Ihh/Wnt5a signalling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis."

The study also mentions CK2 being needed for Nkx3.2.  so CK2 inhibition may be another target for height increase.

Parathyroid Hormone is likely a way to reduce Ihh levels and increase levels of Nkx3.2 and increase height. But is there anyway to increase PTH levels than by the prescription only supplement teriparatide?

Global comparative transcriptome analysis of cartilage formation in vivo.

A complete list of upregulated and downregulated genes is provided.  Mice were used so has very real applications to the LSJL study.  Swiss White Mice were used in contrast to the Sprague Dawley rats in LSJL.

"weak expression of Lepre1{upregulated in LSJL} was observed in the femoral, tibial, and fibular prechondrogenic mesenchymal condensations "

" the gene expression profiles of cartilage pre-condensation tissue at 11.5 dpc, mesenchymal condensations at 12.5 dpc, and differentiated cartilage tissue at 13.5 dpc"

Genes in bold are genes confirmed to have a role in chondrogenesis by the authors of the paper.

Downregulated genes of note: Acp1, barx2{up in LSJL}, bmpr1b{up in LSJL}, CCL21C{up in LSJL}, cops2{down}, CCNG2{down in LSJL}, Col4a1{up}, crabp2{up}, DCAMKL1{up in LSLJL as DCLK1}, Dcx{up}, ebf3{down}, enpp3{down}, fgd4{down}, gas1{up}, glrb{up}, HIF1a(surprising), Hnrph1{down}, hnrpll{down}, jun{up}, Lin7a, Lin28b{up}, l3mbtl3{down}, lrrn{up}, ltb4dh{down}, mdm1{down}, MMP11, Msx2, Notch4, osr1{down}, Pard3{up}, Pax9, Pcdh9{up}, pde7a{down} Pitx1, pkia{up}, Plk2, Plk4, prkir{down}, prrx1{up}, psma6{down}, ptn{up}, ptpn12{down}, rasl11b{down}, rcbtb{down}, Runx1t1, sdpr{down}, slitrk6{up}, Sox7, Sox11, Sox17, Sp4, ssbp1{down}, Sulf1{upregulated by LSJL}, tax1bp1{down}, tes{down}, yme1l1{down}, zadh2{down}, Zfp's 191, 306, 367, 422-rs1, 423, 459, 462, 532, 644, 667.

Upregulated genes of note: ADAMTS 2,3, 12, Agc1{upregulated by LSJL}, Anxa 1,4,5,8{upregulated by LSJL}, 11, Aspn{upregulated by LSJL}, arsi{up}, atp2b1{down}, Bcl 2, 3, 6, BMP5,6,7, Camk4, CSPG4{up}, Cbx6{downregulated by LSJL}, ccrl2{down}, CD9{down}, Cdk6, Cdh11{down}, cgref{up}, Col 1A1{up}, 1A2, 2A1{up}, 6A1{up}, 6A2{up and down}, 6A3{up} 9A1{up}, 9A2, 9A3{up}, 10A1{up}, 11A1{up}, 11A2, 13A1{up}, 14A1{up}, 15A1{up}, 16A1{up}, c1qtnf5{up}, COMP, CTGF, cthrc1{up}, cyr61{up}, ddah1{up}, dhh{up}, dock8{down}, Dlk1, Dlx5, Dsp{down}, Dspg3{up}, Edil3{up}, Egfr{up}, Egr1{up}, Emilin3{up}, Endod1{down}, Epha3{up}, Fkbp10{up}, Fry{down}, Fzd9, GADD45g, GDF10, GPR 17,30,126, HAPLN1{up in LSJL}, Hdlbp{down}, Htra1{up}, IGF2R, Ihh, IL 10rb,16,17d{down}, IRS1{down}, Itga 1,9,11, Itgb4, Lama4{up}, Lepre1{up}, Lmna{up}, Lmo7{down}, Lox{up}, LRP4, Lum{up}, Mafb{down}, MATN 1,2{up},3{up},4{up}, Mbnl1{down}, Metrnl{down}, MMP 9,16, Moxd1{up}, Mtus1{down}, Myl1{up}, Ndph{up}, Ninj1{down}, Npr2, Nt5e{up}, Oxct1{down}, Pcsk6{up}, PDGFA, Plekha6{down}, Prkg2{up}, ptrf{up}, pycr1{down}, RUNX2, S100A4{up}, Saa1{up}, Sct{down}, Sdc2{down}, Sdc4{up}, Sil1{down}, SERPINH1, Slc35a3{down}, Smoc2{up}, Smpd3{down}, Sorbs2{down}, SOCS2, Sox8, Sp7, Sstr4{down}, St8sia6{up}, Steap1{up}, Tcf7{down}, TGFB1, THBS4{up}, TIAM2, TIMP1{up}, TMEM45A{up}, TnC{down}, tnfrsf12a{up}, tnfrsf21{down}, TLR1, Tnn3{down}, Tspan18{down}, Wnt4, Yif1a, Zfp 185,533{up},704, Vcan{up}, Zbtb7c{down}, zcchc5{up}.

Sox9{up} decreased between 11.5 and 12.5 and then increased again between 12.5 and 13.5.

Agc1, COL2A1, Hapln1, cspg4, COL10A1, TnC, MATN2, and MATN3, increased as chondrogenesis progressed.  Lepre1 was only present starting at stage 12.5dpc.  Acp1, bmr1b, Barx2, and Vcan expression went down as chondrogenesis progressed.  Cdh11 was highly expressed throughout chondrogenesis until stage 13.5 when it decreased.  Atp2b1 was only expressed in the laststage of chondrogenesis.  Tcf7 and tnfrsf were decreased between 11.5 and 12.5 and then increased at stage 13.5.

LSJL upregulates ANXA8 3.1 fold.  Dpc stands for days post coitum.  ANXA8 did not increase in expression until about 12 days post coitum.

"Sox4, Sox8, Sox10, Sox11 and Sox13 participate in chondrogenesis in vivo."

Expression of chondrogenic genes by undifferentiated vs. differentiated human mesenchymal stem cells using array technology.

"This study investigated the gene expression profile of human mesenchymal stem cells seeded in collagen sponge for 28 days in three different mediums: (1) basal medium as control containing ITS alone, (2) ITS+TGF-β1 alone or (3) ITS 1% supplemented sequentially by TGF-β1 (D3-D14) followed by BMP-2 (D15-D28). Differential expression of 84 genes implicated in chondrogenic and osteogenic differentiation of MSCs was analyzed at D28"

"TGF-β1 alone down-regulated two genes, CD36 and cathepsin K. Sixteen genes were significantly up-regulated, notably type 2 and type 10 collagens, COMP and Sox9. The sequential combination of TGF-β1 and BMP-2 produced a similar profile with prominent expression of type 2 collagen and the alkaline phosphatase gene. Interestingly, in this in vitro condition, RUNX2 was not up-regulated, suggesting that the sequential combination of TGF-β1/BMP2 enhances the hypertrophic chondrogenic profile without turning towards the osteoblastic pathway."

Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker.

"delta-like1/fetal antigen1 (dlk1/FA1), a transmembrane protein of the Notch/Delta/Serrata family, was shown to be essential for inducing early chondrogenesis. Thus, we investigated the possible use of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1 was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B, a member of TGF-ss family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR{upregulated in LSJL} and VE-cadherin) and SOX9{up in LSJL}. dlk1/FA1(+) cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when cultured as micromass pellets in a xeno-free system containing TGFbeta1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage."

"no co-localization was detected between dlk1/FA1 expression and specific markers for ectoderm (βIII-Tubulin, CD56 and SYP), or endodermal tissue types (HNF4alfa{Down in LSJL} and AFP). Interestingly, dlk1/FA1 was specifically co-expressed with early markers for chondrogenesis in developing cartilage, including type IIA collagen{up in LSJL}, SOX9{up in LSJL} and S100A&B (which is expressed in chondrocytes{S100A4 is upregulated in LSJL}"

"As expected dlk1/FA1+ cells expressed significantly high levels of specific mesoderm gene markers (including MEOX1, MIXL1, ALX4, TBX6{down in LSJL} and FOXF1) compared to dlk1/FA1−"

"dlk1/FA1− cells were unable to differentiate into chondrocytes when cultured as micromass pellets in chondrogenic medium as assessed by their morphology"

"Activin B is one of the first inducers of Dlk1 transcription while other identified factors are known to down-regulate its expression e.g. dexamethasone, growth hormone{down in LSJL}, and cytokine, IL-11"

Interpreted gene expression of human dermal fibroblasts after adipo-, chondro- and osteogenic phenotype shifts.

"Humandermal FB and preadipocytes were isolated from discarded tissue obtained from routine reduction abdomenoplasty. Skin was obtained from both men and women aged between 40 and 65 years of age without any reported skin disease"

"Global gene expression analysis was performed to investigate the changes of the fibroblast phenotype after four-week inductions toward adipocytic, osteoblastic and chondrocytic lineages."

"Human articular CC[chondrocytes] were isolated from the discarded tissue following total knee arthroplasty"

Chondrogenic medium: DMEM, 1% FCS, PEST,1.125 μM insulin, 50 nM A2P and 10 ng/ml TGF-β1

"Cartilage oligomeric protein (COMP), considered a negative marker for dedifferentiation, is down-regulated in C-FB[Chondrogenic fibroblasts] and expressed 5-fold lower in CC compared to FB."

"Targets of BTB and CNC homology 1 (BACH1), a transcriptional repressor responsive to oxidative stress in cartilage, [were over represented]"

"Matrix-related genes fibromodulin (FMOD), biglycan (BGN){up in LSJL} and fibronectin (FN) are highly expressed in FB and not down-regulated in C-FB."

Genes upregulated in both cartilage induced fibroblasts, articular chondrocytes, and LSJL:
CEBPG(down in LSJL)

Genes downregulated in Fibroblasts after differentiation in all groups(chondrocyte, osteoblast, adipocyte) that were also downregulated in LSJL:
CDH13(up in LSJL)
COL15A1(up in LSJL)
GREM2(up in LSJL)

Proteome analysis of rat bone marrow mesenchymal stem cell differentiation.

MSCs were differentiated into chondrocytes by "ascorbic acid, insulin-transferrin-selenous acid, and transforming growth factor-β1 [medium]"

"Protein spots that were determined to be up- or down-regulated when the expression of corresponding spots (between weeks 1 and 2, 1 and 3, 1 and 4) showed an increase (≥2-fold) or decrease (≤0.5-fold) were successfully identified"

"3 [genes are in comon] during chondrogenesis and osteogenesis, and 10 genes [are in common] during adipogenesis and chondrogenesis"

Genes upregulated in chondrogenic line versus other lines[adipogenic, cardiomycygenic, and osteogenic] that were also upregulated in LSJL:

Genes upregulated in adipogenic line(and LSJL):

Molecular differentiation in epiphyseal and physeal cartilage. Prominent role for gremlin in maintaining hypertrophic chondrocytes in epiphyseal cartilage.

"We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development."

Physeal cartilage is growth plate cartilage.  Epiphyseal cartilage is the cartilage used to form the epiphysis before these fuse and only the growth plate regions remain.

Genes upregulated in physeal growth plate cartilage also up in LSJL:
Unbolded genes are upregulated in epiphyseal cartilage as well bolded are differentially regulated
Carbonic Anyhydrase 12

Genes downregulated in physeal growth plate cartilage also down in LSJL:
Differentially(-up) regulated genes in epiphyseal cartilage are bold

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis.

"Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A+) RNA was isolated from the two cell populations and subjected to a gene expression analysis.
A > or =2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. Two transcription factor genes, TWIST and HIF-1alpha, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes.
Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes."

"Culture of [chondrocytes] in monolayers on plastic substrata for prolonged periods or upon repeated passages leads to the loss of their spherical shape and to the acquisition of an elongated fibroblast-like morpholog. These morphologic alterations are accompanied by profound biochemical changes, including loss of the cartilage-specific phenotype, as evidenced by an arrest of the synthesis of the cartilage-specific collagens (types II, IX, and XI) and proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III, and V), and increase in the synthesis of fibroblast-type proteoglycans (versican) at the expense of aggrecan"<-all of these genes are upregulated by LSJL.  Aggrecan is upregulated more strongly than versican in LSJL.  Only Col5a2 is upregulated by LSJL.

Genes upregulated in differentiated versus dedifferentiated chondrocytes also upregulated in LSJL:

Genes downregulated:

Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells.

"we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold."

Genes mostly upregulated between days 7-28 also upregulated by LSJL:

Pbef1{down}-downregulated on day 7 but progressively more upregulated
Ptn-downregulated on day 7
Il17ra{down]-down on day 7
Aspn-greater than hundred fold regulation on day 7 and on sebsequent days tapered to normal levels.

More analysis to be done as the supplementary files cannot be downloaded yet.

Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation.

"Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. 
We separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints.
We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAMα cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen-DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor β3, transforming growth factor β3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAMα cells achieved 8-week survival in vivo.
HAMα cells correspond to MSCs that are highly proliferative and multipotent."

"CD105+ subset of human MSCs from synovium possesses stronger chondrogenic capacity than CD105- subset or the total population of MSCs."

"the CD105+ cells have lost expression of OCT4 and NANOG, the CD105+ MSCs have been suggested to be more differentiated cells toward chondrogenic lineage"

BMP-2 without TGF-Beta3 had the most positive impact on chondrogenic markers.

Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells.

"we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations.
We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5[prime] end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set."

"In order to create an implant which resembles articular cartilage of the knee, we embedded MSCs in alginate discs with the thickness of knee cartilage and exposed these constructs to a differentiation cocktail containing TGFB1, BMP2 and dexamethasone. Evidence of chondrogenic differentiation is shown by upregulation of the chondrocyte marker gene COL2A1 and downregulation of the MSC marker gene CXCL12"

"chondrogenic (COL2A1{up}, COL9A2, COL9A3{up}, COL11A1{up}, COL11A2, ACAN{up}, COMP, SOX5, SOX6 and SOX9{up}) and mesenchymal (CXCL12, IL6{up}, VCAM1{down}, CCL2{up}, PLAU, CLCF1, ADAMTS1{up}, EFEMP1, ACTA2{up} and TPM1) signature genes"

"In MSCs, the promoter region of COL2A1 is marked by wide enrichment in H3K27me3 and no H3K4me3, which just shows a small peak in the first intron. This inactive state changed to a state marked by elevated H3K4me3 around the TSS."

"All the downregulated MSC genes show reduced H3K4me3, H3K36me3 and H3K9ac on day 7 of differentiation, while the genes are still being expressed at moderate levels."

"Of the 488 genes with increased mRNA levels, 90% were associated with increased H3K4me3 around the TSS, with approximately 65% showing a >2-fold increase"

"Although many genes within the general domain of the musculoskeletal system show increased H3K4me3 following 7 days of chondrogenic differentiation, only the genes with the highest increase in H3K4me3 could be correlated with increased gene expression."

"Trimethylation of H3K4 is regulated by several factors, including trithorax group proteins and cyclin-dependent kinase-9 (CDK9) of the positive transcription elongation factor-b complex (P-TEFb)"

"for COL2A1, for instance, that one subset of MSCs [may] lose H3K27me3 and gain H3K4me3 (and H3K36me3) at day 7."

This study will be revisited as not all the data is published yet.

Secretome analysis of human mesenchymal stem cells undergoing chondrogenic differentiation.

"we followed a double stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anti-catabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1."

"Markers of [the chondrogenic] differentiation process in hBMSCs [include] vimentin, gelsolin, fibronectin or cyclophilin A."

Genes upregulated on day 14 related to ECM included "cartilage acidic protein 1 (CRAC1), cartilage oligomeric matrix protein (COMP), lumican (LUMI), Chondroitin sulfate proteoglycan core protein 2 (CSPG2/versican), prolargin (PRELP) or proteoglycan 4 (PRG4/lubricin)."

"the only four proteins detected with a significant reduction in their amounts at day 14 compared to
day 2 were pentraxin-related protein (PTX3), tenascin (TENA), Thy-1 membrane glycoprotein (THY1) and chondroitin sulfate proteoglycan 4 (CSPG4)."

"cartilage-characteristic proteins that were increased at day 14 are matrix Gla protein (MGP), which is thought to act as an inhibitor of bone formation and cartilage calcification, and cartilage oligomeric matrix protein (COMP), which maintains the structural integrity of cartilage via its interaction with
other extracellular matrix proteins and also mediates the interaction of chondrocytes with the cartilage ECM through interaction with cell surface integrin receptors"


  1. Just wondering, is there enough proof to prove that LSJL will induce growth for adults?

  2. how much of the pressure in mice is actually acurate for people? because i seen images of lsjl on mice and the mice had small as well thin bones and very large clamps on their joints in comparison to bone mass, it looked extremely large, i dont want to say that we are trying to go to extreme pressure and cause harm with lsjl but looking at the mice lsjl pictures it just seemed very different in scale to humans.

  3. Interesting point my question is how could we prove or disprove LSJL has the potential to induce adult height growth given that any evidence of success is ancedotal i have grown 3/4 inch LSJL but successful other self reportings remain rare i have all but proven to myself on a personal level but what works for me might not work for somebody else .I think it has been proven in mice and chicks but there growth plates where still active.Has it ever been proven in adult mice/chicks ?.I know it can but it took me 2 years before i noticed any height growth.

  4. @Tyler, What do you think of this as related to Alkoclar formulas?
    He said enhanced CNP expression coupled with fgf-2 receptor stimulation will reactivate growth plates.
    According to many studies this makes a lot of sense, as bfgf/fgf-2 is a potent (many studies call it THE most potent) factor for proliferating MSC's, their differentation to chondrocytes, chondrogenesis and ossification (longitudal bone growth).

    The AOD9021/AOD9201 all trialers got increases IGF-1 receptor sensitivity, FGF-2 and TGF-b. (Alkoclar stated that CNP alone won't reactivate growth plates, FGF-2 does. The CNP will augment the effects of GH to release FGF-2 in the growth plates.
    TGF-b is an important factor for chondrogenesis, it potently elevates the needed SOX9 levels. IGF-1 is supporting almost every step of what we need to reactivate growth plates.

    I'm Interested in your opinion of all of this.
    In an article about FGF's you saw some potential in FGF-2 but knew no way to increase it.
    According to Alkoclar Astragalus (especially Withanolide III) is a potent FGF-2 stimulator.

    I dont know much about LSJL, you are the expert.
    So what do you think would be the benefits of doing LSJL along with highly stimulated FGF-2 receptors?
    Maybe people who dont grow in the trials could take this as an last option to maybe grow taller. Will the enhanced CNP expression inhibit LSJL effects (cGMP -> cAMP pathway?)?

    Please reply