ERG

ERG is downregulated by LSJL

ROLE OF TRANSCRIPTION FACTOR ERG IN SKELETOGENESIS

"Erg is expressed during early synovial joint development along with Gdf5 and Wnt9a and its expression dwindles over time. To test function, we created floxed Erg mice and conditionally ablated Erg in developing joints by mating with Gdf5-Cre mice. Unexpectedly, limb joint development proceeded and the Erg-deficient mice survived to adulthood. To account for absence of a major joint developmental phenotype, we asked whether Fli-1 was co-expressed and in fact it was, and may have compensated for Erg absence. To test possible postnatal roles of Erg, we subjected 2 month-old Erg-deficient mice to knee's medial collateral ligament (MCL) transection to induce experimental osteoarthritis (OA). Strikingly, the Erg-deficient mice developed serious OA-like defects far sooner than operated wild type companions. Indeed, we observed similar severe OA-like defects in aging un-operated 7-11 month-old Erg-deficient mice, while control littermates displayed mild defects. To gain insights into how Erg maintains long-term articular chondrocyte function, we focused on parathyroid hormone-related protein (PTHrP) which is also expressed in developing joints, stabilizes the chondrocyte phenotype and prevents chondrocyte hypertrophy when over-expressed in cartilage (just as transgenic Erg over-expression does). We found that Erg (as well as Fli-1) stimulates PTHrP expression and the PTHrP gene promoter contains several conserved ets binding sites needed for responsiveness. These and other data lead to our central hypothesis is that Erg is essential for the development and long-term stabilization and function of articular chondrocytes and does so in cooperation with Fli-1 and PTHrP. Our Aims are: (1) To uncover the respective roles of Erg and Fli-1 in joint formation and long-term articular cartilage stabilization; (2) To determine how Erg and Fli-1 regulate PTHrP expression; and (3) To determine if transgenic Erg expression protects joints from surgically-induced OA. The work will be carried out using diverse experimental approaches that include transgenic mouse genetics, microsurgery and cell phenotypic expression. It will produce fundamentally new data and insights into the biology and molecular biology of articular chondrocytes and will pave the way to create future repair and regeneration therapies by which function can be restored in articular chondrocytes affected by a variety of adverse conditions including osteoarthritis and natural aging."


Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative erg transgenic mice.

"The Erg gene, is mainly expressed during cartilage formation. We isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression [occurred] in cultured DN-Erg chondrocytes. Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation.  We compared adipocyte presence in wild-type and transgenic mice skeletons. The number of adipocytes [increased] in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone."

"Chondrocytes of murine embryos were isolated from the ribs of 18.5 days post-coitum (E18.5) mice "

"Erg is expressed during the earliest events of skeletal formation and is associated with precartilaginous condensation and chondrogenic differentiation"

"Erg gene is the earliest ETS member family expressed in cartilage during embryonic development followed by Fli1, Ets-2 and Pea3 in a lesser extend"

"Adpn, periplin (Plin), fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), carnitine palmitoyltransferase 1a liver (Cpt-1a), acyl-Coenzyme A oxidase 2 branched chain (Acox2), angiopoietin-like 4 (Angptl4), CD36 antigen (CD36), adipose differentiation related protein (Adfp)) were associated with the ‘PPAR signalling pathway’ and were significantly upregulated in DN-Erg chondrocytes cultured for 20 days"

"Erg protein may be involved in the inhibition of the transdifferentiation of chondrocytes into adipocytes [but] it was not associated with the adipogenic process."

" Pparγ favours the differentiation of mesenchymal stem cells into adipocytes rather than osteoblasts or chondrocytes; Pparγ overexpression has been reported to promote adipogenic differentiation in growth plate chondrocytes"

Genes upregulated 10-fold in wild type chondrocytes on day 20 versus day 0 also upregulated in LSJL:

9930013L23Rik

C1r{down}

Chl1

Cma1

Cxcl5{down}

IL1RL1

IL6

MMP3

Nov

Ptn

Saa1

Saa3

Downregulated 10-fold in wild type chondrocytes:

ATF3{up}

BC064033{up}

Car6{up}

Col11a1{up}

Epyc{up}

Fosb{up}

Fos{up}

Gtf2i

Hapln1{up}

Hist1h4d

Hspa1a{up}

Sgol2

Upregulated Dominant negative Erg day 0 to 20 versus LSJL:

9930013L23Rik

Adam8

Adcy7{down}

Adh1{down}

Anxa3{down}

Apln

Apod

Arhgap30{down} Aspn C1r{down} Ccl2 Ccl7 Cd248 Cdh11{down} Ch25h Chl1 Col5a2 Cpxm2 Cxcl5{down} Egfr Ephx1{down} Folr2 Gpr88{down} Grem2 Hmha1{down} Ifi27{down} Il33 Itgbl1 Lipa{down} Lox Masp1 Mfap4 Mfap5 Mmp3 Myl1 Nov Ostn Plscr2{down} Prrx2 Rasl11b{down} Rgl1{down} Saa1 Saa3 Sla{down} Tagln Tbxas1{down} Thbs4 Tlr7{down} Tmem8{down} Tnmd Vav1{down} Vcam1{down} Vgll3 Wisp2 Xdh{down}

Downregulated:

5430407P10Rik

BC064033{up} Cks2 Cma1{up} Col11a1{up} Cspg4{up} Egr1{up} Egr2{up}

Epyc{up}

Fbp1

Fos{up}

Fosb{up}

Hapln1{up}

Has1{up}

Hmgb2

Hspa1a{up}

Kctd4{up}

Magel2

Mall{up}

Matn3{up}

Ncapg

Nr4a1{up}

Ptgs2{up}

Rps6ka6{up}

Slc38a4{up}

Smpd3

Sox9{up}

Tac1{up}

Hematopoietic stem cells into chondrocytes

Hematopoietic stem cells give rise to osteo-chondrogenic cells.

"To test the ability of hematopoietic stem cells (HSCs) to give rise to osteo-chondrogenic cells, we used a single HSC transplantation paradigm in uninjured bone and in conjunction with a tibial fracture model. Mice were lethally irradiated and transplanted with a clonal population of cells derived from a single enhanced green fluorescent protein positive (eGFP(+)) HSC. Analysis of paraffin sections from these animals showed the presence of eGFP(+) osteocytes and hypertrophic chondrocytes. To determine the contribution of HSC-derived cells to fracture repair, non-stabilized tibial fracture was created. Paraffin sections were examined at 7days, 2weeks and 2months after fracture and eGFP(+) hypertrophic chondrocytes, osteoblasts and osteocytes were identified at the callus site. These cells stained positive for Runx-2 or osteocalcin and also stained for eGFP demonstrating their origin from the HSC. Together, these findings strongly support the concept that HSCs generate bone cells and suggest therapeutic potentials of HSCs in fracture repair."

"transplanted marrow cells that had been transduced with GFP-expressing retroviral vector and observed a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of the recipient mice."

" Recent studies have also identified a population of circulating human osteoblastic cells which expresses osteocalcin or alkaline phosphatase and increases during pubertal growth and during fracture repair. Studies also showed that these osteocalcin positive cells were able to form mineralized nodules in vitro and bone in vivo. This population was subsequently shown to be CD34+, suggesting that it is derived from the HSC."

"In the early process of fracture healing, a hematoma is formed and an inflammatory response occurs at the fracture site within the first 48 h, as demonstrated by the invasion of macrophages, polymorphonuclear leukocytes, and lymphocytes. Osteo-chondrogenic progenitor cells are also recruited to the fracture site within the first week after fracture. Analysis of images taken from within the fracture callus of clonally engrafted animals 7 days after fracture shows a large infiltration of eGFP+ cells. At this stage of fracture repair, the eGFP-expressing cells within the fracture callus are an unorganized, mixed population as demonstrated by their heterogeneous morphology."

"HSCs generate cells with the morphological characteristics of osteoblasts and chondrocytes in the fracture callus 2 weeks after fracture."

"with ossification, both HSC-derived (GFP+) and resident (GFP−) osteoprogenitors occupied the spaces left by apoptosis of hypertrophic chondrocytes. The slow turnover of osteoprogenitors may be one of the mechanisms of the long-term contribution of HSCs to fracture healing."

MSCs have less cell proliferation than HSCs.

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets.

"After expansion in culture, bone marrow–derived MSCs express the surface markers CD29, CD73, CD90, CD105, CD106, CD140b, and CD166, but not CD31, CD45, CD34, CD133, or MHC class II."

"nly adipose tissue–derived MSCs express high levels of CD34, and only amnion-derived MSCs are positive for stage-specific embryonic antigen (SSEA)-4 and tumor rejection antigen (TRA)-1–81. In contrast, bone marrow–derived MSCs, but not placenta-derived MSCs, express CD271 as well as tissue-nonspecific alkaline phosphatase (TNAP)."

CD9 which is downregulated in LSJL is expressed in the synovial membrane.  NT5E(CD73) is a surface marker for bone marrow and it is upregulated in LSJL.  VCAM1(CD106) is a surface marker for bone marrow and umbilical cord and is downregulated by LSJL.

Other Surface Markers altered by LSJL:

CD33{down}

CD8b1{down}{precursor to natural killer cell according to KEGG pathway}

CD22{down}

CD3D{down}{precursor to immune cells}

MS4A1(CD20){down}{B cell marker}

CSF2RA(CD116){down}

ITGA4(CD49D){down}{platelet specific marker}

CD248{up}

CD276{up}{upregulated by axial loading}

CD14{up}{was also upregulated by axial loading}{monocyte lineage specific marker}

Several more stem cell surface markers were altered by LSJL than by axial loading.  LSJL seems to be an immunosuppresant. 

Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach.

"The colony-forming unit-fibroblast frequency was obtained 54 ± 1.33 from 10(3) UC-MSCs at passage 3, and the doubling time was (24.15 ± 0.49) h. Almost 10(10) UC-MSCs were largely produced in about 30 days. By flow cytometry analysis, the adherent cells displayed an abundant presence of CD73{up}, CD90 and CD105 and absence of CD34, CD45 and HLA-DR. When cultured in differentiation media, they can be differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their multidifferentiation related genes were positive. Moreover, stem cell-related transcription factors Nanog, Oct-4 and Sox-2 were positively expressed in UC-MSCs. On the basis of these findings, the single enzyme method is a good method to obtain large-scale production of MSCs from whole human UC in a short time, and the UC can be considered as a novel and convenient source of adult MSCs displaying high expansion potential and primitive pluripotent stem cells"

"To examine chondrogenic differentiation of UC-MSCs, 4 × 105 cells were created as micromass pellets and placed in cube in chondrogenic medium composed of high-glucose DMEM supplemented with 1% ITS, 10 mM l−1 ascorbate-2-phosphate, 10−7 mol l−1 dexamethasone and 10 ng ml−1 TGF-β1. Medium changes were made two to three times per week, and on day 21 of differentiation, cell pellets were harvested, fixed with 10% formalin and then finally embedded in paraffin and stained with 3% Alcian Blue (Sigma). Chondrocyte-specific genes were analysed by RT-PCR, and ACAN and collagen II were detected only in the chondrogenesis-induced cells"

Characterization of human adipose-derived stem cells.

"Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed.

Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73{up}, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers).

It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro."

Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats.

"we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC."

"haematopoietic stem cells may trans-differentiate into epithelial and also mesenchymal lineage cells"

"Osteoblasts, osteocytes and chondrocytes were exclusively ALPP– in bones of BMT rats until the end of study, i.e. 6 months after BMT "

"The failure to detect any ALPP+, BM-derived mesenchymal lineage cells in bone or striated and smooth muscle of BMT rats irrespective of the time after BMT prompted us to ask the question whether stromal precursor cells engraft after BMT with unfractionated BM."

"haematopoietic BMC do not contribute to physiological regeneration of muscle, bone, cartilage or epithelial tissues."

Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.

"mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs."

It seems that hematopoietic stem cells were downregulated and translation into mesenchymal stem cells via Sox9 was initiated in LSJL.

"HSCs have been found preferentially localized in the endosteal region"

"we immunostained femoral sections of Nes-Gfp transgenic mice for haematopoietic lineage markers (anti-Ter119, Gr-1, CD3e, B220 and Mac-1), CD48 and CD150. In agreement with previous studies, CD150+CD48−Lin− HSCs represented a very rare subset (~0.005%) of bone marrow nucleated cells"

Hematopoietic stem cell origin of connective tissues.

"transplantation of 3000 side population cells that are highly enriched for HSCs generated osteoblasts in vivo. Transplanted marrow cells that had been transduced with GFP-expressing retroviral vector and observed a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of the recipient mice."

"Human bone marrow mesenchymal progenitor cells that are capable of adipogenic, osteogenic, and chondrogenic differentiation in culture were shown to express CD13"

"human mesenchymal progenitor cells have been identified from both CD13-expressing bone marrow cells and CD14{up}-positive peripheral blood cells"

Myeloid CD34+CD13+ precursor cells transdifferentiate into chondrocyte-like cells in atherosclerotic intimal calcification.

CD34 is a hematopoietic specific marker.  LSJL upregulates laeverin as 4833403I15Rik which is homologous to CD13.

"Chondrogenic differentiation is pivotal in the active regulation of artery calcification. We investigated the cellular origin of chondrocyte-like cells in atherosclerotic intimal calcification of C57BL/6 LDLr(-/-) mice using bone marrow transplantation to trace ROSA26-LacZ-labeled cells. Immunohistochemical costaining of collagen type II with LacZ and leukocyte defining surface antigens was performed and analyzed by high-resolution confocal microscopy. Chondrocyte-like cells were detected in medium and advanced atherosclerotic plaques accounting for 7.1 +/- 1.6% and 14.1 +/- 1.7% of the total plaque cellularity, respectively. Chimera analysis exhibited a mean of 89.8% LacZ(+) cells in peripheral blood and collagen type II costaining with LcZ revealed an average 88.8 +/- 7.6% cytoplasmatic LacZ(+) evidence within the chondrocyte-like cells. To examine whether hematopoietic stem cells contribute to the phenotype, stem cell marker CD34 and myeloid progenitor-associated antigen CD13 were analyzed. CD34(+) was detectable in 86.9 +/- 8.1% and CD13(+) evidence in 54.2 +/- 7.6% of chondrocyte-like cells, attributable most likely because of loss of surface markers during transdifferentiation. Chondrocyte differentiation factor Sox-9 was detected in association with chondrocyte-like cells, whereas Sm22alpha, a marker for smooth muscle cells, could not be demonstrated. The results show that the majority of chondrocyte-like cells were of bone marrow origin, whereas CD34(+)/CD13(+) myeloid precursors appeared to infiltrate the plaque actively and transdifferentiated into chondrocytes-like cells in the progression of atherosclerosis."

"adult pluripotent cells derived from human peripheral blood monocytes showed transdifferentiation from a monocyte-like structure to a chondrocyte-like structure, indicating that these myeloid cells have the potential to differentiate into collagen type II synthesizing chondrocytes."

"bone marrow derived myeloid CD34+/CD13+ precursors actively infiltrate the plaque where they are capable of transdifferentiating into chondrocytes-like cells in the progression of atherosclerosis."

"Deep cartilage defects are repaired by precursor or stem cells of mesenchymal origin from the subchondral bone marrow. Multipotent mesenchymal stem cells from bone marrow migrate into the defect and differentiate into chondrocyte-like cells with enormous synthesis of proteoglycans and collagen II. There is, thus far, no uniformly accepted clear and specific definitive phenotype or surface marker for the identification of mesenchymal stem cells although lack of the expression of markers including CD34, CD45, CD14{up}, and CD11b have been postulated as criteria of determination."

"use CD34, CD133, and KDR{up} as markers for circulating EPC[endothelial progenitor cells] in human subjects."

Hematopoietic stem cell origin of mesenchymal cells: opportunity for novel therapeutic approaches.

"Human peripheral blood cells that express CD14, a surface protein preferentially expressed on monocytes and macrophages, were shown to generate multiple mesenchymal lineages including osteoblasts, adipocytes, chondrocytes and myocytes in culture "

Endochondral ossification is required for haematopoietic stem-cell niche formation.

"we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation."

"CD105-Thy- populations did not form bones or niches efficiently. Thus far we have not observed marrow formation without bone"

"CD105+Thy1+ progenitors may have lost chondrocyte potential"

Osterix[Sp7] is a transcription factor necessary for endochondral ossification.

"osterix knockdown severely inhibited osteogenesis and abolished niche formation, underscoring the dependence of niche formation on the process of endochondral ossification"

"perichondrial cells and chondrocytes in the developing limb express high levels of VEGF"

"CD105+Thy- mandibular and calvarial progenitors could only form marrowless bones even 60 days after transplantation"

"CD146+ subendothelial cells residing in adult human bone marrow stroma that can generate both bone and marrow when transplanted under the skin of immunodeficient mice"

Since the HSC niche is formed post endochondral ossification, can HSCs play a role in inhibiting the formation of new growth plates?


Bioreactor expansion of human adult bone marrow-derived mesenchymal stem cells.

"we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors, including stem cell factor and interleukin-3 and -6. After 8 days of culture, total cell numbers, Stro-1(+)CD44(+)CD34(-) MSCs, and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-, 29-, and 8-fold, respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin{up in LSJL}, whereas markers associated with lineage differentiation, including osteocalcin (osteogenesis), type II collagen (chondrogenesis), and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis), were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes. The rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs."

"MSCs have been cultured alongside HSCs in spinner flasks without losing their multiple mesenchymal-lineage differentiation potentials"

The bone marrow was from aged individuals 46+.

" MSCs and HSCs, were simultaneously expanded in bioreactor culture"

"After chondrogenic induction for 28 days, the bioreactor-expanded MSCs were positive for type II collagen immunostaining; paraffin-embedded cell pellets stained with alcian blue showed that chondrogenically differentiated cells were encapsulated in the chondrocytic lacunae; TEM examinations of the cell pellets demonstrated typical features of hyaline cartilage, with randomly oriented (collagen) fibrils and (proteoglycan) granules"

" As within the bone marrow cavities, MSCs and HSCs are always in close contact and support each other through the release of various growth factors, cytokines, and chemokines."

So MSCs can undergo chondrogenic differentiation in coculture with HSCs but a pro-chondrogenic factors may be needed to overcome possible HSC chondroinhibitory effects.

This study suggests that HSCs may have a positive effect on chondrogenesis:

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells.

" For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. The presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment."

6-week old male rats were used.

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts.

"KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte-specific cis-regulatory element of the collagen alpha2(XI) gene resulted in enhancement of their chondrogenic nature.  OP9 cells, another marrow stromal cell line, derived from macrophage colony-stimulating factor-deficient osteopetrotic mice and also known to be niche-constituting cells for hematopoietic stem cells expressed chondrocyte-specific or -associated genes such as type II collagen alpha1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF-beta3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen-positive hyaline cartilage within 2 weeks in vivo.  KUM5 and OP9 cells [are in] the same subcategory of "chondroblast," that is, a distinct cell type group. These two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo."

"type II collagen α1, and cartilage oligomeric matrix protein genes were expressed in OP9 cells at more than tenfold higher levels than in 9–15c mesenchymal stem cells, KUSA-O osteo-adipogenic progenitor cells, H-1/A preadipocytes, or even KUM5 chondroblasts."

Genes significantly expressed by OP9 also upregulated by LSJL:

COL2A1

Col9a1

Col11a1

Biglycan

Syndecan 2{down}

Syndecan 4

Sox9

TGFBR1 {down}

VCAM1 {downs}

Genes Absent from OP9 that are downregulated by LSJL:

Col10a1 {up}

Aggrecan {up}

"[in OP9 CELLS] BMP4, BMP6, and BMP7 enhanced the TGF-β3-induced differentiation in a manner. Unlike its effect in KUM5 cells, PDGF did not inhibit TGF-β3- and BMP4-induced differentiation"

"KUM5 cells were positive (more than tenfold compared to the isotype control) for CD9, CD105 (endoglin), Sca-1 and Ly-6C, marginal for CD106 (VCAM-1) and CD140a (PDGFRα), and negative for c-kit (CD117), Flk-1, CD31 (PECAM-1), CD34, CD144 (VE-cadherin), CD45 (leukocyte common antigen), CD49d (integrin α4), CD90 (Thy-1), CD102, CD14, Ly-6G, and CD41. OP9 cells were strongly positive for CD140a, CD106, and CD9, weakly positive for Sca-1, and negative for CD105, c-kit, Flk-1, CD31, CD34, CD144, CD45, CD49d, CD90, CD102, CD14, Ly-6C, Ly-6G, and CD41 "

"OP9 cells are differentiated chondrocytes as a default state while KUM5 cells are oligopotent mesenchymal cells that have a tendency to differentiate into chondrocytes."

"The sequence of enchondral or perichondral ossification by KUM5 and OP9 cells was as follows: deposition of homogeneous matrix surrounding the small nests of the injected cells that subsequently became positive for type II collagen and exhibited metachromasia with toluidine blue staining, trapping them in the secreted homogeneous matrix, and the appearance of small nests of isogenous chondrocytes that probably resulted from repeated cell division. At a later stage, that is, 4–8 weeks after injection, the peripheral region of the generated cartilage became ossified. Importantly, the chondrogenesis by KUM5 and OP9 cells was irreversible and reproducible, and the implanted cells never transformed into malignant cells, formed any abnormal extracellular matrices, or induced any significant inflammatory reactions. It is again noteworthy that the osteogenesis by these two different lines of cells was mediated by chondrogenesis, and it was therefore considered to be chondral ossification."

"OP9 and KUM5 cells are mainly engaged in synthesizing extracellular matrix."<-This is consistent with LSJL with overrepresenation of ECM genes.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells.

" Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]{so both HSCs and chondrocytes can be derived from a common cell line}. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells."

"tissue progenitors in the bone and bone marrow are predetermined to develop into distinct tissue lineages such as osteoblasts and endothelial cells."

Gum Guggl

Gugglesterone doesn't seem like a potential height increasing compound and seems mostly catabolic in effects.

Pharmacological properties of guggulsterones, the major active components of gum guggul.

"ommiphora mukul is a short thorny shrub that is native to the Indian subcontinent. Oleo gum resin extracted by incision of the bark is a very complex mixture of gum, minerals, essential oils, terpenes, sterols, ferrulates, flavanones and sterones. Its active constituents, the Z- and E-guggulsterones, have been demonstrated to exhibit their biological activities by binding to nuclear receptors and modulating the expression of proteins involved in carcinogenic activities. Guggulsterones have also been reported to regulate gene expression by exhibiting control over other molecular targets including transcription factors such as nuclear factor (NF)-κB, signal transducer and activator of transcription (STAT) and steroid receptors."

"Gum guggul is tapped from C. mukul trees by making incisions at the bark."

"guggulsterone is the bioactive principle in gum guggul responsible for its pharmacological action"

"Guggulsterone inhibits the activation of NF-κB and down-regulates the expression of the above-mentioned proinflammatory mediators"<-However NF-kappaB can be pro height increase.  Guggulsterone also inhibits IL-1B.

"guggulsterone blocks RANKL signalling and osteoclastogenesis induced by RANKL by inhibiting the activity of IκBα kinase. Guggulsterone treatment also induces phosphorylation and further degradation of IκBα, which is an inhibitor of NF-κB. Dose-dependent and time-dependent treatment with guggulsterone suggested suppression of monocyte differentiation into osteoclasts"

"Guggulsterone at a concentration of 25 µ m induces S-phase arrest of the cells in the cell cycle through down-regulation of the much-required cyclin D1 and cdc2 and through up-regulation of cyclin-dependent kinase inhibitors p21 and p27 in a time-dependent manner. "

Guggulsterone increases apoptosis.

"Guggulsterone has been found to stimulate the thyroid gland. Of the two hormones, tri-iodothyronine (T3) and serum thyroxine (T4), produced by the thyroid gland, T4 is considered to be active. T4 is converted to T3 in the peripheral tissues. The administration of guggulsterone (10 mg/kg b.w.) in hypothyroid rats could restore the lost thyroid activity. Administration of guggulsterone in albino rats (10 mg/kg b.w.) increased thyroid function. There was an increased uptake of iodine by the thyroid gland. As a result, thyroid peroxidase and protease activities increased in albino rats with a concomitant increase in oxygen consumption"

"guggulsterone activates PR[progesterone receptor] (EC50 = 5.9 µ m compared with progesterone), PXR (half-maximal effective concentration (EC50) = 2.4 µ m compared with rifampicin) and ERα (EC50 = 4.6 μM compared with β-eostradiol)."

"Guggulsterone prevents phosphorylation of STAT-3, which suppresses protein tyrosine kinases activation. Guggulsterone stimulates the transcription of mRNA of SHP-1 and its translation product, tyrosine protein phosphatase. Moreover, guggulsterone was found to down-regulate the expression of antiapoptotic, proliferative and angiogenic gene products regulated by STAT-3. Further study suggested its use in the suppression of cell proliferation, accumulation and induction of apoptosis"

Gugglsterone also inhibits phosphorylation of MAPK and Akt.

Sesamin

Scivation Sesamin 180 Sci-Gels.

Sesamin is in sesame seeds.

Sesamin Stimulates Osteoblast Differentiation Through p38 and ERK1/2 MAPK Signaling Pathways.

"Sesamin [is] a major lignan compound found in Sesamun indicum Linn.
Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 hour of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 hour of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stain with Alizarin Red S staining. MAPK signaling pathway activation was observed by using western blotting.
Sesamin promoted the expression COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19.
The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the MAPK signaling pathway (p38 and ERK) and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts."

It's possible that sesamin has effects on chondrogenesis too.

Chondroprotective and anti-inflammatory effects of sesamin.

"[Sesamin] has an anti-inflammatory effect by specifically inhibiting Δ5-desaturase in polyunsaturated fatty acid biosynthesis. The chondroprotective effects of sesamin were thus studied in a porcine cartilage explant induced with interleukin-1beta (IL-1β) and in a papain-induced osteoarthritis rat model. With the porcine cartilage explant, IL-1β induced release of sulfated-glycosaminoglycan (s-GAG) and hydroxyproline release, and this induction was significantly inhibited by sesamin. This ability to inhibit these processes might be due to its ability to decrease expression of MMP-1, -3 and -13, which can degrade both PGs and type II collagen, both at the mRNA and protein levels. Interestingly, activation of MMP-3 might also be inhibited by sesamin. Moreover, in human articular chondrocytes (HACs), some pathways of IL-1β signal transduction were inhibited by sesamin: p38 and JNK{this is in contrast to osteoblasts where p38 was activated}. In the papain-induced OA rat model, sesamin treatment reversed the following pathological changes in OA cartilage: reduced disorganization of chondrocytes in cartilage, increased cartilage thickness, and decreased type II collagen and PGs loss. Sesamin alone might increase formation of type II collagen and PGs in the cartilage tissue of control rats."

We should mainly look at the control rats as osteoarthritis alters a lot of biological processes.

"Sesamin exposure inhibited IL-1β signals through p38 and JNK but did not act through ERK1/2"

" rats treated with 1 and 10 μM sesamin showed strong and diffuse collagen intensity in the extracellular matrix of the cartilage tissue sections in the control groups"

Does being stressed stunt growth?

The effect of acute and chronic stress on growth.

"Impaired bone growth is observed in many children exposed to stress. The growth plate is specifically targeted by stress through many different mechanisms, including increased serum concentrations of proinflammatory cytokines and cortisol, as well as impaired actions of the growth hormone (GH)-insulin-like growth factor-1 (IGF-1) axis. Both glucocorticoids, such as cortisol, and proinflammatory cytokines adversely affect several aspects of chondrogenesis in the growth plate, and these effects can be ameliorated[improved] by raising local IGF-1 concentrations. However, this intervention does not completely normalize growth. In children with stress related to chronic inflammation, the cornerstone of improving stress-impaired growth remains the judicious use of glucocorticoids while ensuring effective control of the disease process. Specific immunomodulatory therapy that targets the actions of tumor necrosis factor-α (TNFα) is at least partially effective at rescuing linear growth in many children with juvenile idiopathic arthritis (JIA). Patients who do not respond to anti-TNF treatment may be candidates for therapeutic agents that target other proinflammatory cytokines and for GH intervention. Although GH treatment rescues linear growth in some patients with JIA, it is unknown whether GH can rescue growth in those patients who do not respond to anticytokine therapy."

"Acute and chronic stress may influence many mechanisms involved in the regulation of bone growth. Many of the effects can be linked to increased serum concentrations of the proinflammatory cytokines tumor necrosis factor–α (TNFα), interleukin-1β (IL-1β), and IL-6. These cytokines affect the production of many hormones, such as leutenizing hormone (LH), follicle stimulating hormone (FSH), sex steroids, GH, IGF-1, insulin-like growth factor binding proteins (IGFBPs), and glucocorticoids "

" receptors for both IL-1 (IL-1R1) and TNF (TNF-R1) are widely expressed in the growth plate, verifying that these cytokines have the potential to act directly on growth plate chondrocytes. When fetal rat metatarsal bones were cultured with either IL-1β or TNFα, alone, longitudinal bone growth was unaffected. In contrast, the combination of IL-1β and TNFα markedly inhibited bone growth, confirming that these cytokines act synergistically on the growth plate"

"combined treatment with IL-1β and TNFα induces massive chondrocyte apoptosis in cultured fetal rat metatarsal bones. Growth plate chondrocytes are effectively rescued from undergoing apoptosis when IGF-1 is added to the bones exposed to IL-1β and TNFα"<-IGF-1 activates Akt which protects cells from apoptosis.

"Anakinra (Kineret) is an IL-1 receptor antagonist (IL-1Ra), and etanercept (Enbrel) is a soluble TNF receptor."

"we measured femur growth in young male and female rabbits treated with glucocorticoid eye drops for 8 weeks. These experiments demonstrated that animals that received eye drops containing the synthetic glucocorticoid dexamethasone (Dexam) had significantly impaired femur growth when compared to vehicle-treated controls. These animal data show that glucocorticoids have the potential to impair longitudinal bone growth even when locally administered in the eye."

"no catch-up growth occurs after dexamethasone injections are discontinued."

"dexamethasone induces cell death throughout the growth plate. Furthermore, the abundance of the proapoptotic protein caspase 3 increased, whereas the abundance of the antiapoptotic protein Bcl-2 decreased in growth plates from dexamethasone-treated rats."

e2f1

E2F1 is downregulated by LSJL.

Constitutive E2F1 overexpression delays endochondral bone formation by inhibiting chondrocyte differentiation.

"endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation."

"First, the relative size of the proliferating zone was increased in the growth plate [of E2F1 transgenic mice] compared to the wild-type control. Second, the maturing prehypertrophic and hypertrophic layers were significantly reduced in size. The reduced amount of hypertrophic chondrocytes was also evident in the growth plates of the humerus, ulna, and radius"

"loss of E2F1 does not appear to inhibit either proliferation or cellular differentiation during embryonic development, overexpression has been shown to prevent terminal differentiation in different cell lineages."

"Overexpression of the AP-1 transcription factor c-Fos, a positive regulator of cell proliferation, also inhibits chondrocyte differentiation in ATDC5 cells with markedly reduced α1 (II) collagen and aggrecan gene expression"

Transcriptional regulation of fibrillin-2 gene by E2F family members in chondrocyte differentiation.

"Mutation in fibrillin-2, a major structural component of extracellular microfibrils in connective tissue, results in the autosomal dominant disease congenital contractural arachnodactyly. This genetic disease is characterized by dolichostenomelia and arachnodactyly, in addition to contractures of the large joints and abnormal pinnae formation, thus indicating the significance of fibrillin-2 in chondrogenesis. In this study, we investigated the transcriptional regulation of fibrillin-2 in chondrogenic differentiation. Although mRNA expression of fibrillin-1, a highly homologous protein to fibrillin-2, remained almost unchanged during chondrogenesis of mouse ATDC5 cells, fibrillin-2 mRNA expression varied. Fibrillin-2 was highly expressed at the early stage and declined progressively during differentiation. The 5'-flanking region of the fibrillin-2 gene contains potential binding sites for E2F, Runx, AP-2, and Sox transcription factors. The promoter activity of fibrillin-2 decreased markedly following deletion and mutagenesis of the E2F binding site between -143 and -136 bp. Overexpression of E2F1 resulted in a marked increase in its promoter activity, whereas expression of other transcription factors including AP-2alpha and Runx2 had no effect. The increase in promoter activity by E2F1 was completely suppressed by the coexpression of E2F4. E2F2 and E2F3 had positive effects on the promoter activity. Although ATDC5 cells expressed transcripts for the E2F family genes at all stages of differentiation, the expression profiles differed. E2F1 expression remained almost unchanged, whereas E2F4 expression increased markedly at the late stage of differentiation."

"ATDC5 cells constitutively expressed E2F1 mRNA except for the decrease of E2F1 expression at a late stage. E2F2 and E2F3 mRNA levels increased slightly during early chondrogenic differentiation and then finally diminished. In contrast, E2F4 transcripts were not detected in undifferentiated cells, but increased markedly during chondrogenic differentiation. Moreover, the expression changes of E2F1 and E2F4 protein were coincident with that of each mRNA during differentiation. It is highly likely that the expression of fibrillin-2 is upregulated by transactivators (E2F1, E2F2, and E2F3) during the early phase of chondrogenesis and subsequently downregulated as a result of rapid increases in transrepressor (E2F4). This hypothesis was supported by our finding that forced expression of E2F4 inhibited the E2F1-mediated increase in fibrillin-2 promoter activity in a dose-dependent manner"

Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis, cell cycling, adhesion and invasion.

"Chondrosarcoma develops as a result of overgrowth of chondrocytes and overproduction of cartilage matrix. It is currently surgically treated, although non-invasive methods are being sought. In this report, pigment epithelium-derived factor (PEDF) induced apoptosis in the chondrosarcoma cell line - JJ012, with upregulation of Bax, Fas, caspase-3 and -6 and downregulation of Bcl-2. Cell cycling was also decreased with decreased expression of p38, p-Akt, p-Erk and JNK1 and increased expression of p73 and E2F1. Furthermore, PEDF increased adhesion of cells to collagen-I, with decreased expression of p-Fak, RhoA and cdc42. Invasion of cells through collagen-I was also reduced by PEDF, with decreased expression of uPAR, MMP-14 and increased expression of PAI-1. These findings seminally indicate that PEDF may have potential as an anti-cancer agent against chondrosarcoma."

"E2F1 has been found to be responsible for promoting the G1 to S-phase progression of the cell cycle, thereby promoting increased cell cycling. However, E2F1 also possesses the ability to induce apoptosis in a p53-dependent or –independent manner, mainly through p73, thereby giving E2F1 the ability to trigger both cell proliferation and apoptosis"


Loss of pRB and p107 disrupts cartilage development and promotes enchondroma formation.

"The pocket proteins pRB, p107 and p130 have established roles in regulating the cell cycle through the control of E2F activity. The pocket proteins regulate differentiation of a number of tissues in both cell cycle-dependent and -independent manners. Prior studies showed that mutation of p107 and p130 in the mouse leads to defects in cartilage development during endochondral ossification, the process by which long bones form. Despite evidence of a role for pRB in osteoblast differentiation, it is unknown whether it functions during cartilage development. Here, we show that mutation of Rb in the early mesenchyme of p107-mutant mice results in severe cartilage defects in the growth plates of long bones. This is attributable to inappropriate chondrocyte proliferation that persists after birth and leads to the formation of enchondromas[cartilage cysts] in the growth plates as early as 8 weeks of age. Genetic crosses show that development of these tumorigenic lesions is E2f3 dependent. These results reveal an overlapping role for pRB and p107 in cartilage development, endochondral ossification and enchondroma formation that reflects their coordination of cell-cycle exit at appropriate developmental stages"

c-Myc

c-Myc protein in the rabbit growth plate. Changes in immunolocalisation with age and possible roles from proliferation to apoptosis.

"Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes."

Full study stated to be available but couldn't find it.

Zuogui Pill

[Effects of zuogul pill on the gene expressions of Type- II collagen and proteoglycan during the differentiation of mesenchymal stem cells towards chondrocytes].

"To study the effects of zuogui pill (ZP) contained serum on the gene expressions of type-II collagen and proteoglycan during the differentiation of mesenchymal stem cells (MSCs) towards chondrocytes.
MSCs isolated from rat bone marrow were in vitro induced differentiation towards chondrocytes and stimulated with high- (57 g/kg), middle- (28.5 g/kg), and low-dose (9.5 g/kg) ZP contained serums and serum of blank rats. The proliferation of MSCs was analyzed by CCK-8 method. The 3rd-passage MSCs were divided into the blank control group (by adding serum of the blank group rats), the induction control group (by adding the induction fluid and serum of the blank group rats), and the ZP contained serum group (by adding the induction fluid and middle-dose ZP contained serum). The expressions of type-II collagen and proteoglycan were determined using reverse transcriptase PCR, Real-time PCR, and immunohistochemistry.
Compared with the blank control group, the proliferation of MSCs could be promoted by ZP contained serum at different doses (P < 0.05), with the most obvious effect shown in the middle-dose ZP contained serum group (P < 0.05). The mRNA and protein expressions of type-II collagen could be identified in the induction control group and the ZP contained serum group on the 21st day of the induction. Of them, the mRNA expression of type-II collagen in ZP contained serum groups was obviously higher than that of the induction control group. Results of Real-time PCR showed that on the 21st day of the induction, the mRNA expression quantitation of proteoglycan in ZP contained serum groups was about 16-fold and 3-fold of the levels on the 7th day and the 14th day (P < 0.05), obviously higher than those of the induction control group (P < 0.05).
ZP contained serum could induce MSCs proliferation, the gene expressions of type- II collagen and proteoglycan."

This website has more information: http://www.activeherb.com/zuogui/.

iPSCs to chondrocytes

iPSCs were also discussed here.

Chondrogenic Differentiation in vitro of Murine Two-Factor Induced Pluripotent Stem Cells is Comparable to Murine Embryonic Stem Cells.

"Differentiation of embryonic stem (ES) cells via embryoid bodies has been established as an appropriate model to study the development of various cell types in vitro. Here, we show that murine induced pluripotent stem (iPS) cells, reprogrammed by exogenous expression of the two transcription factors Oct4 and Klf4 (2F OK iPS), differentiate into chondrocytes in vitro characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes and proteins. Quantitatively, the chondrogenic differentiation potential of 2F OK iPS and ES cells was found to be similar. Further, we demonstrate the induction of chondrogenic iPS cell differentiation by certain members of the transforming growth factor-β family (BMP-2, TGF-β(1)). The number of Alcian blue-positive nodules and the expression of the cartilage marker molecule collagen type II increased after application of BMP-2, whereas simultaneous treatment with both BMP-2 and TGF-β(1) showed no significant effect on gene expression."

"Oct4 together with Klf4 is sufficient to reprogram neural stem cells into pluripotent stem cells, so-called two-factor (2F) OK iPS cells"

"Growing on mitotically inactivated MEF undifferentiated murine iPS cells of line 2F OK, clone F-4, (2F OK iPS) formed colonies very similar to ES cells of line D3 (ES1) but smaller in size"

"Examination of the proliferation rate of 2F OK iPS cells with ES1 cells demonstrated that the analyzed ES cell line proliferated approximately 1.9-fold faster than the iPS cells. At the time of assessment, 40% of the iPS cells had entered the S-phase of the cell cycle, in contrast to 74% in the case of the ES cells"

iPS had lower Nanog expression but higher Oct4 expression.

"subtle differences observed between the analyzed iPS and ES cells may be due to clonal variation."

"2F OK iPS cell-derived EBs expressed somewhat higher levels of collagen type II compared to ES1 cell-derived EBs."

"At 5 + 14 days, BMP-2-treated 2F OK iPS cell-derived EBs expressed about 2.6-fold higher levels of collagen type II than the control EBs."

"Treatment of 2F OK iPS cell-derived EBs with 10 ng/ml BMP-2 and 2 ng/ml TGF-β1 during the suspension phase (2–5 days) resulted in an even higher and statistically significant increase in Alcian blue-positive nodules"

"the induction of chondrogenesis by simultaneous application of BMP-2 and TGF-β1 could not be confirmed by real-time PCR analysis"

"Various iPS cell lines generated from different cell types in fact show remarkable differences with respect to pluripotency marker expression. In general, differences among iPS cells and between ES and iPS cells have been reported which are most probably caused by epigenetic marks of the cell type of origin"

"murine iPS cells can be differentiated into various cell types using the same protocols used for ES cells but often less efficiently compared to ES cells"

"iPS cells retain an epigenetic memory of their tissue of origin that might hinder or even limit their differentiation propensity, favoring differentiation along lineages not related to the donor cell type"

[Direct cell reprogramming to chondrogenic cells from dermal fibroblast culture].

"Since development of iPS cells, it has become possible to convert cell types through altering epigenome by transducing plural kye transcription factors into cells. We found that transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) into mouse dermal fibroblasts results in direct induction of chondrogeni cells. Directly induced chondrogenic cells showed highly methylated promoter sequences of fibroblastic markers, type I collagen genes. These induced cells, when transplanted into nude mice, produced hyaline cartilage without expressing type I collagen, suggesting that type I collagen genes were silenced in these induced chondrogenic cells in vivo ."

Mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration.

"the induced pluripotent stem cells (iPS cells), allowing creation of patient- and disease-specific stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental stem cell an attractive source of mesenchymal stem cells for tissue regeneration."

"PS cells resemble human ESC and can differentiate into advanced derivates of all three primary germ layers. Unlike ESC, iPS cell technology can derive patient-specific stem cells allowing derivation of tissue-matched differentiation donor cells for basic research, disease modeling, and regenerative medicine"

"mesenchymal stem cells are delivered into root canal spaces during regenerative endodontic procedures in immature teeth with open apices"

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors.

"in vitro de-differentiation of chondrocytes into fibrochondrocytes [occurs], which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells."

"Fibrocartilage is a type of scar tissue that expresses types I and II collagen; hyaline cartilage, in contrast, does not express type I collagen"

"The expression of a defined set of factors (Oct3/4, Sox2, c-Myc, and Klf4, as well as Nanog and human LIN28) can fully reprogram dermal fibroblasts into iPS cells"

"Primary chondrocytes expressed a small amount of Col1a1 and Col1a2, probably due to minor contamination of fibroblasts during the harvesting procedure or de-differentiation of chondrocytes"

"The cytosine guanine (CpG) dinucleotides in the promoters of the fibroblast-associated genes Col1a1 and Col1a2 were highly methylated in MK cell lines, but were unmethylated in parental MDFs. This suggests that expression of c-Myc, Klf4, and SOX9 induced silencing of the Col1a1 and Col1a2 genes in MDF culture. The Col1a1 promoter was moderately methylated in primary chondrocytes. Despite the low expression levels of Col1a2, its promoter was not methylated in primary chondrocytes prepared from neonatal rib cartilage. These results suggest that fibroblast marker gene promoters were excessively methylated in induced cells as compared with those in primary chondrocytes."

"the absence of type I collagen is prerequisite for proper function of hyaline cartilage"

Cell sources for cartilage repair; contribution of the mesenchymal perivascular niche.

"Tissue and cell sources for cartilage repair are revised, including: 1) cartilage and subchondral bone (auto and allografts; single or multiple/mosaicplasty grafts), 2) cultured chondrocytes (autologous/ACI, characterized/CCI, matrix assisted/MAC, or allogenic), 3) adult mesenchymal stem cells (MSCs), 4) progenitor cells from perichondrium and periosteum, 5) embryonic and prenatal stem cells, 6) induced pluripotent stem cells, and 7) genetically modified cells. We consider the biological mechanisms that explain usage and possible complications, advantages and limitations, emerging technologies and possible modulations on extracellular matrix properties and on migration, proliferation, de-differentiation, re-differentiation, morphology, function and integration of the cells. The study of MSC role involve: a) identification, b) location (perivascular niche hypothesis, pericytes as progenitor cells), c) lineage (myoadipofibrogenic system: transit amplifying cells, fibroblast/myofibroblasts, chondrocytes, osteoblasts, odontoblasts, vascular smooth muscle cells and adipocytes), and d) use in cartilage repair, comprising: 1) MSCs recruited from neighbouring tissues (bone marrow stimulation, MSCs based "in situ" cartilage repair, microfracture) and 2) MSCs cultured and expanded from bone marrow, adipose tissue, synovial membrane or granulation tissue."

"The criteria for identification of human MSCs include the following: a) adherence to plastic in standard culture conditions, b) expression of at least CD-73, CD-90, and CD-105, while CD-11b, CD-14, CD-19, CD-34, CD-45, and CD-79a are negative, and c) "in vitro" differentiation into chondroblasts, adipocytes, and osteoblasts"

"n the bone marrow microvasculature there is a continuous layer of subendothelial pericytes, which acquires a reticular morphology (reticular cells [type III collagen expressing cells]) in the venous side. Therefore, the marrow pericytes may be the same entity as the bone marrow stromal cells, since they share features such as: a) similar location of pericytes and stromal cells, b) expression of similar markers, such as SMA, PDGFR beta, EGFR, and CD146, and c) similar response to growth factors"

" the repair sequence includes stages of granulation tissue formation: hematoma (fibrin-deposition binding of platelets), macrophage recruitment, angiogenesis (neovascularization), recruitment and proliferation of multipotent mesenchymal stromal cells, re-absorption of the fibrin clot, and development of a vascularized scar-like tissue"

"spontaneous differentiation and remodelling mainly result in a fibrocartilaginous repair tissue, which may be subjected to excessive deformation with mechanical failure and degeneration after 20-48 weeks"

"the new collagen does not project into or intermingle with the native cartilage, thus hampering the integration and adherence of the newly generated cartilage {maybe because the cells have a different origin}. Indeed, the results after microfracture in the knee and their comparison with ACI demonstrate problems regarding the durability of the repair tissue in major defects and in defects located in areas other than the femoral condyles. Covers that trap the cells in the initial stages of granulation tissue formation (preventing escape of cells and anabolic cell mediators from the site of repair, since fibrin deposition contains the highest percentage of migrating mesenchymal stem cells) have been developed (e.g. collagen matrix) "

"scaffolds (e.g. poli (DL) lactide-coglycoide or alginate-gelatin biopolymer hydrogel), cell-free or seeded with autologous chondrocytes, with osteochondral regenerative potential, have been developed experimentally, with restoration of hyaline cartilage and bone"

"Bone marrow aspirate[refers to removal of a sample of bone marrow via a needle] contains very few MSCs, which can be isolated by means of Stro-1+ antibody recognition "<-Adipose tissues tend to have more MSCs than bone marrow.

"differentiation (expression levels of the chondrocyte specific genes Sox9, collagen type II, aggrecan, and cartilage olygomeric matrix protein) was more prominent in cells cultured in collagen type II hydrogel and that it increased in a time dependent manner. In this way, to induce and maintain chondrogenesis, transforming growth factors (TGF) b1 and b3, fibroblast growth factor, bone morphogenic proteins (BMPs)-2, -6, and -9, and insulin-like growth factors, may be used "

"we implanted perforated rigid tubes in the rat soft tissue, generating a peritubular granulation[fibrous connective] tissue, which progresses through the holes reaching tube light. The granulation tissue evolved into connective  and adipose tissues, except in the intratubular zone near the tube wall close to the holes (in the angle formed between the inner surfaces of the hole and the tube wall), where, during contraction, the granulation tissue presses onto the rigid material, the cells differentiating into chondrocytes"

"The perichondrium and periosteum share the same origin, regulatory mechanisms, and some morphological and functional characteristics. The perichondrium may differentiate into the periosteum. Both perichondrium and periosteum produce multiple positive and negative factors regulating the differentiation of the underlying skeletal elements (e.g. regulating gene expression in the underlying chondrocytes). Both structures have two distinct morphologic layers, an outer fibrous layer and the cambium or inner cellular layer. The inner cellular layer contains fibroblasts and chondroprogenitor / osteoprogenitor cells (Multipotent periosteum cells). Therefore, the periosteum can promote new cartilage and its chondrogenic potential decreases with age"

"When the perichondrium or the periosteum were activated, and the pericytes of the local postcapillary venules were labelled with an exogenous marker, the process of cartilage and bone formation from chondrogenic and osteoprogenitor cells already present in the perichondrium and the periosteum was augmented by proliferation and differentiation of the labelled pericytes, which contributed a supplementary population of newly formed chondrocytes and osteoblasts"

"perichondrium and periosteum not only provide chondrogenic and osteoprogenitor cells but act as inducers of proliferation and differentiation of cells with mesenchymal capacity."

"scaffolds that facilitate environment for chondrogenesis [include] natural (collagen, fibrin, alginate, hyaluronan, agarose, chitosan) or synthetic materials that provide a biodegradable matrix with biochemical properties, supporting neomatrix deposition by chondrocytes, b) several signalling pathways and transcription factors (e.g.: Wnt, transforming growth factor β/bone morphogenetic protein signalling, PDGF, IGF-1, EGF, HGF), which act in migration (PDGF, IGF-1, EGF, HGF, TGF β), proliferation (EGF, PDG,F TGF β), and differentiation (dexamethasone, TGF β). Some may be locally introduced, modulating cell differentiation into cartilage, c) procedures to prevent escape of cells and anabolic cell mediators from the site of repair, d) strategies for cartilage integration, e) inhibition of cartilage degeneration and inflammation (TNF-x and IL-1 application), and f) gene transfer for optimization of cell chondrogenic capacity."

"cells expressing bone formation cytokines, including over-expression of BMPs, have been developed"

Bone tissue engineering: current strategies and techniques--part II: Cell types.

"Sox9, Sox5, and Sox6 induce the differentiation of MSCs to chondrocytes."

"In the process of OB differentiation, Runx2, Sp7, and canonical Wnt signaling also inhibit the differentiation of mesenchymal cells into chondrocytes."

"osteogenic potential appears to be one of the last lineage commitment phenotypes to be lost [with passaging i.e. cell divisions]"

"fluid flow is also sufficient to induce BMSCs differentiation. Interstingly, investigators have demonstrated that even in the absence of dexamethasone, fluid flow enhances BMSC proliferation, ALP activity, osteopontin secretion, and calcium deposition compared with static controls."

"when cultured in the presence of estrogen, BMSCs were shown to have a marked decrease in apoptosis [due to elevated BCL2] and colonies become more robust and lived longer."

"BMSCs contain an EPC[endothelial progenitor cell] subpopulation [and] are capable of recruiting vasculogenic progenitors, endothelial cells, and pericyte-like cells when seeded onto a ceramic scaffold."

"PSC[Periosteal Stem Cells] proliferated faster than BMSCs and that BMSCs were more osteogenic than PSC."<-thus perhaps PSCs are more chondrogenic.

Generation of human induced pluripotent stem cells from osteoarthritis patient-derived synovial cells.

"Human synovial cells isolated from two 71-year-old women with advanced OA were characterized and reprogrammed into induced PSCs by ectopic expression of 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc). The pluripotency status of each induced PSC line was validated by comparison with human embryonic stem cells (ESCs).
We found that OA patient-derived human synovial cells had human mesenchymal stem cell (MSC)-like characteristics, as indicated by the expression of specific markers, including CD14-, CD19-, CD34-, CD45-, CD44+, CD51+, CD90+, CD105+, and CD147+. "

"19,771 genes (44.96%) showed ≥2-fold differences in expression level in human induced PSCs [versus] human MSCs."

"Human ESC–specific genes (TDGF3, CLDN6, DPPA4, LEFTY1, SOX2, ZFP42, CKMT1B, POU5F1, and Nanog) were significantly up-regulated by ≥180 times in human induced PSCs compared to MSCs, while lineage-specific genes were down-regulated in human induced PSCs"

"human MSCs from patients with OA showed rapid induction of the hypertrophic marker type X collagen (COL10A1) "

"expression of aggrecan mRNA in chondrogenically differentiated human induced PSCs was markedly higher than that in human ESCs."

Genes upregulated in iPSCs versus MSCs also upregulated in LSJL:

GNG4

Sema6b{down}

Slain1{down}

Ucp2{down}

F11r{down}

Vav3{down}

Ras11b{down}

Downregulated:

Gls

Col3a1{up}

Postn{up}

Shc1

Fer1l3{up}

Adamts1{up}

Nt5e{up}

Col12a1{up}

In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells.

"We established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α(+)/Flk-1(-) population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α(+)/KDR(-) population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α(+)/KDR(-) population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors."

"Activin A prevented apoptosis [of iPS cells]"

"the addition of high dose of BMP4 enhanced cell proliferation [and] the absence of BMP4 resulted in large apoptosis"

"LiCl enhances Wnt signaling by translocation of β-catenin from cytoplasm to nucleus followed by inhibiting the activity of GSK3β"

"Ectopic cartilage was covered with a capsule and did not form part of the teratoma since it did not contain any other tissues. the ectopic cartilage was derived from engrafted cells that expressed DsRed."

"we transplanted both PDGFR-α+ and PDGFR-α− cells (derived from DsRed/iPS cells) into the tibial anterior (TA) muscle of immunodeficient mice. Four weeks after transplantation, tumor formation was only observed in TA muscle engrafted with PDGFR-α− cells (n = 3)"  Go to the study and 5B to see the ectopic cartilage.

"The PSP and DP populations gave rise to Alcian Blue-positive chondrocytes, while the DN and KSP populations had very low chondrogenic potentials"

"Expression profile of PDGFR-α and KDR in differentiated human iPS cells on day 6. DP, double-positive population; DN, double-negative population; PSP, PDGFR-α single-positive population; KSP, KDR single-positive population." So you want PDGFRA positive for chondrogenesis.

"the addition of LiCl, which activates Wnt signaling via inhibition of GSK3β, dramatically increased the proportion of PDGFR-α+ cells in serum-free culture."

Cartilage tissue engineering using differentiated and purified iPSCs

"iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells.  Increased microscale elastic moduli [was] associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage."

From John Friedline:
"Micromass cell cultures of iPSCs with BMP-4 and dexamethasone.  Then the Col2 promoter/enhancer was utilized to enhance production of green fluorescent protein (GFP), which served as a marker for chrondrogenic differentiation within the iPSC population. Those iPSCs which did not chondrogenically differentiate(despite BMP-4 and dexamethasone exposure) were GFP negative.  The GFP+ cells expressed type II cartilage, Col2 and aggrecan typical of chondrocytes, whereas GFP- cells expressed very little of these markers.  Subsequently both the GFP+ and GFP- iPSCs were induced to proliferate in monolayer using FBS and bFGF.  After monolayer expansion, both sets of cells were centrifuged to form pellets and were cultured for 21 days with TGF-beta3, which enhanced production of GAG (glycosaminoglycans) in the GFP+ cells. "

"the inner half of the cartilagenous matrix from GFP+ cells had a much higher degree of elastic modulus than that produced by GFP- cells."

The iPSCs had an interstitial deletion from 2D to 2F3 in chromosome 2 and the loss of the Y sex chromosome.  Which is interesting considering that the pro-chondrogenic gene Sox9 is a sex-linked gene. According to Table S1, Sox9 was much more homologously expressed between GFP+ and GFP- cells than Col2, Agc, Col10, Col1, and Nanog. Col2, ColX, and Agc ranged from 10 to 100 fold difference whereas Sox9 was only 2 fold different.  Perhaps Sox9 was not the driving transcription factor behind chondrogenesis, Klf4 is a likely suspect.
According to Generation of Porcine-Induced Pluripotent Stem Cells by Using OCT4 and KLF4 Porcine Factors., BMP4 is a stem-cell like marker and BMP4 may activate Myc.  This may explain why BMP4 was most effective at inducing chondrogenesis given the transgenic expression of the four key stemness genes(Oct4, Sox2, Klf4, Myc).
Other BMP's were related to the stemness cluster according to String.Embl but BMP4 was the closest.  The alternative protein they mention TGFB3 was not able to be connected to the stemness cluster even at numerous steps away.
The scientists showed that the elastic modulus was higher for the GFP+ than the GFP- cells. Since both cells were in the same culture and exposed to the same stimulii,  perhaps the elastic modulus is indicative of actin cytoskeleton differences.  According to Actin filaments play a primary role for structural integrity and viscoelastic response in cells., a decrease in actin cytoskeleton organization is associated with a decrease in elasticity and viscosity.  But this difference was only for the inner region of pellets and not the outer region.  Structural indentation stiffness was higher for GFP+ over GFP- cells in bulk measurements.

Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells.

"We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2{Lefty2 is downregulated by LSJL}). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin{up} and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells."

"SB431542 inhibits activation of the activin receptor-like kinase (ALK) receptors 4, 5, and 7, key members of the TGF-β signaling pathway, and is thought to induce hESC differentiation by inhibiting SMAD2/3 phosphorylation"

"exogenous transcription factors used to induce iPSC generation (OCT4, SOX2, and cMyc) typically suppress TGF-β superfamily signaling and KLF4"<-This is is consistent as KLF4 has similarities to Sox9 which is a target of TGFB.

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones, not Cell Type of Origin.

"Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.

The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation."

"[DF] cells were separately transfected with pMXs vectors harboring four human Yamanaka factors (OCT3/4, SOX2, KLF4, and c-MYC) with FuGENE 6 transfection reagent"

"Using probe sets of differentially methylated regions (DMR) between DFs and BMSCs (6,176 in 485,531 probes), the overall methylation patterns of iPSC clones were similar to each other and those of ESCs, and distinct from those of original DFs and BMSCs"

Genes upregulated in BMSC's versus dermal fibroblasts also upregulated in LSJL:
NEXN{down}
VCAM1{down}
EBF3{down}
TSPAN18{down}
ENDOD1{down}
GPRC5A
COL4A2
COL4A1
ACAN
CNN1
HAS1
TM4SF20
SORBS2{down}
Edil3
HBEGF
TES{down}
BAIAP2L1
Fzd3
Tgfbr1{down}
ECM2

Downregulated:
DPT{up}
Grem2{up}
CH25H{up}
CPXM2{up}
MMP3{up}
WDHD1
RAD51
Wdr76
MFAP4{up}
Hoxd10{up}
DPP4
DSN1
CCRL1
SLCO2A1{up}
LRRC15{up}
APOD{up}
THBS4{up}
HIST1H1A
MOXD1{up}
GLI3{up}
BAALC{up}
DNM1{up}

Erbb3

Erbb3 is upregulated by LSJL.


Requirement for ErbB2/ErbB signaling in developing cartilage and bone.

"Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter-driven expression in limb cartilage and bone. The limb skeletal elements of Prx1-Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1-Herstatin limb skeletal elements is markedly reduced."

"Only ErbB1 and ErbB4 can form functional ligand-activated homodimers, as ErbB3 is kinase-inactive, and ErbB2 lacks a soluble ligand and a ligand binding pocket revealed by the crystal structure of its ectodomain"

"Herstatin blocks tyrosine phosphorylation and activation of ErbB2, ErbB1 and ErbB3 and suppresses expression of ErbB4"

"By day 18.5, the lengths of the limb skeletal elements of Prx1-Herstatin embryos were comparable to littermate wild type controls, indicating that the effects of Herstatin were overcome with time."<-So Erbb inhibition via herstatin may only slow growth rate rather than adult height.

"endogenous ErbB2 is expressed by immature and maturing chondrocytes as well as periosteal bone of developing mouse skeletal elements."

"the functional ErbB requirement blocked by Herstatin expression during endochondral ossification is compensated for over time"

The EGFR network in bone biology and pathology.

"the epidermal growth factor receptor (EGFR) system plays important roles in skeletal biology and pathology. This network, including a family of seven growth factors - the EGFR ligands - and the related tyrosine kinase receptors EGFR (ERBB1){up in LSJL}, ERBB2, ERBB3 and ERBB4, regulates aspects such as proliferation and differentiation of osteoblasts, chondrocytes and osteoclasts, parathyroid hormone-mediated bone formation and cancer metastases in bone."

Egfr knockout results in impaired endochondral ossification.  EGF overexpression results in height loss.  BTC{up in LSJL over 3 fold} overexpression reduces height.

"EGF was shown to be one of the factors required for bone marrow stromal fibroblast colony formation"

"EGF increases IGFI-R expression in growth plate chondrocytes, enhancing IGF-I-stimulated mitotic activity and proteoglycan synthesis; furthermore, systemic treatment of rats and minipigs with EGF reduced circulating levels of IGF-I and modulated the levels of various IGF-binding proteins"

High cortical bone mass phenotype in betacellulin transgenic mice is EGFR dependent.

"Signaling through the epidermal growth factor receptor (EGFR) by ligands such as epidermal growth factor (EGF), transforming growth factor alpha (TGFA), [BTC], and amphiregulin (AREG) has been reported to have effects on skeletal growth. transgenic mice overexpressing BTC ubiquitously under the control of the chicken beta-actin promoter (BTC-tg) exhibited stunted growth and disproportionately sized long bones. In this study, we performed a detailed phenotypic analysis of BTC-tg mice at 3, 6, and 9 wk of age. Osteoblastic cells from transgenic mice showed strong expression of BTC as determined by Western blots and by immunohistochemistry on bone sections. In femurs of male and female BTC-tg mice, we found reduced longitudinal bone growth and a pronounced increase in total volumetric BMD. The increased femoral BMD was mainly caused by augmented endocortical bone apposition and subsequent cortical bone thickening. In contrast, vertebral BMD was reduced in BTC-tg mice of both sexes. An overall similar phenotype was found in 6-mo-old BTC-tg mice. The increase in cortical bone mass in the appendicular skeleton of BTC-tg mice was largely blocked when they were crossed into the Egfr (Wa5) background characterized by a dominant negative EGFR."

"Btc expression was increased ∼1000-fold{much more than 3} in bones from BTC-tg mice. It was reported previously that overexpression of individual EGF ligands can upregulate the expression of other EGF ligands. However, the only statistically significant change in mRNA abundance of EGF ligands in bones from BTC-tg relative to WT mice was a 33% reduction in Egf transcripts"

"nonsignificant trends toward increased expression of Ereg and downregulated expression of Tgfa mRNAs [occurred] in BTC-tg mice"

BMPs

Gradients in bone morphogenetic protein-related gene expression across the growth plate.

"In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation."

"Mice deficient in both BMP receptor-1a and -1b in cartilage lack the majority of skeletal elements that form through endochondral ossification"<-LSJL upregulates BMPR1B.

"Inhibitors of BMP signaling, BMP-3, GDF-10, gremlin, and chordin were expressed early in the differentiation pathway."

"BMPR1a was detected at similar levels throughout the growth plate, whereas BMPR-1b was found to be expressed approximately eightfold higher in the HZ than in the PZ "

"In the growth plate, we found that BMP-2 and -6 are expressed primarily in the hypertrophic zone at levels similar to or greater than those found in the trabecular bone of the metaphysis. In contrast, BMP-7, previously shown to inhibit growth plate chondrocyte differentiation in cultured fetal metatarsal bones, was found to be expressed at the highest levels in PZ."

"treatment with Noggin, a BMP antagonist, inhibits proliferation of resting zone chondrocytes and hypertrophy of proliferative zone chondrocytes"

"Overexpression of a constitutively active BMPR1a in mice accelerates hypertrophic differentiation, but has no effect on proliferation"

"BMPR1a may mediate the effects of BMPs earlier in the differentiation pathway, especially in the transition from resting to proliferative phenotype, whereas BMPR1b may preferentially mediate the effects of BMPs on hypertrophic differentiation."

"BMP agonists were expressed primarily in HZ, whereas BMP antagonists were expressed primarily in the resting zone."

Bovine GDF10 gene polymorphism analysis and its association with body measurement traits in Chinese indigenous cattle.

"The objective of this research was to detect bovine GDF10 gene polymorphism and analyze its association with body measurement traits (BMT) of animals sampled from 6 different Chinese indigenous cattle populations. The populations included Xuelong (Xl), Luxi (Lx), Qinchuan (Qc), Jiaxian red (Jx), Xianang (Xn) and Nanyang (Ny). Blood samples were taken from a total of 417 female animals stratified into age categories of 12-36 months. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was employed to find out GDF10 single polymorphism nucleotide (SNPs) and explore their possible association with BMT. Sequence analysis of GDF10 gene revealed 3 SNPs in total: 1 in exon1 (G142A) and 2 in exon3 (A11471G, and T12495C). G142A and T12495C SNPs are both synonymous mutation. They showed 2 genotypes namely respectively (GG, GA) and (PP and PB). A11471G SNP is a missense mutation leading to the change of Alanine to Threonine amino acid. It showed three genotypes namely AA, BB and AB. Analysis of association of polymorphism with body measurement traits at the three locus showed that there were significant effects on BMT in Qc, Jx and Ny cattle population. These results suggest that the GDF10 gene might have potential effects on body measurement traits in the above mentioned cattle populations and could be used for marker-assisted selection."

Body Length(BL)

Height at Hips(HH)

A PB mutation at exon 3 increased body length.

"GDF10 has been discovered in rat and human femur tissue"

"BMP4 and GDF9 were found to be associated with litter size in goat "

Bone morphogenetic protein-3b (BMP-3b) inhibits osteoblast differentiation via Smad2/3 pathway by counteracting Smad1/5/8 signaling.

"Despite the involvement of BMP-3b (also called GDF-10) in osteogenesis, embryogenesis and adipogenesis, the functional receptors and intracellular signaling of BMP-3b have yet to be elucidated. In the present study, we investigated the cellular mechanism of BMP-3b in osteoblast differentiation using mouse myoblastic C2C12 cells. BMP-3b stimulated activin/TGF-β-responsive promoter activities. The stimulatory actions of BMP-3b on activin/TGF-β-responsive activities were suppressed by co-treatment with BMP-2. BMP-responsive promoter activities stimulated by BMP-2 were significantly inhibited by treatment with BMP-3b. BMP-3b suppressed the expression of osteoblastic markers including Runx2, osteocalcin and type-1 collagen induced by BMP-2, -4, -6 and -7. BMP-2-induced Smad1/5/8 phosphorylation and mRNA levels of the BMP target gene Id-1 were suppressed by co-treatment with BMP-3b, although BMP-3b failed to activate Smad1/5/8 signaling. Of interest, the BMP-3b suppression of BMP-2-induced Id-1 expression was not observed in cells overexpressing Smad4 molecules. On the other hand, BMP-3b directly activated Smad2/3 phosphorylation and activin/TGF-β target gene PAI-1 mRNA expression, while BMP-2 suppressed BMP-3b-induced Smad2/3 signal activation. BMP-2 inhibition of BMP-3b-induced PAI-1 expression was also reversed by overexpression of Smad4. Analysis using inhibitors for BMP-Smad1/5/8 pathways revealed that these BMP-3b effects were mediated via receptors other than ALK-2, -3 and -6. Furthermore, results of inhibitory studies using extracellular domains for BMP receptor constructs showed that the activity of BMP-3b was functionally facilitated by a combination of ALK-4 and ActRIIA. Collectively, BMP-3b plays an inhibitory role in the process of osteoblast differentiation, in which BMP-3b and BMP-2 are mutually antagonistic possibly by competing with the availability of Smad4."

"Sca-1, a stem cell marker having a regenerative capacity, maintains the growth and invasive characteristics of tumor cells in part by suppressing the expression of BMP-3b, leading to inhibition of TGF-β signaling through BMP-3b"

BMP-6 and BMPR-1a are up-regulated in the growth plate of the fractured tibia.

In the joint loading study, the non-loaded limb was compared to the loaded limb so it's possible that BMP-6 and BMPR-1a were upregulated in both limbs thus accounting for the growth increase in the contralateral versus control limbs.

"Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long-term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture-animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague-Dawley rats (male, 4 weeks old), were subjected to a unilateral mid-diaphyseal tibial fracture. Kinetic expression of physeal BMP-2, -4, -6, -7, and BMP receptor-1a (BMPR-1a) was analyzed in a monthly period by quantitative real time-polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post-fracture, no changes in physeal BMPs gene-expression were detected. Twenty-nine days post-fracture, when the fracture was consolidated, physeal expression of BMP-6 and BMPR-1a was significantly upregulated in the growth plate of the fractured and contra-lateral intact bone compared to control (p < 0.005)[or it could be that the duration was not long enough, gene expression after joint loading was done after 49 hours but an increase in contralateral bone length was still observed after studies taking place only 10 days]. This study demonstrates a late role of BMP-6 and BMPR-1a in fracture-induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory "cross-talk" mechanism between the lower limbs whose function could be to limit leg-length-discrepancies following the breakage of growing bones. "

"An average overgrowth following shaft fracture of the femur has been defined to be almost 1 cm (range 0.4–2.7 cm) in a review of 74 patients under the age of 13."

They also detected a change in BMP-7.

"Postnatally, [BMPs] are expressed at the physis in resting (BMP-3), proliferating (BMP-7) and pre-/hypertrophic chondrocytes (BMP-2 and -6), as well as in the perichondrium and periosteum (BMP-2, -3, -4, -5, and -7)."

" BMP-6, -7, and -receptor 1a showed a tendency towards a lower expression in physeal tissue of broken bones compared to the contra-lateral and the control group during the acute inflammatory phase of fracture healing (Day 1 and Day 3). BMP-4 mRNA levels were lowered only on Day 1 compared to control animals and to the contra-lateral non-fractured bon. Ten and 14 days after fracture, no significant differences in physeal gene-expression could be observed between the different groups. Thus, as no significant differential physeal expression of BMP-2, -4, -6, -7, and BMPR-1a was observed during inflammatory, fibrogenic, and early osteogenic stages of fracture healing, our results indicate that BMP-2, -4, -6, and -7 do not account for an enhanced proliferation rate of physeal chondrocytes, nor an up-regulation of mitogenic genes seen shortly after fracture."

"apoptosis rates of physeal chondrocytes achieved their maximum on Day 29 post diaphyseal fracture."

"BMP-6 and BMPR-1a were deregulated in both the broken and contra-lateral non-broken tibiae 29 days post-fracture"

"BMP-6, redundantly expressed by hypertrophic chondrocytes, contributes to posttraumatic overgrowth by exerting its stimulating effect on chondrogenesis and osteogenesis via BMPR-1a activation during later stages of bone healing."

I sent a comment to the author that contralateral overgrowth was induced in LSJL studies before day 29 and that no evidence was seen of an upregulation of BMPR-1a and BMP-6 in the LSJL studies however it's possible that these two BMP's were upregulated in both limbs thus canceling each other out.

Here was her response:

"I am aware that tensile dynamic loading applied to growing bones stimulates growth and therefore might be used to overcome leg length discrepancies..."

Although more loading is involved in LSJL than tensile dynamic loading like hydrostatic pressure and fluid flow.

"Our paper however addressed to analyze the input of a diaphyseal fracture on the nearby growth plate and showed that BMP -6 and the revozier 1a are upregulated at later stages of fracture healing which was also observed at the contralateral side on day 29 but not at the earlier investigated time-points, with the last one before, being day 14th.
I don't think that it is possible to compare our results with a joint loading study, as the fractured bone in our study was not loaded at all during early stages of feacture healing due to instable fracture conditions.

Nevertheless, I think, that we can draw the connection in the context that the contralateral side does respond to any kind of trauma or external stimulus, eventhough the bone is kept untouched in the experiments.
Bmp -6 seems to be one target gene of the whole process which certainly involves several signalling pathways including systemic growth hormons."

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways.

"we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a(CKO)) and Bmpr1a(CKO);Bmpr1b+/- (Bmpr1a(CKO);1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. The balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1."

"there was a significant increase in activated STAT1- and STAT5a-positive chondrocytes in the proliferative zones in Bmpr1aCKO;1b+/- mice"

"FGFR1 levels are increased in Bmpr1aCKO and Bmpr1aCKO;1b+/- growth plates "

"the Ihh promoter is responsive to SMAD1 in chondrocytes, suggesting that BMPs induce Ihh, and that this regulation may be direct. "

"BMP signaling partially inhibits ERK1/2 MAPK."