Monday, January 2, 2012

What's the typical gene expression of bone marrow?

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

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

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

"Adult Lewis rats were used"

Genes upregulated in MSCs versus ASCs also upregulated in LSJL:


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Gene expression increased during 14 days of chondrogenic differentiation versus day 2 that were also up-(or down) regulated by LSJL:
COL6A2(both up- and down-)


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Supplementary Material

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

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

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

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

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

Genes upregulated in PDLSCs versus WJCMSCs also upregulated in LSJL:

-2fold cutoff detailed analysis TBD

Genes downregulated in PDLSCs versus WJCMscs also downregulated in LSJL:

-2 fold cutoff

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

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

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

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

The youngest donor for MSCs was 29.

Pro-chondrogenic genes expressed in BM-MSCs at a higher fold than Adipose Tissue MSCs:

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

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


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

A mesenchymal stromal cell gene signature for donor age.

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

HMGA2 expression also increases with age.

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

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

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

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

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

Geometric cues for directing the differentiation of mesenchymal stem cells.

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

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

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

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

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

In basal media

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

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

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

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

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

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

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

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

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

Genes upregulated in MSCs versus repair cartilage also up in LSJL:

Genes downregulated:
Col6a2{up and down}

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

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

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

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

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

In osteoblast category:
Bhlhb2{also upregulated in LSJL and known as DEC1}
Cdh11{down, actually an osteoblast related gene}

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

In muscle category:

In fibroblast:



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

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

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

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

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

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

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

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

Genes upregulated:

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

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

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

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

Genes upregulated during chondrogenic differentiation also up in LSJL:

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