Recapitulation of mesenchymal condensation enhances in vitro chondrogenesis of human mesenchymal stem cells
"Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. Exposing hMSCs to hypoxia (2% O2) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O2) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks. The benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9{up} and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X{up}, [RUNX2], and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O2, resulted in the most efficient chondrogenesis. While hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation."
"hMSCs expanded under hypoxic conditions (2% O2 tension) exhibited greater proliferation and expression of connexin-43, and higher fibronectin and type I collagen (COL I) production compared to normoxia (20% O2 tension)"
"hypoxia diminishes chondrogenesis of adipose-derived mesenchymal stem cells"
"for hMSC chondrogenesis, the transient activation of NOTCH signaling for the first 5 days is necessary, but needs to be inactivated afterwards, otherwise continued NOTCH signaling inhibits chondrogenesis"
"The benefits of 1 week of normoxic preconditioning observed in our study falls close to the initial 5 days of required NOTCH signaling. NOTCH signaling is known to be activated by the cell–cell contact"
"TGF-β-3 used in chondrogenic differentiation medium is known to enhance the transcriptional activation of SOX-9 by inhibiting NOTCH signaling"
All for one and one for all: condensations and the initiation of skeletal development
"Extracellular matrix molecules, cell surface receptors and cell adhesion molecules, such as fibronectin, tenascin{LSJL upregulates Tenascin N}, syndecan{LSJL upregulates Syndecan 4 and downregulates Syndecan 2}, and N-CAM, initiate condensation formation and set condensation boundaries. Hox genes (Hoxd-11-13) and other transcription factors (CFKH-1, MFH-1, osf-2), modulate the proliferation of cells within condensations. Cell adhesion is ensured indirectly through Hox genes (Hoxa-2, Hoxd-13), and directly via cell adhesion molecules (N-CAM and N-cadherin). Subsequent growth of condensations is regulated by BMPs, which activate Pax-2, Hoxa-2 and Hoxd-11 among other genes. Growth of a condensation ceases when Noggin inhibits BMP signalling, setting the stage for transition to the next stage of skeletal development, namely overt cell differentiation. "
"Condensations can be complex temporally; the subunits of a condensation may persist for different lengths of time before making the transition to overt cell differentiation"
"prechondrogenic cells in condensations [have an affinity] for peanut agglutinin (PNA)"
"The four phases of the development of a skeletal element are: migration of preskeletal cells to
the site of future skeletogenesis, which is always associated with an epithelium and epithelial basement membrane; interactions of those cells with epithelial cell products resulting in initiation of a condensation; and overt differentiation of chondroblasts or osteoblasts"
Epithelium genes involved in LSJL:
UP: Frem2
"Thrombospondin-4{up}, one of five related glycoproteins, is expressed transiently in mesenchyme associated with both chondrogenic and osteogenic condensations."
"Condensations form as a result of one or a combination of three processes: (1) enhanced mitotic activity{LSJL increases cell proliferation}; (2) aggregation of cells toward a center{LSJL creates a pressure gradient moving the cells to one location}; and (3) failure of cells to disperse from a center{Hydrostatic pressure may eventually inhibit cell movement}."
"condensation [results] from epithelial-mesenchymal interactions controlled by TGF-b, BMP-2{up}, Msx-1, and tenascin."
"Levels of intracellular cAMP increase when prechondrogenic cells condense. The increase is associated with phosphorylation of p35, the nuclear substrate for cAMP-dependent protein kinase (PKA). Indeed, within the chondrogenic pathway, PKA is found only in the nuclei of condensing cells and not in chondrocyte nuclei; PKA is imported into the nucleus at condensation. The cell-to-cell interactions concomitant with condensation and which elevate cAMP levels may in turn mediate upregulation of chondrogenic genes, thereby providing a mechanism for initiating condensation and skeletal development."<-LSJL alters the gene expression of several cAMP related protions and inhibitors.
"Pax-1{up} and Pax-9, genes that share the paired-type DNA binding homeodomain and encode nuclear transcription factors, have their strongest expression at the condensation stage, consistent with a role in condensation"
"Pax-2, regulated by BMP-7, is an important player regulating condensation size"
"Epithelial-mesenchymal interactions in avian skeletogenesis are mediated, in part, by Prx-1{up as Prrx1} and Prx-2{up as Prrx2}"
"Mice in which Hoxa-2, Dlx-2, MHox, Otx, or the retinoic acid receptor have been knocked-out display ectopic or apparently duplicated skeletal elements."
MicroRNA-375, a new regulator of cadherin-7, suppresses the migration of chondrogenic progenitors.
"Endochondral
bone formation requires a complex interplay among immature mesenchymal
progenitor cells to form the cartilaginous anlagen, involving migration,
aggregation and condensation. Even though condensation of chondrogenic
progenitors is an essential step in this process, the mechanism(s) by
which this occurs has not been well studied. Here, we investigated the
involvement of microRNAs (miRNAs) in this process and found that the
expression of miR-375 decreased upon chondrogenic differentiation of
limb mesenchymal cells. Blockade of miR-375 via peptide nucleic acid
(PNA)-based antisense oligonucleotides (ASOs) increased the migration of
chondrogenic progenitors, the formation of precartilage condensations
and the expression level of cadherin-7. Furthermore, miR-375 was
necessary and sufficient to down-regulate cell migration through
negative regulation of cadherin-7 by the direct interaction with 3' UTR
of cadherin-7. In addition, miR-375 is also involved in the cell
migration and precartilage condensation mediated by p38MAPK, a positive
signaling in the chondrogenic differentiation. Collectively, our results
suggest that miR-375 negatively modulates cell migration and subsequent
precartilage condensation by targeting cadherin-7."
"Mesenchymal condensation results from either
increased mitotic activity or from increased cell movement and
subsequent mesenchymal cell packing density without increased cell
proliferation"
"cadherin-7 directly controls cell migration in chick limb bud mesenchymal cells"
Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation.
"Matrix elasticity and cell seeding density are important factors in hMSCs differentiation. We cultured hMSCs at different seeding densities on polyacrylamide hydrogels with different stiffness corresponding to Young's moduli of 1.6 ± 0.3 and 40 ± 3.6 kPa. The promotion of osteogenic marker expression by hard gel is overridden by a high seeding density. Cell seeding density, however, did not influence the chondrogenic marker expressions induced by soft gel. The promotion of osteogenic differentiation on hard matrix was shown to be mediated through the Ras pathway. Inhibition of Ras (RasN17) significantly decreased ERK, Smad1/5/8 and AKT activation, and osteogenic markers expression. However, constitutively active Ras (RasV12) had little effect on osteogenic marker expression, suggesting that the Ras pathways are necessary but not sufficient for osteogenesis."
Chondrogenic gene expression was greatest on soft gels with a high density. Chondrogenic gene expression was possible on hard gels with a high density. Since bone is a hard tissue it would likely be more akin to a hard gel. Chondrogenic gene metrics did not increase at all on glass slides.
"For hMSCs cultured on soft matrix, cell aggregation was more predominant than cells cultured on hard matrix, hence we speculate that the aggregation-induced cell–cell contact on soft matrix, regardless of cell seeding density, may mimic 3D culture to enhance the chondrogenic processes."<-thus why high density may overcome a hard matrix.
" ERK and AKT activations were not affected by matrix elasticity during chondrogenesis"
Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation.
"Matrix elasticity and cell seeding density are important factors in hMSCs differentiation. We cultured hMSCs at different seeding densities on polyacrylamide hydrogels with different stiffness corresponding to Young's moduli of 1.6 ± 0.3 and 40 ± 3.6 kPa. The promotion of osteogenic marker expression by hard gel is overridden by a high seeding density. Cell seeding density, however, did not influence the chondrogenic marker expressions induced by soft gel. The promotion of osteogenic differentiation on hard matrix was shown to be mediated through the Ras pathway. Inhibition of Ras (RasN17) significantly decreased ERK, Smad1/5/8 and AKT activation, and osteogenic markers expression. However, constitutively active Ras (RasV12) had little effect on osteogenic marker expression, suggesting that the Ras pathways are necessary but not sufficient for osteogenesis."
Chondrogenic gene expression was greatest on soft gels with a high density. Chondrogenic gene expression was possible on hard gels with a high density. Since bone is a hard tissue it would likely be more akin to a hard gel. Chondrogenic gene metrics did not increase at all on glass slides.
"For hMSCs cultured on soft matrix, cell aggregation was more predominant than cells cultured on hard matrix, hence we speculate that the aggregation-induced cell–cell contact on soft matrix, regardless of cell seeding density, may mimic 3D culture to enhance the chondrogenic processes."<-thus why high density may overcome a hard matrix.
" ERK and AKT activations were not affected by matrix elasticity during chondrogenesis"
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