Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells

Abstract

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian-blue staining, qRT-PCR, and quantification of alkaline phosphatase (ALP) activity. Pre-differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)-beta was always required for chondrogenic differentiation and deposition of a collagen-type-II-positive extracellular matrix, while bone morphogenetic protein (BMP)-2, -4, -6, -7, aFGF, and IGF-I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF-beta, however, without preventing collagen type X expression. bFGF or parathyroid hormone-like peptide (PTHrP) inhibited the TGF-beta-responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up-regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor-independent pathway. While TGF-beta was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II(+)/collagen type X(-) cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes.

Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis

“…At 1,000 pM PTHrP(1-40) appears to inhibit chondrogenesis and is therefore out of therapeutic considerations if applied throughout the four weeks of differentiation as described here. Similar to our results, Weiss et al [14] could show inhibition of early and late stages of MSC chondrogenesis by PTHrP(1-34) without selective inhibition of hypertrophy markers. Both the expression of chondrogenic markers (type II collagen) and hypertrophic markers (type X collagen and ALP) were suppressed by PTHrP.…”

Human articular chondrocytes secrete parathyroid hormone–related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis

“…In light of the improved chondrogenesis in HACconditioned medium, as opposed to the down-regulation of COL2A1 in the presence of 10 ng/ml of PTHrP observed in our previous study (33), we believe that PTHrP is certainly not the only factor involved in the attenuation of hypertrophy by HAC-conditioned medium. Most likely, factors that support anabolic differentiation act in synergy with more than one negative regulator; however, PTHrP is a strong candidate negative regulator.…”

“…This would require the prevention of PTHrP actions in HAC culture supernatants by a specific inhibitor that would block PTHrP. Known inhibitors, such as PTHrP , target the PTHrP receptor PTHR-1, and our previous results (33) suggest that PTHrP exerts receptor-independent effects during early chondrogenesis, when PTHR-1 mRNA is not yet detected (33,40).…”

“…We therefore performed assays for FGF-2 and PTHrP, both of which are negative regulators of chondrocyte differentiation (32) and MSC chondrogenesis (33). FGF-2 and PTHrP have been shown to severely reduce type X collagen expression, AP activity, and cell enlargement of lower sternal chondrocytes from immature chicken (34,35), and both molecules are soluble factors produced by articular chondrocytes (36)(37)(38).…”

Effects of Low Intensity Ultrasound on the Chondrogenic Differentiation of Adult Stem Cells From Adipose Tissue

“…This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. that in vitro MSCs chondrogenesis protocols may result in expression of hyaline cartilage components associated with early onset of hypertrophic markers such as collagen type X and alkaline phosphatase [20][21][22][23]. MSCs that were differentiated by common in vitro protocols and transplanted to ectopic sites underwent alterations related to endochondral ossification instead of a stable chondrogenic phenotype [24].…”