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bioRxiv · 10.1101/2024.03.12.584629

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: A revision of the prohypertrophic signalling network concept

Diederichs, S.·Dreher, S. I.·Nueesch, S.-A.·Schmidt, S.·Merle, C.·Richter, W.
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Abstract

BackgroundIn vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-{beta} is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-{beta} can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN 212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-{beta} receptors during MSC in vitro chondrogenesis. MethodsStandard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-{beta} and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. ResultsAll groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. ConclusionsOverall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-{beta} that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-{beta}-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

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Diederichs, S., Dreher, S. I., Nueesch, S.-A., Schmidt, S., Merle, C., Richter, W.. 2024-03-13. Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: A revision of the prohypertrophic signalling network concept. https://doi.org/10.1101/2024.03.12.584629

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