[en] Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behavior of serum expanded progenitor cells, but the underlying mechanism and how this is reflected in vivo remains unknown. Herein, the cellular and molecular changes associated with a cellular phenotype shift induced by serum-free preconditioning of human periosteum-derived cells were investigated. Following BMP-2 stimulation, preconditioned cells displayed enhanced in vivo bone forming capacity, associated with an adapted cellular metabolism together with an elevated expression of BMPR2. Single-cell RNA sequencing confirmed the activation of pathways and transcriptional regulators involved in bone development and fracture healing, providing support for the augmentation of specified skeletal progenitor cell populations. The reported findings illustrate the importance of appropriate in vitro conditions for the in vivo outcome. In addition, BMPR2 represents a promising biomarker for the enrichment of skeletal progenitor cells for in vivo bone regeneration.
Disciplines :
Engineering, computing & technology: Multidisciplinary, general & others
Author, co-author :
Bolander, Johanna ; Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium.
Herpelinck, Tim; Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium.
Chaklader, Malay; Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium.
Gklava, Charikleia; Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium.
Geris, Liesbet ; Université de Liège - ULiège > GIGA > GIGA In silico medecine - Biomechanics Research Unit ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium. ; Biomechanics Section, KU Leuven, Leuven, Belgium.
Luyten, Frank P; Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium.
Language :
English
Title :
Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity.
Langer R, Vacanti J. Tissue engineering. Science. 1993;260:920-926.
Ma J, Both SK, Yang F, et al. Concise review: cell-based strategies in bone tissue engineering and regenerative medicine. Stem Cells Translational Medicine. 2014;3:98-107. https://doi.org/10.5966/sctm.2013-0126.
Ho-Shui-Ling A, Bolander J, Rustom LE, Johnson AW, Luyten FP, Picart C. Bone regeneration strategies: engineered scaffolds, bioactive molecules and stem cells current stage and future perspectives. Biomaterials. 2018;180:143-162. https://doi.org/10.1016/j.biomaterials.2018.07.017.
Lenas P, Moos M, Luyten FP. Developmental engineering: a new paradigm for the design and manufacturing of cell-based products. Part I: from three-dimensional cell growth to biomimetics of in vivo development. Tissue Eng Part B Rev. 2009;15:381-394. https://doi.org/10.1089/ten.TEB.2008.0575.
Ingber DE, Mow VC, Butler D, et al. Tissue engineering and developmental biology: going biomimetic. Tissue Eng. 2006;12:3265-3283. https://doi.org/10.1089/ten.2006.12.3265.
Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev Rheumatol. 2015;11:45-54. https://doi.org/10.1038/nrrheum.2014.164.
Colnot C. Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration. J Bone Miner Res. 2009;24:274-282. https://doi.org/10.1359/jbmr.081003.
Colnot C, Zhang X, Knothe Tate ML. Current insights on the regenerative potential of the periosteum: molecular, cellular, and endogenous engineering approaches. J Orthop Res. 2012;30:1869-1878. https://doi.org/10.1002/jor.22181.
Bolander J, Chai YC, Geris L, et al. Early BMP, Wnt and Ca(2+)/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates. Biomaterials. 2016;86:106-118. https://doi.org/10.1016/j.biomaterials.2016.01.059.
Bolander J, Ji W, Geris L, et al. The combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells. Eur Cells Mater. 2016;31:11-25.
Bolander J, Ji W, Leijten J, et al. Healing of a large long-bone defect through serum-free Iin vitro priming of human periosteum-derived cells. Stem Cell Rep. 2017;8:758-772. https://doi.org/10.1016/j.stemcr.2017.01.005.
Salazar VS, Capelo LP, Cantù C, et al. Reactivation of a developmental Bmp2 signaling center is required for therapeutic control of the murine periosteal niche. Elife. 2019;8:e42386. https://doi.org/10.7554/eLife.42386.
Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol. 2016;12:203-221. https://doi.org/10.1038/nrendo.2016.12.
International Stem Cell Initiative Consortium, Akopian V, Andrews PW, et al. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim. 2010;46:247-258. https://doi.org/10.1007/s11626-010-9297-z.
Verbeeck L, Geris L, Tylzanowski P, Luyten FP. Uncoupling of in-vitro identity of embryonic limb derived skeletal progenitors and their in-vivo bone forming potential. Sci Rep. 2019;9:5782. https://doi.org/10.1038/s41598-019-42259-x.
Yang PJ, Yuan WX, Liu J, et al. Biological characterization of human amniotic epithelial cells in a serum-free system and their safety evaluation. Acta Pharmacol Sin. 2018;39:1305-1316. https://doi.org/10.1038/aps.2018.22.
Trapnell C, Cacchiarelli D, Grimsby J, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol. 2014;32:381-386. https://doi.org/10.1038/nbt.2859.
Platzer A. Visualization of SNPs with t-SNE. PLoS One. 2013;8:e56883. https://doi.org/10.1371/journal.pone.0056883.
Vento-Tormo R, et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature. 2018;563:347-353. https://doi.org/10.1038/s41586-018-0698-6.
Aibar S, et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods. 2017;14:1083-1086. https://doi.org/10.1038/nmeth.4463.
Kim S, et al. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. Genes Dev. 2003;17:1979-1991. https://doi.org/10.1101/gad.1119303.
Featherall J, Robey PG, Rowe DW. Continuing challenges in advancing preclinical science in skeletal cell-based therapies and tissue regeneration. J Bone Miner Res. 2018;33:1721-1728. https://doi.org/10.1002/jbmr.3578.
Berger MG, et al. Cell culture medium composition and translational adult bone marrow-derived stem cell research. Stem Cells. 2006;24:2888-2890. https://doi.org/10.1634/stemcells.2006-0387.
Dimarakis I, Levicar N. Cell culture medium composition and translational adult bone marrow-derived stem cell research. Stem Cells. 2006;24:1407-1408. https://doi.org/10.1634/stemcells.2005-0577.
Lowery JW, Rosen V. Bone morphogenetic protein-based therapeutic approaches. Cold Spring Harb Perspect Biol. 2018;10:a022327. https://doi.org/10.1101/cshperspect.a022327.
Knuth CA, Witte-Bouma J, Ridwan Y, Wolvius EB, Farrell E. Mesenchymal stem cell-mediated endochondral ossification utilising micropellets and brief chondrogenic priming. Eur Cell Mater. 2017;34:142-161. https://doi.org/10.22203/eCM.v034a10.
Eyckmans J, et al. Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors. Biomaterials. 2013;34:4612-4621. https://doi.org/10.1016/j.biomaterials.2013.03.011.
Yamashita A, et al. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Stem Cell Rep. 2015;4:404-418. https://doi.org/10.1016/j.stemcr.2015.01.016.
Colciago A, et al. In vitro effects of PDGF isoforms (AA, BB, AB and CC) on migration and proliferation of SaOS-2 osteoblasts and on migration of human osteoblasts. Int J Biomed Sci. 2009;5:380-389.
Fiedler J, Etzel N, Brenner RE. To go or not to go: migration of human mesenchymal progenitor cells stimulated by isoforms of PDGF. J Cell Biochem. 2004;93:990-998. https://doi.org/10.1002/jcb.20219.
Godwin SL, Soltoff SP. Extracellular calcium and platelet-derived growth factor promote receptor-mediated chemotaxis in osteoblasts through different signaling pathways. J Biol Chem. 1997;272:11307-11312.
Canalis E, Varghese S, McCarthy TL, Centrella M. Role of platelet derived growth factor in bone cell function. Growth Regul. 1992;2:151-155.
Li G, Cui Y, McIlmurray L, Allen WE, Wang H. rhBMP-2, rhVEGF(165), rhPTN and thrombin-related peptide, TP508 induce chemotaxis of human osteoblasts and microvascular endothelial cells. J Orthop Res. 2005;23:680-685. https://doi.org/10.1016/j.orthres.2004.12.005.
Deveza L, Ortinau L, Lei K, Park D. Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells. PLoS One. 2018;13:e0190909. https://doi.org/10.1371/journal.pone.0190909.
Benn A, Bredow C, Casanova I, Vukicevic S, Knaus P. VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling. J Cell Sci. 2016;129:206-218. https://doi.org/10.1242/jcs.179960.
Eyckmans J, Roberts SJ, Schrooten J, Luyten FP. A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling. J Cell Mol Med. 2010;14:1845-1856. https://doi.org/10.1111/j.1582-4934.2009.00807.x.
Mao AS, Shin JW, Mooney DJ. Effects of substrate stiffness and cell-cell contact on mesenchymal stem cell differentiation. Biomaterials. 2016;98:184-191. https://doi.org/10.1016/j.biomaterials.2016.05.004.
Nohe A, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem. 2002;277:5330-5338. https://doi.org/10.1074/jbc.M102750200.
Dorpholz G, et al. IRS4, a novel modulator of BMP/Smad and Akt signalling during early muscle differentiation. Sci Rep. 2017;7:8778. https://doi.org/10.1038/s41598-017-08676-6.
Onishi T, Ishidou Y, Nagamine T, et al. Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats. Bone. 1998;22:605-612.
Riddle RC, Clemens TL. Bone cell bioenergetics and skeletal energy homeostasis. Physiol Rev. 2017;97:667-698. https://doi.org/10.1152/physrev.00022.2016.
Stegen S, et al. HIF-1alpha promotes glutamine-mediated redox homeostasis and glycogen-dependent bioenergetics to support postimplantation bone cell survival. Cell Metab. 2016;23:265-279. https://doi.org/10.1016/j.cmet.2016.01.002.
Chan CKF, et al. Identification of the human skeletal stem cell. Cell. 2018;175:43-56.e21. https://doi.org/10.1016/j.cell.2018.07.029.
