Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7-related osteopetrosis

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Uta Rössler - , Charité – Universitätsmedizin Berlin (Author)
  • Anna Floriane Hennig - , Charité – Universitätsmedizin Berlin, Free University of Berlin, University of Göttingen (Author)
  • Nina Stelzer - , Charité – Universitätsmedizin Berlin (Author)
  • Shroddha Bose - , Free University of Berlin (Author)
  • Johannes Kopp - , Charité – Universitätsmedizin Berlin, Free University of Berlin, Max Planck Institute for Molecular Genetics (Author)
  • Kent Søe - , University of Southern Denmark (Author)
  • Lukas Cyganek - , University of Göttingen, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK) (Author)
  • Giovanni Zifarelli - , National Research Council of Italy (CNR) (Author)
  • Salaheddine Ali - , Charité – Universitätsmedizin Berlin, Max Planck Institute for Molecular Genetics (Author)
  • Maja von der Hagen - , Department of Paediatrics, Division of Neuropediatrics (Author)
  • Elisabeth Tamara Strässler - , Charité – Universitätsmedizin Berlin, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK) (Author)
  • Gabriele Hahn - , Institute and Polyclinic of Diagnostic and Interventional Radiology (Author)
  • Michael Pusch - , National Research Council of Italy (CNR) (Author)
  • Tobias Stauber - , Free University of Berlin, MSH Medical School Hamburg (Author)
  • Zsuzsanna Izsvák - , Max Delbrück Center for Molecular Medicine (MDC) (Author)
  • Manfred Gossen - , Charité – Universitätsmedizin Berlin, Helmholtz-Zentrum Hereon (Author)
  • Harald Stachelscheid - , Charité – Universitätsmedizin Berlin (Author)
  • Uwe Kornak - , Charité – Universitätsmedizin Berlin, University of Göttingen, Max Planck Institute for Molecular Genetics (Author)

Abstract

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases.

Details

Original languageEnglish
Pages (from-to)1621-1635
Number of pages15
JournalJournal of bone and mineral research
Volume36
Issue number8
Publication statusPublished - Aug 2021
Peer-reviewedYes

External IDs

PubMed 33905594

Keywords

Sustainable Development Goals

Keywords

  • CLCN7, hiPSCs, OSTEOCLASTS, OSTEOPETROSIS

Library keywords