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

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Uta Rössler - , Charité – Universitätsmedizin Berlin (Autor:in)
  • Anna Floriane Hennig - , Charité – Universitätsmedizin Berlin, Freie Universität (FU) Berlin, Georg-August-Universität Göttingen (Autor:in)
  • Nina Stelzer - , Charité – Universitätsmedizin Berlin (Autor:in)
  • Shroddha Bose - , Freie Universität (FU) Berlin (Autor:in)
  • Johannes Kopp - , Charité – Universitätsmedizin Berlin, Freie Universität (FU) Berlin, Max Planck Institut für Molekulare Genetik (Autor:in)
  • Kent Søe - , University of Southern Denmark (Autor:in)
  • Lukas Cyganek - , Georg-August-Universität Göttingen, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK) (Autor:in)
  • Giovanni Zifarelli - , National Research Council of Italy (CNR) (Autor:in)
  • Salaheddine Ali - , Charité – Universitätsmedizin Berlin, Max Planck Institut für Molekulare Genetik (Autor:in)
  • Maja von der Hagen - , Klinik und Poliklinik für Kinder- und Jugendmedizin, Abteilung für Neuropädiatrie (Autor:in)
  • Elisabeth Tamara Strässler - , Charité – Universitätsmedizin Berlin, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK) (Autor:in)
  • Gabriele Hahn - , Institut und Poliklinik für diagnostische und interventionelle Radiologie (Autor:in)
  • Michael Pusch - , National Research Council of Italy (CNR) (Autor:in)
  • Tobias Stauber - , Freie Universität (FU) Berlin, MSH Medical School Hamburg (Autor:in)
  • Zsuzsanna Izsvák - , Max-Delbrück-Centrum für Molekulare Medizin (MDC) (Autor:in)
  • Manfred Gossen - , Charité – Universitätsmedizin Berlin, Helmholtz-Zentrum Hereon (Autor:in)
  • Harald Stachelscheid - , Charité – Universitätsmedizin Berlin (Autor:in)
  • Uwe Kornak - , Charité – Universitätsmedizin Berlin, Georg-August-Universität Göttingen, Max Planck Institut für Molekulare Genetik (Autor:in)

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

OriginalspracheEnglisch
Seiten (von - bis)1621-1635
Seitenumfang15
FachzeitschriftJournal of bone and mineral research
Jahrgang36
Ausgabenummer8
PublikationsstatusVeröffentlicht - Aug. 2021
Peer-Review-StatusJa

Externe IDs

PubMed 33905594

Schlagworte

Ziele für nachhaltige Entwicklung

Schlagwörter

  • CLCN7, hiPSCs, OSTEOCLASTS, OSTEOPETROSIS

Bibliotheksschlagworte