Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7‐related osteopetrosis
Creators: name: Rössler, Uta
name: Hennig, Anna Floriane
name: Stelzer, Nina
name: Bose, Shroddha
name: Kopp, Johannes
name: Søe, Kent
name: Cyganek, Lukas
name: Zifarelli, Giovanni
name: Ali, Salaheddine
name: Hagen, Maja Von Der
name: Strässler, Elisabeth Tamara
name: Hahn, Gabriele
name: Pusch, Michael
name: Stauber, Tobias
name: Izsvák, Zsuzsanna
name: Gossen, Manfred
name: Stachelscheid, Harald
name: Kornak, Uwe
Publisher: name: Charité - Universitätsmedizin Berlin
Year: 2021
Subjects: subject: CLCN7
subject: hiPSCs
subject: OSTEOCLASTS
subject: OSTEOPETROSIS
subject: 600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit

description: 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. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).