Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells

Abstract

In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or genetically manipulated mouse lines. These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.

p62/sequestosome 1 deficiency accelerates osteoclastogenesis in vitro and leads to Paget’s disease–like bone phenotypes in mice

“…Culture plates were washed with water (two times), stained with 5% aqueous AgNO 3 solution in combination with UV light treatment for 30 min, and washed again with water (three times). Resorption areas were quantified as described previously (31).…”

Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology

“…Finally, the RAW 264.7 cell line is broadly used to obtain osteoclasts in vitro; however, the endogenous production of M-CSF by these cells differs largely from the primary osteoclast culture [10]. The ER-Hoxb8 cell line is a murine, conditionally immortalized, monocyte precursor cell line that has recently been shown to differentiate toward functional osteoclasts in vitro [11,12]. Hoxb8 is a member of the Homeobox gene family, a family of transcription factors that controls various aspects of development.…”

Genetic engineering of Hoxb8-immortalized hematopoietic progenitors – a potent tool to study macrophage tissue migration

“…Besides the exploration of gene function by deletion, CRISPR/ Cas9-mediated genome editing of Hoxb8-immortalized hematopoietic precursor cells also provides the opportunity to easily perform multiple gene deletions, gene overexpression or expression of specific mutants or reporter genes (Salsman and Dellaire, 2017). Added to the capacity of Hoxb8 cell progenitors to differentiate into a large scale of hematopoietic cells, such as DCs (Hammerschmidt et al, 2018), granulocytes (Wang et al, 2006), osteoclasts (Zach et al, 2015) or even B and T lymphocytes (Redecke et al, 2013), it represents a potent, rapid and simple model that opens unlimited possibilities for analysis of leukocyte biological functions including tissue migration.…”

“…Expansion of murine hematopoietic precursors that were transiently immortalized with a retrovirus-delivered and estrogeninducible form of the transcription factor Hoxb8 have been described (Wang et al, 2006) and validated for the study of hematopoietic cell biology (Cabron et al, 2018;Chu et al, 2019;Di Ceglie et al, 2017;Gurzeler et al, 2013;Hammerschmidt et al, 2018;Lee et al, 2017;Rosas et al, 2011;Wang et al, 2006;Witschi et al, 2010;Zach et al, 2015). The recent coupling of this long-term hematopoietic progenitor cell line to the CRISPR/Cas9 technology (Hammerschmidt et al, 2018;Roberts et al, 2019) has enabled the creation of new genetically modifiable cell models.…”