Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

Abstract

The directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocytemacrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-B ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. IntroductionThe embryonic development of the mammalian skeleton and the maintenance of skeletal and calcium homeostasis during adult life is dependent on the continuous interaction between osteoclasts, which degrade or resorb bone, and osteoblasts, which form bone. 1 The origins of these cells are well established: whereas osteoblasts are derived from mesenchymal stem cells, osteoclasts are highly specialized, multinucleated cells that are derived from hematopoietic stem cells, specifically from cells of the monocyte-macrophage lineage. 2,3 A large proportion of diseases that affect bone metabolism, such as osteoporosis, rheumatoid arthritis, multiple myeloma, and metastatic bone disease, is characterized by pathologic osteoclastic bone resorption. These cells are therefore very important targets for therapeutic intervention of bone diseases characterized by bone loss.The bulk of what is known about the transcription factors, signaling molecules, and genes that control osteoclast differentiation and activation has come largely from studying mammalian osteopetroses with the use of natural and targeted mutations in mice. [2][3][4][5] The breakthroughs in the past 10 years after the discovery of the tumor necrosis factor (TNF) family member, receptor activator of nuclear factor-B ligand (RANKL), which binds to its receptor RANK and stimulates osteoclast differentiation from macrophage colony-stimulating factor (M-CSF)-dependent precursors, 2,3,6,7 have made it relatively straightforward to generate osteoclasts efficiently from murine hematopoietic precursors.However, the differentiation of human osteoclasts from circulating precursors present in peripheral blood mononuclear cells (PBMCs) is less efficient. Although it is known that osteoclast precursors reside in the CD14 ϩ monocyte fraction from PBMCs, high donor variability, sample heterogeneity, low precursor frequency, and unpredictable responsiveness to different batches of serum for in vitro culture have provided the biggest obstacles to establishing defined, efficient, and reproducible system...

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