Human Mesenchymal Stem Cells Promote Human Osteoclast Differentiation from CD34+ Bone Marrow Hematopoietic Progenitors*

Abstract

Interactions between osteoclast progenitors and stromal cells derived from mesenchymal stem cells (MSCs) within the bone marrow are important for osteoclast differentiation. In vitro models of osteoclastogenesis are well established in animal species; however, such assays do not necessarily reflect human osteoclastogenesis. We sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human marrow-derived MSCs (hMSCs) and CD34+ hematopoietic stem cells (HSCs). After 3 weeks, coculture of hMSCs and HSCs resulted in an increase in hematopoietic cell number with formation of multinucleated osteoclast-like cells (Ocls). Coculture of hMSCs with HSCs, transduced with a retroviral vector that expresses enhanced green fluorescent protein, produced enhanced green fluorescent protein+ Ocls, further demonstrating that Ocls arise from HSCs. These Ocls express calcitonin and vitronectin receptors and tartrate-resistant acid phosphatase and possess the ability to resorb bone. Ocl formation in this assay is cell contact dependent and is independent of added exogenous factors. Conditioned medium from the coculture contained high levels of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), and macrophage-colony stimulating factor. IL-6 and LIF were present at low levels in cultures of hMSCs but undetectable in cultures of HSCs alone. These data suggest that coculture with HSCs induce hMSCs to secrete cytokines involved in Ocl formation. Addition of neutralizing anti-IL-6, IL-11, LIF, or macrophage-colony stimulating factor antibodies to the coculture inhibited Ocl formation. hMSCs seem to support Ocl formation as undifferentiated progenitor cells, because treatment of hMSCs with dexamethasone, ascorbic acid, and beta-glycerophosphate (to induce osteogenic differentiation) actually inhibited osteoclastogenesis in this coculture model. In conclusion, we have developed a simple and reproducible assay using culture-expanded hMSCs and purified HSCs with which to study the mechanisms of human osteoclastogenesis.

Hurler Disease Bone Marrow Stromal Cells Exhibit Altered Ability to Support Osteoclast Formation

“…Based on these findings, we hypothesized that a possible increase of the osteoclast number in MPS I growth plates could result from abnormalities in the levels of RANKL/OPG locally produced by BMSC. To test our hypothesis, we evaluated the capacity of MPS I versus HD BMSC to support the generation of osteoclasts from normal CD34 positive cells in vitro [33]. The number of osteoclasts differentiated in the presence of MPS I BMSC was significantly higher.…”

“…To investigate the osteoclastogenic capacity of BMSC derived from MPS IH patients, we established a co-culture system between MPS I and HD BMSC and CD34 + HD BMderived cells (purity > 95%, by flow cytometry), as previously described by Mbalaviele et al [33], to evaluate the formation of osteoclast-like cells. After 3 weeks, we observed multinucleated cells in both groups of co-cultures (Fig.…”

Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

“…A) Purification of osteoclast precursors from PBMNC with CD14 and CD34 antibodies was reported [5,6]. This is not necessary in our method, making the protocol simpler and cheaper.…”

Mesenchymal stem cells from bone marrow show a stronger stimulating effect on megakaryocyte progenitor expansion than those from non-hematopoietic tissues

“…In coculture experiments, bone marrow-derived MSCs form cell clusters with CD41 þ megakaryocytes [9]. At the same time as providing physical support for HSCs, they constitutively secrete cytokines that are important for megakaryocytic differentiation [7,10,11]. Therefore, MSCs play a role in regulating megakaryocytopoiesis.…”