Sidestream Smoke Extracts from Harm-Reduction and Conventional Camel Cigarettes Inhibit Osteogenic Differentiation via Oxidative Stress and Differential Activation of intrinsic Apoptotic Pathways.

Epidemiological studies suggest cigarette smoking as a probable environmental factor for a variety of congenital anomalies, including low bone mass, increased fracture risk and poor skeletal health. Human and animal in vitro models have confirmed hypomineralization of differentiating cell lines with sidestream smoke being more harmful to developing cells than mainstream smoke. Furthermore, first reports are emerging to suggest a differential impact of conventional versus harm-reduction tobacco products on bone tissue as it develops in the embryo or in vitro. To gather first insight into the molecular mechanism of such differences, we assessed the effect of sidestream smoke solutions from Camel (conventional) and Camel Blue (harm-reduction) cigarettes using a human embryonic stem cell osteogenic differentiation model. Sidestream smoke from the conventional Camel cigarettes concentration-dependently inhibited in vitro calcification triggered by high levels of mitochondrially generated oxidative stress, loss of mitochondrial membrane potential, and reduced ATP production. Camel sidestream smoke also induced DNA damage and caspase 9-dependent apoptosis. Camel Blue-exposed cells, in contrast, invoked only intermediate levels of reactive oxygen species insufficient to activate caspase 3/7. Despite the absence of apoptotic gene activation, damage to the mitochondrial phenotype was still noted concomitant with activation of an anti-inflammatory gene signature and inhibited mineralization. Collectively, the presented findings in differentiating pluripotent stem cells imply that embryos may exhibit low bone mineral density if exposed to environmental smoke during development.

Human Pluripotent Stem Cells to Assess Developmental Toxicity in the Osteogenic Lineage.

Musculoskeletal birth defects are frequent, yet their causes remain insufficiently investigated. Aside from genetic factors, exposure to environmental toxicants is suspected to contribute to the etiology of skeletal malformations. However, most chemicals in the environment are insufficiently characterized for their potential to cause harm to the differentiation of osteoblasts, the bone-forming cells and thereby the development of the skeleton.This lack of information primarily stems from animal testing being prohibitively expensive and time-consuming, which has prompted the development of predictive in vitro alternative methods. With the advent of mouse embryonic stem cells, which represent cells with the potential to become any of the 200 cell types in the body, among them osteoblasts, the past 15 years have borne suitable opportunities to assess chemicals in vitro. However, with an increasing understanding of the differences between mouse and human embryonic development, a need for human-specific developmental toxicity testing has risen. This chapter provides a detailed protocol on how to differentiate human embryonic stem cells into the osteogenic lineage, how to assess differentiation inhibition and how to evaluate such findings in relation to the mitochondrial activity of human embryonic stem cells and human fibroblasts, while exposed to a potential toxicant. Together, these endpoints allow for a human-specific screening of developmental toxicity specifically related to the osteogenic lineage.

microRNA Regulation of Skeletal Development.

PURPOSE OF REVIEW: Osteogenesis is a complex process involving the specification of multiple progenitor cells and their maturation and differentiation into matrix-secreting osteoblasts. Osteogenesis occurs not only during embryogenesis but also during growth, after an injury, and in normal homeostatic maintenance. While much is known about osteogenesis-associated regulatory genes, the role of microRNAs (miRNAs), which are epigenetic regulators of protein expression, is just beginning to be explored. While miRNAs do not abrogate all protein expression, their purpose is to finely tune it, allowing for a timely and temporary protein down-regulation. RECENT FINDINGS: The last decade has unveiled a multitude of miRNAs that regulate key proteins within the osteogenic lineage, thus qualifying them as "ostemiRs." These miRNAs may endogenously target an activator or inhibitor of differentiation, and depending on the target, may either lead to the prolongation of a progenitor maintenance state or to early differentiation. Interestingly, cellular identity seems intimately coupled to the expression of miRNAs, which participate in the suppression of previous and subsequent differentiation steps. In such cases where key osteogenic proteins were identified as direct targets of miRNAs in non-bone cell types, or through bioinformatic prediction, future research illuminating the activity of these miRNAs during osteogenesis will be extremely valuable. Many bone-related diseases involve the dysregulation of transcription factors or other proteins found within osteoblasts and their progenitors, and the dysregulation of miRNAs, which target such factors, may play a pivotal role in disease etiology, or even as a possible therapy.