MicroRNA biogenesis and regulation of bone remodeling

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression. This review will highlight our current understanding of miRNA biogenesis and mechanisms of action, and will summarize recent work on the role of miRNAs, including the miR-29 family, in bone remodeling. These studies represent the first steps in demonstrating the importance of miRNAs in the control of osteoblast and osteoclast differentiation and function. An in-depth understanding of the roles of these regulatory RNAs in the skeleton will be critical for the development of new therapeutics aimed at treating bone loss and perhaps facilitating fracture repair

The Role of microRNAs in Bone

miRNAs are short, non-coding RNAs that negatively regulate gene expression by binding to specific sequences within a target mRNA. Families of miRNAs can work coorperatively to regulate complex cellular processes, including differentiation (Chapter 1). The purpose of these studies was to understand the molecular mechanisms by which miRNAs regulate osteoblast differentiation. ^ miR-29 Suppression of Osteonectin in Osteoblasts: Regulation During Differentiation and by Canonical Wnt Signaling (Chapter 3). Osteonectin is expressed by osteoblasts and regulates collagen fibril formation. In osteoblasts, osteonectin expression during matrix deposition decreases with matrix maturation/mineralization. Osteonectin is a target for miR-29, and miR-29 levels are low during matrix deposition, and increase as osteoblasts mature. Canonical Wnt signaling increases as osteoblasts mature, providing a potential mechanism for downregulating osteonectin during differentiation. Further, Wnt signaling rapidly induces miR-29 and decreases osteonectin. ^ Single nucleotide polymorphisms in the human osteonectin/SPARC 3\u27 UTR mediate differential regulation of gene expression: regulation by microRNAs (Chapter 4). Previously, haplotypes consisting of 3 SNPs in the 3\u27UTR of osteonectin, at cDNA bases 1046(C/G), 1599(C/G) and 1970(T/G), are found to be associated with bone mass in a cohort of men with idiopathic osteoporosis. Interestingly, the osteonectin 3\u27 UTR haplotype found at the highest frequency in the most severely affected osteoporosis patients allowed the lowest level of gene expression. We demonstrated that SNPs at 1599 and 1970 differentially regulate gene expression, and characterized miRNAs that may selectively interact with SNP regions. Selective regulation of osteonectin by miRNAs could be one mechanism contributing to variation in bone mass. ^ miR-29a modulates canonical Wnt signaling in human osteoblasts through a positive feedback loop (Chapter 5). We found that miR-29a transcription is induced by canonical Wnt signaling. miR-29a promotes the expression of osteoblastic markers, and directly targets Dkk1, Kremen2, and sFRP2, inhibitors of Wnt signaling. This results in miR-29a potentiating Wnt signaling, to drive osteoblast differentiation.^ The study of miRNAs in bone cells is novel and miRNA-based therapeutics may be promising for the treatment of bone diseases (Chapter 6).

Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders

The abbreviated pluripotent cell cycle

Human embryonic stem cells (hESCs) and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory, and structural. The primary temporal context that the pluripotent self-renewal cell cycle of hESCs is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naive transcriptome of hESCs, which is competent to stringently regulate the embryonic stem cell (ESC) cell cycle. This supports the requirements of pluripotent cells to self-propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated ESC cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle

The architectural organization of human stem cell cycle regulatory machinery

Two striking features of human embryonic stem cells that support biological activity are an abbreviated cell cycle and reduced complexity to nuclear organization. The potential implications for rapid proliferation of human embryonic stem cells within the context of sustaining pluripotency, suppressing phenotypic gene expression and linkage to simplicity in the architectural compartmentalization of regulatory machinery in nuclear microenvironments is explored. Characterization of the molecular and architectural commitment steps that license human embryonic stem cells to initiate histone gene expression is providing understanding of the principal regulatory mechanisms that control the G1/S phase transition in primitive pluripotent cells. From both fundamental regulatory and clinical perspectives, further understanding of the pluripotent cell cycle in relation to compartmentalization of regulatory machinery in nuclear microenvironments is relevant to applications of stem cells for regenerative medicine and new dimensions to therapy where traditional drug discovery strategies have been minimally effective