The cranial sutures are the primary sites of bone formation during skull growth. Morphogenesis and phenotypic maintenance of the cranial sutures are regulated by tissue interactions, especially those with the underlying dura mater. Removal of the dura mater in fetuses causes abnormal suture development and premature suture obliteration. The dura mater interacts with overlying tissues of the cranial vault by providing: (1) intercellular signals, (2) mechanical signals and (3) cells, which undergo transformation and migrate to the suture. The intercellular signaling governing suture development employs the fibroblast growth factors (FGFs). In rats during formation of the sutures in the fetus, FGF-1 is localized mainly in the dura mater, while other FGFs are expressed in the overlying tissues. By birth, FGF-2 largely replaces FGF-1 in the dura mater. FGFs present in the calvaria bind either the IIIb or IIIc mRNA splice variants of the FGF receptors (FGFRs) 1, 2, or 3. Monoclonal antibodies to the b variant of FGFR2 were used to determine the distribution of FGFR2IIIb during suture development and its extracellular localization. FGFR2IIIb is present in association with mature osteoblasts and osteogenic precursor cells of the suture in the fetus. Ectodomains of FGFR2IIIb, the products of proteolytic cleavage of the receptors, were present throughout the extracellular matrix of sutures resisting obliteration (coronal and sagittal), but absent from the core of sutures undergoing normal fusion (posterior intrafrontal). This observation is consistent with a possible mechanism, in which truncated receptors bind FGFs, thus regulating free FGF available to nearby cells. Mechanical signaling in the calvaria results from tensional forces in the dura mater generated during rapid expansion of the neurocranium. Posterior intrafrontal sutures of rats, which fuse between days 16 and 24, were subjected to cyclical tensional forces in vitro. Significant delay in the timing of suture fusion and increases in the expression domains of FGFR1 and 2 were observed, demonstrating the sensitivity of suture patency to mechanical signals and a possible role of the FGF system in mediating such stimuli. Finally, cells of the dura mater beneath the intrafrontal and sagittal sutures were observed to undergo a morphological transformation to a dendritic morphology and migrate into the suture mesenchyme between days 10 and 16 of development. This process may participate in suture and bone morphogenesis and influence the patency of the sutures along the anterior-posterior axis.
Regulated secretion requires the developmental coupling of neuronal or hormonal stimuli to an exocytotic response, a multistep pathway whose appearance may be linked with cellular adhesion to the newly formed exocrine cell basement membrane. We screened for adhesion-associated coupling activity using lacrimal acinar cells and have identified "BM180", a novel basement membrane protein enriched in guanidine HCl extracts of lacrimal and parotid exocrine secretory glands. BM180 resides primarily in a previously inexamined lower molecular-mass basement membrane peak (peak 2) that contains cell adhesion activity inhibitable with the anti-BM180 monoclonal antibody 3E12. Removal of peak 2 by gel filtration or preincubation of basement membrane with 3E12 decreased regulated peroxidase secretion by one-half without affecting constitutive secretion or the amount of cellular peroxidase available for release. Adding back peak 2 restored regulated secretion in a dose-dependent and 3E12-inhibitable manner and suggested a synergistic relationship between BM180 and laminin 1. BM180 has a mobility of 180 and 60 kDa in the absence or presence of dithiothreitol, respectively, and shows no immunological identity by competitive enzyme-linked immunosorbent assay with laminin 1, collagen IV, entactin, fibronectin, BM-40, perlecan, or vitronectin. We propose that BM180 is an important resident of certain glandular basement membranes where it interacts with the cell surface, thereby possibly signaling the appearance of a transducing element in the stimulus-secretion coupling pathway.
While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2–6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.
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