Low Osteogenic Yield in Human Pluripotent Stem Cells Associates with Differential Neural Crest Promoter Methylation

Abstract

SUMMARY Human pluripotent stem cell-derived osteoblasts possess great potential for utilization in bone disorder elucidation and repair; yet, while the general ability of human pluripotent stem cells to differentiate into osteoblasts and lay down bone-specific matrix has been shown, previous studies lack the complete characterization of the process whereby such osteoblasts are derived as well as a comparison between the osteogenic efficiency of multiple cell lines. Here, we compared the osteogenic potential of two human induced pluripotent stem cell lines (RIV9 and RIV4) to human H9 embryonic stem cells. Generally capable of osteogenic differentiation, the overall osteogenic yield was lower in the RIV9 and RIV4 lines and correlated with differential expression of osteocalcin (OCN) in mature cultures and PAX7 and TWIST1 during early differentiation. In the undifferentiated cells the promoters of the latter two genes were differentially methylated potentially explaining the variation in differentiation efficiency. Furthermore, the expression signatures of selected neural crest and mesodermal genes and proteins suggested that H9 cells preferentially gave rise to neural crest-derived osteoblasts, whereas the osteoblasts in the RIV9 cultures were generated both through a mesodermal and a neural crest route albeit each at a lower rate. These data suggest that epigenetic dissimilarities between multiple PSC lines may lead to differences in lineage derivation and mineralization. Since osteoblast progenitors from one origin inadequately repair a defect in the other, these data underscore the importance of screening human pluripotent stem cells lines for the identity of the osteoprogenitors they lay down.

WNT/β-CATENIN modulates the axial identity of ES derived human neural crest

“…However, a powerful alternative has emerged through modeling human NC development using human embryonic stem cells (hESCs) (Pomp et al, 2005). Initial efforts relied on co-cultures, the use of serum or serum-replacement cocktails, and either embryoid bodies or neural rosettes (Bajpai et al, 2010;Brokhman et al, 2008;Curchoe et al, 2010;Jiang et al, 2009;Lee et al, 2007;Liu et al, 2012;Rada-Iglesias et al, 2012;Sparks et al, 2018). Simpler models, where cell density was controlled along with defined media components, have enabled a better analysis of contributing factors during human NC development from hESCs (Fukuta et al, 2014;Hackland et al, 2017;Huang et al, 2016;Leung et al, 2016;Menendez et al, 2011).…”

Expression of miRNAs from the Imprinted DLK1/DIO3 Locus Signals the Osteogenic Potential of Human Pluripotent Stem Cells

“…Different studies have also demonstrated the potential of hPSC to phenocopy osteogenic defects associated with genetic disorders such as progressive fibrodysplasia ossificans or Marfan syndrome [39,40]. Nonetheless, these different applications of hPSCs are hindered by the differentiation inconsistency of individual hPSC lines and the fact that the molecular mechanism underlying the interline variability has remained elusive [41]. Based on the analysis of a dozen hPSC lines that replicated the observed variability in osteogenic capacity, we demonstrated that a reduced expression of miRNAs belonging to the imprinted DLK1/DIO3 locus was associated with a low osteogenic yield of hPSC lines.…”

Video-based kinetic analysis of calcification in live osteogenic human embryonic stem cell cultures reveals the developmentally toxic effect of Snus tobacco extract

“…H9 human embryonic stem cells (hESCs) were obtained from WiCell and were cultured on Matrigel (BD Biosciences) treated culture plates in mTeSR 1 medium (Stem Cell Technologies) as feeder-free cultures at 37°C with 5% CO 2 as described [32]. Colonies were passaged every 5 days using accutase treatment (2-4 min at room temperature) and a cell scraper to dislodge cell clumps from the plastic.…”

“…This process of calcification is facilitated by the osteoblast, which secretes a collagenous matrix as the frame work for the hydroxyapatite formation as well as all other extracellular matrix (ECM) proteins necessary for bone function [26]. Among these, osteocalcin (OCN) is uniquely found only in bone tissue, which is why its presence is often used as a biomarker for osteogenesis [27][28][29][30][31][32]. The ability of osteoblasts to calcify the ECM can also be confirmed by calcium specific stains, such as von Kossa or Alizarin Red [28,30,[32][33][34], but this type of assessment is often qualitative.…”