scholarly journals miR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jaeeun Lim ◽  
Eiko Sakai ◽  
Fuminori Sakurai ◽  
Hiroyuki Mizuguchi
Keyword(s):  
Transcriptional Repression ◽  
Inhibitory Effect ◽  
Definitive Endoderm ◽  
Bmp Signaling ◽  
Marker Genes ◽  
Hepatic Differentiation ◽  
Early Differentiation ◽  
Common Precursor ◽  
Micro Rnas ◽  
Induced Pluripotent

AbstractHuman induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development

Development ◽  
1999 ◽  
Vol 126 (18) ◽  
pp. 4005-4015 ◽  
Author(s):  
M. Weaver ◽  
J.M. Yingling ◽  
N.R. Dunn ◽  
S. Bellusci ◽  
B.L. Hogan
Keyword(s):  
Cell Types ◽  
Surfactant Protein ◽  
Bmp Signaling ◽  
Dominant Negative ◽  
Clara Cell ◽  
Mouse Lung ◽  
Marker Genes ◽  
Distal Marker ◽  
Morphogenetic Protein

In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693–1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.

Continuous inhibition of Sonic hedgehog signaling effectively leads to differentiation of human-induced pluripotent stem cells into functional insulin-producing β cells

2020 ◽  
Author(s):  
Song Lee ◽  
Jae Hyun Joo ◽  
Ju Yun Oh ◽  
Eun Ha Seo ◽  
Yang Hee Kim ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Small Molecules ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Definitive Endoderm ◽  
Patient Specific ◽  
Diabetic Patients ◽  
Glucose Stimulation ◽  
Sonic Hedgehog Signaling ◽  
Induced Pluripotent

Abstract Background: Human-induced pluripotent stem cell (iPSC)-derived insulin-producing cells (IPCs) can be used for islet cell transplantation in type 1 diabetic patients and as patient-specific cells for the development of novel anti-diabetic drugs. Therefore, it is necessary develop a method for generating functional IPCs from iPSCs and simplifying the stepwise protocol. Methods: We compared combinations of small molecules that could efficiently induce the differentiation of cells into a definitive endoderm, and preferentially into islet precursor cells. IPCs, generated using the optimal combination of small molecules, were confirmed to demonstrate insulin secretion in response to glucose stimulation. Finally, we re-constructed spheroid IPCs and verified the optimized culture and maturation conditions. Results: It was confirmed by quantitative polymerase chain reaction that definitive endoderm-specific markers were expressed differently depending on the combination of the small molecules used. Small molecule SANT-1 induced the differentiation of cells into functional IPCs by acting as an inhibitor of Sonic hedgehog signaling. Images of 2D culture showed that IPCs were spheroid-shaped from day 5 and demonstrated sustained insulin secretion. We developed a simple differentiation method using small molecules that produced functional IPCs that responded efficiently to glucose stimulation in a relatively short time. Conclusions: We posit that this method along with a method that refines the process of differentiation can be used for growing IPCs that can be employed in clinical trials.

scholarly journals In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells

2020 ◽  
Vol 21 (2) ◽  
pp. 581 ◽  
Author(s):  
Phoonsuk Limraksasin ◽  
Takeru Kondo ◽  
Maolin Zhang ◽  
Hiroko Okawa ◽  
Thanaphum Osathanon ◽  
...  
Keyword(s):  
Cartilage Tissue ◽  
Marker Genes ◽  
Induction Medium ◽  
Mechanical Stimuli ◽  
Tissue Formation ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Chondrogenic Marker ◽  
Induced Pluripotent

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

scholarly journals Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium

2020 ◽  
Vol 55 (8) ◽  
pp. 763-774
Author(s):  
Yasufumi Koterazawa ◽  
Michiyo Koyanagi-Aoi ◽  
Keiichiro Uehara ◽  
Yoshihiro Kakeji ◽  
Takashi Aoi
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Retinoic Acid Receptor ◽  
Marker Genes ◽  
Acid Receptor ◽  
All Trans Retinoic Acid ◽  
Human Esophagus ◽  
Induced Pluripotent

Abstract Background The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. Methods We established a novel stepwise protocol with transwell culture and an air–liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. Results We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. Conclusion We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

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