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Human-Induced Pluripotent Stem Cells: Derivation
Published in Deepak A. Lamba, Patient-Specific Stem Cells, 2017
Uthra Rajamani, Lindsay Lenaeus, Loren Ornelas, Dhruv Sareen
Nanog: Nanog is known to be a master regulator of stem cell pluripotency (29), which is expressed in the inner cell mass, the primordial germ cell, and the epiblast in early embryos (30). The name of the Nanog gene was chosen by Professor Ian Chambers, taking a cue from Irish mythology legend, “Tír na nÓg,” which signifies immortality conferred by Nanog upon the stem cells. Nanog plays a key role in suppressing Cdx2, which in turn leads to the specification of the fate of the inner cell mass (31). Nanog also works in synchrony with Oct4 and Sox2 in maintaining the pluripotency of stem cells (32,33). Although Dr. Yamanaka and his group proved that the initial derivation of iPSCs was possible without the ectopic expression of Nanog (2,3), Dr. Thomson’s group showed that Nanog is a key component of the reprogramming factor cocktail. It is, nonetheless, an important marker for the fully reprogrammed germ line-competent human iPSC state (34–36).
Bio-interactive nanoarchitectonics with two-dimensional materials and environments
Published in Science and Technology of Advanced Materials, 2022
Xuechen Shen, Jingwen Song, Cansu Sevencan, David Tai Leong, Katsuhiko Ariga
Song et al. effected tunable alignment of FNWs assembled through the LB approach. Improved control over nanoarchitecture enabled more detailed correlation between FNW alignment and cell behaviours. High alignment promoted self-renewal and retention of multipotency in human mesenchymal stem cells (hMSCs) (Figure 12) [199]. FNWs (m-xylene and IPA) of different aspect ratios were fabricated by controlling addition rate (of IPA to C60/m-xylene solution). Adjusting the composition of FNWs (high aspect ratio: low aspect ratio) tuned their alignment in the resulting LB film. With increasing alignment, hMSCs exhibited increasing unidirectional orientation (along FNWs) and elongation. Upregulation of stemness genes OCT4, SOX2, NANOG and corresponding markers correlated with increasing alignment. Hallmarks of stemness persisted over 2 weeks, indicating long-term multipotency retention through symmetrical self-renewal. This result may become significant for in vitro stem cell expansion, which is desirable in tissue engineering and clinical applications. The hydrophobic and nano-topographic surface of aligned FNWs provided appropriate cues to restrict focal adhesion growth and activate mechanotransducive Yes-Associated Protein (YAP) signalling pathways; YAP nuclear translocation promoted upregulation of stemness genes, modulating cell fate.
Derivation of induced pluripotent stem cell lines from New Zealand donors
Published in Journal of the Royal Society of New Zealand, 2022
Jin Kyo Oh, Aneta Przepiorski, Hao-Han Chang, Rachel C. Dodd, Veronika Sander, Brie Sorrenson, Jen-Hsing Shih, Jennifer A. Hollywood, Janak R. de Zoysa, Peter R. Shepherd, Alan J. Davidson, Teresa M. Holm
We used the integration-free Sendai virus system to reprogram dermal fibroblasts from two adult donors towards pluripotency. iPSC colonies emerged 3–4 weeks post-transduction and were picked to generate individual lines. We obtained multiple clones of the MANZ-2 and MANZ-4 lines, which could be stably passaged and expanded while exhibiting the morphology characteristic of human ESCs (Figure 1A). Six iPSC lines per donor were randomly selected for karyotyping. MANZ-2-2 and MANZ-4-37 lines showed no chromosome abnormalities and were selected for further characterisation (Figure 1B). STR analysis for 16 loci confirmed the genetic integrity of the MANZ lines (Supplementary Figure 3). Using qPCR, we measured that transcript levels of the pluripotency genes OCT4, NANOG and SOX2 were highly induced in both MANZ lines compared to the corresponding fibroblasts (Figure 1C). cMYC induction was less prominent, presumably due to a high base expression of cMYC in the corresponding fibroblasts (Waters et al. 1991). Sendai virus RNA was not detectable by RT–PCR (Supplementary Figure 4), indicating that our lines successfully maintained pluripotency without viral transgene expression. Immunocytochemistry confirmed the presence of the pluripotency cell-surface-antigen markers SSEA-4, TRA-1-60 and TRA-1-81 (Figure 1D), whereas the differentiation marker SSEA-1 was undetectable in both MANZ lines (not shown).
Down-regulation of pluripotency and expression of SSEA-3 surface marker for mesenchymal Muse cells by in vitro expansion passaging
Published in Egyptian Journal of Basic and Applied Sciences, 2019
Ali M. Fouad, Mahmoud M. Gabr, Elsayed K. Abdelhady, Sahar A. Rashed, Sherry M. Khater, Mahmoud M. Zakaria
Total RNA was extracted from all passages by Direct-Zol RNATM Mini Prep kit (ZYMORESEARCH, USA). Three micrograms of total RNA was converted by RT2 First Standard Kit (Qiagen, Germany) to cDNA. Gene expression was evaluated for pluripotent genes; Nanog Homeobox (Nanog), SRY-box2 (Sox2), and POU Class 5 Homeobox 1 (POU5F1 or OCT-4) as referred in Table 1. MSCs gene expression for targeted genes served as control and for mathematical calculations. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was included as a reference gene and for normalization the results. Amplifications were performed in a total 25 µL reaction volume in each well that contains 12.5 µL 2X SYBR Green Rox Master Mix (Qiagen), 100 ng of cDNA template, 10 pmol primers and nuclease-free water. The plate array was inserted in CFX96 real-time system (Bio-Rad, USA) and programmed according to manufacturer instructions by using the cycling parameters of the PCR amplification as follows: initial denaturation at 95ºC for 3 min, followed by 40 cycles of amplification (denaturation at 94°C for 10 s, annealing and extension at 60°C for 30 s). For each sample, the procedure was carried out in triplicate. A mathematical model introduced by M. Pfaffl was used for the relative quantification of target genes [10].