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The Promise of Therapy with Embryonic Stem Cells
Published in Howard Green, Therapy with Cultured Cells, 2019
For analysis of the development of keratinocytes from human embryonic stem cells it is useful to study the appearance of markers in cells migrating from cultured embryoid bodies (Green et al., 2003). The order of marker succession is as follows: Loss of Oct4. This loss is seen in cells migrating away from an embryoid body (Fig. 30). Appearance of keratinocytes containing p63, K14 and basonuclin, markers of the basal layer of the keratinocytes. This is seen in Figure 31. Multiplication of the keratinocytes and their concentration close to the migration front. This is seen in Figure 32. The presence of basonuclin is known to be associated with proliferative capacity of the keratinocyte (Tseng and Green, 1994). Terminal differentiation. After many days, there appeared scattered squame-like structures containing involucrin, a precursor of the cross-linked envelope characteristic of terminal differentiation (Fig. 33).
Blood–Retinal Barrier
Published in Glenn J. Jaffe, Paul Ashton, P. Andrew Pearson, Intraocular Drug Delivery, 2006
David A. Antonetti, Thomas W. Gardner, Alistair J. Barber
Gene-deletion experiments have demonstrated a more complex role for occludin in tight junction barrier formations. Embryonic stem cells from occludin null mice formed cystic embryoid body structures with an outermost layer of epithelial cells, similar to wild-type embryonic cells (58). Ultrastructural analysis revealed no changes in the tight junctions; the tight junction protein ZO-1 exhibited normal localization at apical junctional regions in the outermost layer of epithelial cells and no change in barrier properties was observed in the occludin null cells. However, the adult occludin homozygous null mice, although viable, possessed a host of abnormalities (59). Occludin-deficient mice exhibited postnatal growth retardation, male knockout mice were infertile, and female knockout mice were unable to suckle their litters. Overall, these mice exhibited abnormalities in the testis and salivary gland, thinning of compact bone, calcium deposits in the brain, chronic gastritis, and hyper-plasia of the gastric epithelium. In addition, recent studies using siRNA to occludin demonstrate that occludin forms a barrier to organic acids up to 6.96 Å, such as arginine and choline (60). Thus, these studies have led to the hypothesis that occludin contributes to the regulation of barrier properties by creating a doorway or regulated pore through the tight junction.
Next Generation Tissue Engineering Strategies by Combination of Organoid Formation and 3D Bioprinting
Published in Naznin Sultana, Sanchita Bandyopadhyay-Ghosh, Chin Fhong Soon, Tissue Engineering Strategies for Organ Regeneration, 2020
Shikha Chawla, Juhi Chakraborty, Sourabh Ghosh
Embryoid bodies are prepared using embryonic stem cells and induced pluripotent cells, using selective cocktails of cytokines. In several cases, preparation of embryoid bodies is a crucial first step in lineage-specific differentiation protocols for stem cells (Brickman and Serup 2017). In this process, relatively large numbers of cells (>1000 to 1 million cells per well) are aggregated, using microwells and hanging drops, and induced to differentiate towards specific cell type. Gradually with increasing culture time, within the embryoid body, differentiated cells seem to assume different morphologies, to develop disorganized cell mass. Reaggregation of embryoid bodies, followed by cellular self-assembly, leads to development of organoids. Recently, self-assembled embryonic stem cells based organoid formation has been acknowledged as one of the key in vitro methods to generate mini organs like mini-gut, brain-like organoids (Warmflash et al. 2014) and liver organoids (Takebe et al. 2013) etc. Morphogen signaling gradient plays a critical role in positioning and patterning of the whole embryos and tissues during normal development, leading to final body parts compartmentalization. Thus, quest for modalities to include combination of cytokines to reproduce such embryonic development based on spatio-temporal morphogen signaling is the need of current developmental biology inspired tissue engineering. Apart from cellular self-assembly tissue, organ formation may involve other complex phenomenon, for instance, varied affinity amongst distinct elements (expression of hemophilic or heterophilic adhesion molecules), hypoxia or mass transport issues, cellular rearrangements and active cell migration. Several organoid formation strategies have been utilized in the past to develop mini-organs. Few examples include the development of two layered optic cup-like organoid using 3D aggregates of self-assembled mouse pluripotent embryonic stem cells (Eiraku et al. 2011). Nelson et al. developed a 3D in vitro model of mouse mammary epithelial tubules; they utilized micropatterning technology to guide the 3D structure of the developed organoid. They successfully demonstrated that the geometry of multicellular tubules, in addition to the availability of the spatially controlled levels of autocrine inhibitory morphogen concentration, can regulate the branch positioning during mammary epithelial cells and primary organoids morphogenesis (Nelson et al. 2006). Another interesting example of morphogen induced control of organoid formation was demonstrated by Montesano et al. (Montesano et al. 1991). Using 3D culture of Madin–Darby canine kidney cells, Montesano et al. demonstrated voluntary tubular formation from the epithelial cyst in the presence of hepatocyte growth factor. Thus, conclusively, developmental biology inspired morphogen signaling in 3D organoids tissue and organ is a promising approach for developing tissue-engineered equivalents (Fig. 4.2). It is pertinent to mention here that since this strategy is scaffold/biomaterial-free, there is no concern how polymer/biomaterial will change cell behavior and thus may closely mimic the in vivo like self-driven cellular/tissue morphogenesis.
Retinoic acid signaling is critical for generation of pancreatic progenitors from human embryonic stem cells
Published in Growth Factors, 2023
Niloufer P. Dumasia, Aparna P. Khanna, Prasad S. Pethe
hESCs were cultured in a defined serum-free media consisting of basal Essential eight media supplemented with Essential eight supplement and penicillin–streptomycin. This media was changed daily. Cells were maintained in Vitronectin (0.5 µg/cm2)-coated dishes. hESCs were passaged every 3–4 days at a split ratio of 1:4–1:6 using 0.5 mM EDTA (Sigma-Aldrich, MO, USA). All cells were cultured at 37 °C with 5% CO2 in a humidified atmosphere. Unstained cells were photographed using an inverted light, phase-contrast microscope (Carl Zeiss AG, Axio Vert.A1, Oberkochen, Germany). Prior to differentiation the KIND1 cells were characterized using immunofluorescence and reverse transcription PCR for pluripotency factors, as well as analyzed using embryoid body formation assay (see Supplementary Information Figure S2).
A review of protein-protein interaction and signaling pathway of Vimentin in cell regulation, morphology and cell differentiation in normal cells
Published in Journal of Receptors and Signal Transduction, 2022
Danial Hashemi Karoii, Hossein Azizi
Vimentin intermediate filaments (VIFs), the most diverse component of the Metazoa cytoskeleton, are made up of one or more highly conserved intermediate filament (IF) proteins expressed by about 70 genes [1]. It is the most often utilized IF protein as a developmental marker of normal cells and tissues, and it is widely recognized as a classic marker of the epithelial-mesenchymal transition (EMT) [2]. The VIF network covers the cytoplasm, from the plasma membrane to the nucleus surface, assisting in the localization of the nucleus and organelles. VIFs assemble to form a complex network that surrounds the nucleus, which is one characteristic of their organization. Previous experiments investigated limiting Vimentin to decrease embryonic stem cells, and spermatogonia stem cells [3]. Our previous study indicated that the absence of Vimentin impairs the spontaneous differentiation of spermatogonia stems cells to the spermatogonia phenotype [4]. A recent study demonstrated that mouse embryonic stem cells from Vimentin –/– mice reduce embryoid body processes. Mouse embryonic fibroblasts lacking Vimentin are challenging to differentiate [5]. Another study discovered morphological changes in glial cells, decreased dilation caused by vascular resistance flow, impaired wound healing due to defects in fibroblast migration, which reflects a role in the mechanical transmission of shear stress, a lack of integration into the vascular endothelium, and disruption of leukocytes in lymph nodes.
The plant hormone abscisic acid stimulates megakaryocyte differentiation from human iPSCs in vitro
Published in Platelets, 2022
Weihua Huang, Haihui Gu, Zhiyan Zhan, Ruoru Wang, Lili Song, Yan Zhang, Yingwen Zhang, Shanshan Li, Jinqi Li, Yan Zang, Yanxin Li, Baohua Qian
Hematopoietic stem cells (HSCs), embryonic stem cells, induced pluripotent stem cells (iPSCs) and adipose tissue-derived stromal cells (ADSCs) have already been used in differentiation protocols for MKs and platelets [8, 9]. Human iPSCs (hiPSCs) are considered to be an inexhaustible source of MK and platelet generation to overcome the shortage of clinical platelet supplies. Nakamura[10] developed a technique to progenitors once they had undergone (low purity) directed differentiation to increase the number of MKs, while Moreau [11] developed a technique to induce a high level of MK differentiation, and these cells still underwent apoptosis after ~110 days. These results suggest the importance of increasing the input of MKs (forward programming) and reducing the loss of MKs (through apoptosis) from the pool of viable MKs. The “spin-embryoid body (EB)” formation differentiation system[12] is relatively easy to operate and has high repeatability, but the efficiency of platelet differentiation is relatively low, so further research and optimization are needed.