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Cell therapy is one of the promising approaches used against type 1 diabetes. Efficient generation of human embryonic stem cell (hESC)-derived pancreatic progenitors (PPs) is of great importance. Since signaling pathways underlying human pancreas development are not yet fully understood, various differentiation protocols are conducted, each considering variable duration, timing, and concentrations of growth factors and small molecules. Therefore, we compared two PP differentiation protocols in static suspension culture. We tested modified protocols developed by Pagliuca et al. (protocol 1) and Royan researchers (protocol 2) until early PP stage. The morphological changes of hESC aggregates during differentiation, and also gene and protein expression after differentiation, were evaluated. Different morphological structures were formed in each protocol. Quantitative gene expression analysis, flow cytometry, and immunostaining revealed a high level of PDX1 expression on day 13 of Royan's differentiation protocol compared to protocol 1. Our data showed that using protocol 2, cells were further differentiated until day 16, showing higher efficiency of early PPs. Moreover, protocol 2 is able to produce hESCs-PPs in a static suspension culture. Since protocol 2 is inexpensive in terms of media, growth factors, and chemicals, it can be used for massive production of PPs using static and dynamic suspension cultures. [ABSTRACT FROM AUTHOR]

Objective: The ability to generate lung alveolar epithelial type II (ATII) cells from pluripotent stem cells (PSCs) enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm (mESC-DE) cells towards ATII cells. Materials and Methods: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 (RB20) was induced to differentiate into DE (6 days) and then into ATII cells (9 days) by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation (SFD) media and induced them with 200 nM small molecule inducer of definitive endoderm 2 (IDE2). For days 7-15 (9 days) of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor (FGF2) (group F), 0.5 μg/ml hydrocortisone (group H), and A549 conditioned medium (A549 CM) (group CM) in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step. Results: Although both F and H (alone and in combination) promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C (SP-C) expressing cells (~37%) were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies (LB) in the ATII-like cells. Conclusion: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2- induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies. [ABSTRACT FROM AUTHOR]

137 Cell J, Vol 23, No 1, April-June (Spring) 2021 Correction Hydrocortisone Promotes Differentiation of Mouse Embryonic Stem Cell-Derived Definitive Endoderm toward Lung Alveolar Epithelial Cells Mohammad Reza Mokhber Dezfouli, D.V.M., D.V.Sc. Citation: Mokhber Dezfouli MH, Sadeghian Chaleshtori S, Moradmand A, Basiri M, Baharvand H, Tahamtani Y. Hydrocortisone promotes differentiation of mouse embryonic stem cell-derived definitive endoderm toward lung alveolar epithelial cells. [Extracted from the article]

Objective: Motor neuron differentiation from human embryonic stem cells (hESCs) is a goal of regenerative medicine to provide cell therapy as treatments for diseases that damage motor neurons. Most protocols lack adequate efficiency in generating functional motor neurons. However, small molecules present a new approach to overcome this challenge. The aim of this research is to replace morphogen factors with a cocktail of efficient, affordable small molecules for effective, low cost motor neuron differentiation. Materials and Methods: In this experimental study, hESCs were differentiated into motor neuron by the application of a small molecule cocktail that consisted of dorsomorphin, A8301, and XAV939. During the differentiation protocol, we selected five stages and assessed expressions of neural markers by real-time polymerase chain reaction (PCR), immunofluorescence staining, and flow cytometry. Motor neuron ion currents were determined by whole cell patch clamp recording. Results: Immunofluorescence staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) indicated that they were positive for NESTIN (92.68%), PAX6 (64.40%), and SOX1 (82.11%) in a chemically defined adherent culture. The replated (hESC)-derived NE differentiated cells were positive for TUJ1, MAP2, HB9 and ISL1. We evaluated the gene expression levels with real-time reverse transcriptase-PCR at different stages of the differentiation protocol. Voltage gated channel currents of differentiated cells were examined by the whole-cell patch clamp technique. The hESC-derived motor neurons showed voltage gated delay rectifier K+, Na+ and Ca2+ inward currents. Conclusion: Our results indicated that hESC-derived neurons expressed the specific motor neuron markers specially HB9 and ISL1 but voltage clamp recording showed small ionic currents therefore it seems that voltage gated channel population were inadequate for firing action potentials. [ABSTRACT FROM AUTHOR]

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs). Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies. [ABSTRACT FROM AUTHOR]

Objective: In vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs), researchers have yet to discover universal, efficient and cost-effective protocols. Materials and Methods: In this experimental study, we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd- A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100). Results: Gene expression analysis showed up regulation of DE-specific marker genes (SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (p<0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs. Conclusion: These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs. [ABSTRACT FROM AUTHOR]

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid., Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs)., Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1 GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus., Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.