Effects of various culture conditions on pluripotent stem cell derivation from chick embryos

Farzaneh, Maryam; Zare, Masoumeh; Hassani, Seyedeh‐Nafiseh; Baharvand, Hossein

doi:10.1002/jcb.26761

Cited by 13 publications

(11 citation statements)

References 33 publications

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“…After validating the ES-cell clones, the undifferentiated state of the mutant-ES cells were determined. Although the cultivation of ES cells in medium supplemented with R2i efficiently maintains pluripotency state of the ES cells (30, 31), in order to ensure the undifferentiated state of our knockout-ES cells after genetic manipulation process and several passages, the expression of stemness markers were investigated, which successfully confirmed the ground state pluripotency of mutant ES cells. Efficient cleavage and wide-range of indels, such as biallelic mutations, in all mutant ES cell clones can be generated using CRISPR/Cas9 system with highly active and correctly-designed sgRNAs.…”

Section: Discussionmentioning

confidence: 99%

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

et al. 2019

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Background Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences. Objectives The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time. Materials and Methods Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously. Results CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression. Conclusions Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.

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Section: Discussionmentioning

confidence: 99%

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

et al. 2019

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“…The use of small molecules as substitutes for growth factors or various cytokines can reduce side effects and media costs [137,138]. Further studies are necessary to understand which genetic or epigenetic alternations improve the terminal differentiation of iPSC-derived erythroid cells [139].…”

Section: Primary Technical Challenges For the Clinical Application Ofmentioning

confidence: 99%

Differentiation of human induced pluripotent stem cells into erythroid cells

et al. 2020

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During the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

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“…Se observó que en la condición de cultivo 1, un mayor número de fibroblastos gingivales presentaron este marcaje lisosomal (Figura 4B), mientras que en los fibroblastos gingivales en contacto con la condición de cultivo 2, el marcaje de LAMP-1 estuvo presente, pero solo en ciertas células (Figura 4E). Estudios precedentes realizados en diferentes tipos celulares en presencia o ausencia de suero fetal bovino han permitido constatar cómo este componente afecta el rendimiento de los cultivos celulares (Gürdal et al 2018;Farzaneh et al 2018). El medio de cultivo DMEM hace posible el crecimiento celular in vitro, proporcionando los nutrientes esenciales requeridos por los fibroblastos gingivales.…”

Section: Resultados Y Discusiónunclassified

Efecto de la ausencia de suero fetal bovino sobre la morfología y organelas de células fibroblásticas

Simancas-Escorcia

Caballero

2020

Rev. U.D.C.A Act. & Div. Cient.

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Los fibroblastos son células constituyentes de los tejidos conectivo. Los fibroblastos gingivales (FGs), células responsables de la síntesis de la matriz extracelular (MEC) en el tejido conectivo gingival, participan en la regulación de los procesos de cicatrización y de reparación de la encía. Debido a su potencial regenerativo, los FGs podrían ser células capaces de contribuir a mejorar los procesos de cicatrización, a nivel local y sistémico e, incluso, ser utilizadas como un modelo celular útil en la comprensión de los aspectos fisiopatológicos de la cavidad oral. El objetivo del presente trabajo fue describir el impacto de la concentración del suero fetal bovino (SFB), en la supervivencia, el crecimiento y la expresión de marcadores celulares en los FGs. Cultivos celulares de FGs fueron realizados durante 7 días, utilizando medio de cultivo DMEM (Dulbecco’s Modified Eagle’s médium), en ausencia y presencia de 10% de SFB. Análisis morfológicos e inmunohistoquímicos de la actina, mitocondrias, lisosomas y retículo endoplasmático (RE) fueron usados para evaluar el impacto de la concentración del SFB sobre los FGs. Los resultados indican que los FGs cultivados en presencia de 10% de SFB tuvieron una tasa de crecimiento más elevada en comparación con los FGs, cultivados en ausencia de SFB. El marcaje de los elementos celulares indica la ausencia de alteraciones en las organelas celulares de los FGs, cuando son cultivados en ausencia de SFB. En conclusión, los FGs son capaces de sobrevivir, proliferar y conservar sus características morfológicas, cuando son cultivados en presencia y ausencia de SFB.

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Effects of various culture conditions on pluripotent stem cell derivation from chick embryos

Cited by 13 publications

References 33 publications

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

Differentiation of human induced pluripotent stem cells into erythroid cells

Efecto de la ausencia de suero fetal bovino sobre la morfología y organelas de células fibroblásticas

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