Viable mice produced from three-factor induced pluripotent stem (iPS) cells through tetraploid complementation

In a suspension culture of iPS cells, the shear stress generated during mixing is expected to promote differentiation of induced pluripotent stem (iPS) cells. The stress on the cells can be controlled by rotational rate and shape of impeller. However, it is difficult to optimize these operative parameters by experiments. Therefore, we have developed a numerical model to obtain the average and the maximum shear stress in two kinds of stirred tanks and an orbital shaking cylindrical container. The present results showed that the shear stress strongly depended on the type of mixing and lesser extent on the shape of the impeller. The average shear stress is larger in the shaking mode than that in the stirring mode. In contrast, the maximum shear stress is much smaller in the shaking than the stirring. These results suggest that stirring and shaking should be selectively used depending on the application

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Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

PLoS ONE ◽

10.1371/journal.pone.0260852 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0260852

Author(s):

Meryem Ozgencil ◽

Julian Barwell ◽

Marc Tischkowitz ◽

Louise Izatt ◽

Ian Kesterton ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Ips Cells ◽

Variants Of Uncertain Significance ◽

Cellular Models ◽

Pathogenic Variants ◽

Damage Repair ◽

Uncertain Significance ◽

Induced Pluripotent ◽

The Impact

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

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Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

Current Issues in Molecular Biology ◽

10.3390/cimb43030147 ◽

2021 ◽

Vol 43 (3) ◽

pp. 2124-2134

Author(s):

Hyun Soo Lee ◽

Jeewon Mok ◽

Choun-Ki Joo

Keyword(s):

Progenitor Cells ◽

Dermal Fibroblast ◽

Ips Cells ◽

Human Dermal Fibroblast ◽

Corneal Epithelial ◽

Specific Medium ◽

Morphogenetic Protein ◽

Human Ips Cells ◽

Limbal Stem Cell ◽

Induced Pluripotent

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

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RT-qPCR analyses on the osteogenic differentiation from human iPS cells: An investigation of reference genes

10.1101/2020.05.27.118000 ◽

2020 ◽

Author(s):

Kensuke Okamura ◽

Yusuke Inagaki ◽

Takeshi K. Matsui ◽

Masaya Matsubayashi ◽

Tomoya Komeda ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Reference Gene ◽

Reference Genes ◽

Ips Cells ◽

Expression Stability ◽

Differentiation Markers ◽

Osteogenic Differentiation Medium ◽

Need To Evaluate ◽

Induced Pluripotent ◽

Beta 2 Microglobulin

AbstractReverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem (iPS) cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days. RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein (TBP) in the first place, followed by transferrin receptor (TFRC), ribosomal protein large P0 (RPLP0), and finally, beta-2-microglobulin (B2M), which was revealed as the least stable. Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.

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Use of a Sendai virus-based vector for effcient transduction of pinniped fbroblasts

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj18.445 ◽

2019 ◽

Vol 22 (8) ◽

pp. 1020-1025 ◽

Cited By ~ 1

Author(s):

V. R. Beklemisheva ◽

A. G. Menzorov

Keyword(s):

Transgene Expression ◽

Viral Vectors ◽

Lentiviral Vector ◽

Sendai Virus ◽

Homo Sapiens ◽

Fluorescent Proteins ◽

American Mink ◽

Ips Cells ◽

Canis Lupus Familiaris ◽

Induced Pluripotent

Generation of induced pluripotent stem (iPS) cells expanded possibilities of pluripotency and early development studies. Generation of order Carnivora iPS cells from dog (Canis lupus familiaris), snow leopard (Panthera uncia), and American mink (Neovison vison) was previously reported. The aim of the current study was to examine conditions of pinniped fbroblast reprogramming. Pinnipeds are representatives of the suborder Caniformia sharing conservative genomes. There are several ways to deliver reprogramming transcription factors: RNA, proteins, plasmids, viral vectors etc. The most eﬀective delivery systems for mouse and human cells are based on viral vectors. We compared a lentiviral vector which integrates into the genome and a Sendai virusbased vector, CytoTune EmGFP Sendai Fluorescence Reporter. The main advantage of Sendai virusbased vectors is that they do not integrate into the genome. We performed delivery of genetic constructions carrying ﬂuorescent proteins to fbroblasts of seven Pinnipeds: northern fur seal (Callorhinus ursinus), Steller sea lion (Eumetopias jubatus), walrus (Odobenus rosmarus), bearded seal (Erignathus barbatus), Baikal seal (Pusa sibirica), ringed seal (Phoca hispida), and spotted seal (Phoca largha). We also transduced American mink (N. vison), human (Homo sapiens), and mouse (Mus musculus) fbroblasts as a control. We showed that the Sendai virusbased transduction system provides transgene expression onetwo orders of magnitude higher than the lentiviral system at a comparable multiplicity of infection. Also, transgene expression after Sendai virusbased transduction is quite stable and changes only slightly at day four compared to day two. These data allow us to suggest that Sendai virusbased vectors are preferable for generation of Pinniped iPS cells.

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