Induced Pluripotent Stem Cells in Asian Bats

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Questions and Answers

What is the primary purpose of introducing Yamanaka factors into somatic cells?

To enhance their immune response

To induce pluripotency (correct)

To increase their lifespan

To strengthen their viral resistance

Which technique was traditionally used to deliver reprogramming factors into target cells?

Microinjection

Nanoparticle delivery

Electroporation

Retroviruses or lentiviruses (correct)

What is a significant risk associated with using viral vectors for reprogramming?

Increased cost of the procedure

Reduced cell viability

Integration into the host genome (correct)

Lack of cell type specificity

Which of the following is NOT one of the Yamanaka factors used for reprogramming?

p53 Signup and view all the answers

What advancement has been made in reprogramming techniques recently?

Development of non-integrating methods Signup and view all the answers

Which of the following is an example of a non-integrating method for delivering reprogramming factors?

Protein delivery systems Signup and view all the answers

What is the main function of the Yamanaka factors in the reprogramming process?

To reset gene expression patterns Signup and view all the answers

What characteristic of non-integrating methods enhances the safety profile of iPSC generation?

Avoidance of permanent genetic modifications Signup and view all the answers

What dye was used to counterstain the nuclei during the experiment?

DAPI Signup and view all the answers

What was the dilution factor for the secondary antibodies used in the experiment?

1:200 Signup and view all the answers

Which method was employed to assess the resultant teratoma histologically?

Haematoxylin and eosin staining Signup and view all the answers

What is one of the indicators that would lead to the termination of the experiment?

Eating limitations due to tumor Signup and view all the answers

What is the initial cell type used in the reprogramming process described?

Bat fetal fibroblast cells Signup and view all the answers

Which of the following factors are carried by the Sendai viral particles used in the study?

Sox2, Oct3/4, Klf4, c-Myc Signup and view all the answers

Which staining technique was used to examine muscle fibers or mesoderm derivatives?

Masson's trichrome stain Signup and view all the answers

What was used for RNA extraction in the quantitative real-time PCR process?

TRIzol® reagent Signup and view all the answers

What medium was used for culturing the transferred colonies?

Stempan E14 GMEM or TeSR™-E8™medium Signup and view all the answers

What step follows the fixation of cell colonies in paraformaldehyde for alkaline phosphatase detection?

AP staining Signup and view all the answers

How many BEFs were seeded onto the gelatin-coated wells prior to viral transduction?

3 × 10^5 Signup and view all the answers

What was used to ensure the exclusion of autofluorescence in the experiment?

Using controls without primary antibodies Signup and view all the answers

What solution was used to dissociate the colonies for passaging?

0.1% Trypsin/1 mM EDTA Signup and view all the answers

At what confluency were individual cells expanded onto 10 cm dishes?

60–70% Signup and view all the answers

Who approved the animal treatment and maintenance in the study?

Institutional Animal Care and Use Committee (IACUC) Signup and view all the answers

Which chemical supplier is mentioned in the study for obtaining materials?

Sigma-Aldrich (Merck KGaA) Signup and view all the answers

What type of cell lines were established using the integration-free CytoTune® iPS 2.0 Sendai Reprogramming Kit?

Bat-induced pluripotent stem cells Signup and view all the answers

Which of the following factors had the highest amino acid identity compared to humans?

Sox2 Signup and view all the answers

What potential outcome is anticipated from introducing BatiPSCs into nude mouse embryos?

Regeneration of thymic tissue Signup and view all the answers

What is a significant advantage of the Sendai virus-based system for producing iPSCs?

It maintains genomic integrity Signup and view all the answers

What was observed regarding chimeric formation in the study?

No chimeric embryos were formed Signup and view all the answers

What specific stage of stem cells is necessary for creating chimeras between bats and mice?

Intermediate-stage differentiated stem cells Signup and view all the answers

Which reprogramming factor had the lowest amino acid identity when compared to humans?

Myc Signup and view all the answers

What is one of the risks associated with methods that integrate into the host genome?

Insertional mutagenesis Signup and view all the answers

What method was used to dissociate BatiPSCs into single cells?

0.1% trypisin/1 mM EDTA solution Signup and view all the answers

What was the primary purpose of washing the colonies three times with PBS?

To eliminate non-adherent cells Signup and view all the answers

What was the incubation temperature and CO2 concentration specified for the humidified incubator?

37°C with 5% CO2 Signup and view all the answers

What was used as a fluorescent signal to detect the target cDNA amounts during RT-qPCR?

TOPreal qPCR 2 PreMIX (SYBR green with low ROX) Signup and view all the answers

What was the final step in the thermal cycling protocol for RT-qPCR?

40 seconds at 72°C Signup and view all the answers

How much total RNA was isolated for conversion to cDNA?

200 ng Signup and view all the answers

What was the purpose of replacing the medium with a fresh differentiation medium after 24 hours?

To eliminate non-adherent cells Signup and view all the answers

What is the density at which the cells were plated onto the petri dishes?

2 × 10^6 cells per dish Signup and view all the answers

What are induced pluripotent stem cells primarily used for?

Predictive toxicology as an alternative to animal testing Signup and view all the answers

What is a potential advantage of using stem cell organoids in research?

They provide a more accurate representation of human diseases Signup and view all the answers

Which of the following is a limitation of utilizing stem cell technologies?

High potential for ethical dilemmas Signup and view all the answers

What does the data availability statement imply about the study's findings?

The data can be accessed by request from the author Signup and view all the answers

Who authored the study on using stem cells as alternatives to animal experimentation?

Kim T-W, Che J-H, Yun J-W Signup and view all the answers

Which publication discusses the availability and applications of induced pluripotent stem cells?

World J Stem Cells Signup and view all the answers

What does the reference to bat-induced pluripotent stem cells suggest about the scope of stem cell research?

It includes research across diverse animal species Signup and view all the answers

What is one of the primary goals of replacing animal testing with stem cell approaches?

To improve the predictive accuracy of toxicological assessments Signup and view all the answers

Study Notes

Induced Pluripotent Stem Cells from Asian Bats

Bats play crucial roles in ecosystems, including insect control, pollination, and seed dispersal
Researchers successfully generated induced pluripotent stem cells (iPSCs) from two Asian bat species (Hypsugo alaschanicus and Pipistrellus abramus)

iPSC Generation Methods

Sendai reprogramming kit was used
Somatic cells were reprogrammed into a pluripotent state using Yamanaka factors (Oct4, Sox2, Klf4, c-Myc)
Retroviruses or lentiviruses are often used as vectors, but non-viral methods such as episomal vectors, mRNA transfection, and protein delivery are newer and safer options

iPSC Characterization

iPSCs displayed expression of key pluripotency markers: Oct4, Sox2, and Nanog
Myc proto-oncogene expression was lower than in other induced pluripotent stem cell sources.
iPSCs were able to differentiate into three germ layers (endoderm, mesoderm, and ectoderm) as demonstrated by teratoma formation in nude mice.
Researchers successfully generated GFP-expressing iPSCs and used to create chimeric mouse models. Slight GFP signals were detectable in the neck region of mouse fetuses.

Culture Conditions

Two common culture media (TeSR™-E8 and Stempan E14 GMEM) were examined
LIF (leukemia inhibitory factor) was crucial for maintaining the pluripotency of iPSCs

Applications

iPSCs are valuable for disease modeling, drug development, and potentially for conservation of endangered species
iPSCs can be used as an alternative to animal models for studying disease progression, evaluating therapeutics and vaccines, and guiding public health interventions.

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Description

This quiz explores the generation and characterization of induced pluripotent stem cells (iPSCs) derived from Asian bat species. It outlines the methods used, including reprogramming techniques and the expression of key pluripotency markers. Test your knowledge on the significance of iPSCs in research and their potential applications.