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Related Concept Videos

Induced Pluripotent Stem Cells01:13

Induced Pluripotent Stem Cells

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Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have differentiated into a specific cell type are post-mitotic—that is, they no longer divide. However, scientists have found a way to reprogram these mature cells so that they “de-differentiate” and return to an unspecialized, proliferative state. These cells are also pluripotent like embryonic stem cells—able to produce all cell types—and are therefore...
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Induced Pluripotent Stem Cells01:06

Induced Pluripotent Stem Cells

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Stem cells are undifferentiated cells that divide and produce different cell types. Ordinarily, cells that have differentiated into a specific cell type are terminally differentiated; however, scientists have found a way to reprogram these mature cells so that they dedifferentiate and return to an unspecialized, proliferative state. These cells are pluripotent like embryonic stem cells—able to produce all cell types—and are called induced pluripotent stem cells (iPSCs).
Somatic...
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Embryonic Stem Cells00:58

Embryonic Stem Cells

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Embryonic stem (ES) cells are undifferentiated pluripotent cells, meaning they can produce any cell type in the body. This gives them tremendous potential in science and medicine since they can generate specific cell types for use in research or to replace body cells lost due to damage or disease.
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Embryonic Stem Cells00:57

Embryonic Stem Cells

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Embryonic stem (ES) cells were first discovered in mice in 1981 by Martin Evans. In 1998, James Thomson identified a method to isolate embryonic stem cells from humans. Human embryonic stem cells (hESCs) are obtained from 3-5 day old embryos that remain unused after an in vitro fertilization procedure.
ES cells are grown in a culture medium where they can divide indefinitely, creating ES cell lines. Under certain conditions, ES cells can differentiate, either spontaneously into a variety of...
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Adult Stem Cells01:33

Adult Stem Cells

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Stem cells are undifferentiated cells that divide and produce more stem cells or progenitor cells that differentiate into mature, specialized cell types. All the cells in the body are generated from stem cells in the early embryo, but small populations of stem cells are also present in many adult tissues including the bone marrow, brain, skin, and gut. These adult stem cells typically produce the various cell types found in that tissue—to replace cells that are damaged or to continuously...
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Reproductive Cloning01:27

Reproductive Cloning

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Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT).
Somatic Cell Nuclear Transfer
In SCNT, an egg cell is taken from an animal and its nucleus is removed, creating an enucleated egg. Then a somatic...
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Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells
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Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells.

Amar M Singh1

  • 1Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, University of Georgia, Athens, GA, United States.

Frontiers in Cell and Developmental Biology
|February 16, 2019
PubMed
Summary
This summary is machine-generated.

This study presents an efficient method for single-cell cloning of human pluripotent stem cells (hPSCs) using fluorescent-assisted cell sorting (FACS). This technique streamlines gene editing by improving cloning efficiency and reducing manual labor.

Keywords:
cloning protocolhuman embryonic stem cellsinduced pluripotent stem cellspluripotent stem cellssingle-cell

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Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells
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Area of Science:

  • Stem Cell Biology
  • Genomic Engineering
  • Molecular Biology

Background:

  • Genomic manipulation of human pluripotent stem cells (hPSCs) is crucial for genetic modification and reporter line development.
  • Single-cell cloning is a bottleneck in hPSC gene editing, often inefficient and labor-intensive with manual colony picking.
  • Fluorescent-assisted cell sorting (FACS) for hPSC isolation is challenging, limiting its application in cloning.

Purpose of the Study:

  • To develop and present a streamlined protocol for single-cell cloning of hPSCs using FACS.
  • To improve the efficiency and reduce the time and labor associated with hPSC gene editing.
  • To provide a fundamental method for researchers working with hPSCs.

Main Methods:

  • Utilizing FACS for the direct isolation of single hPSCs post-transfection.
  • Sorting single cells onto a feeder layer of mouse embryonic fibroblasts (MEFs).
  • Employing a defined stem cell medium supplemented with Knockout Serum Replacement (KSR) and a ROCK inhibitor.

Main Results:

  • Achieved single-cell cloning efficiencies approaching 40% within 7-10 days.
  • Enabled FACS-based single-cell isolation as early as 1-2 days after transfection.
  • Streamlined the overall gene editing process for hPSCs.

Conclusions:

  • The presented FACS-based protocol significantly enhances the efficiency of hPSC single-cell cloning.
  • This method offers a time- and labor-saving alternative to manual colony picking.
  • The protocol provides a valuable tool for researchers engaged in hPSC genomic manipulation and gene editing studies.