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Method for single-cell sorting and expansion of genetically modified human embryonic stem cells
Cory R Nicholas1, Renee A Reijo Pera
1Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304-5542, USA.
CSH Protocols
|March 2, 2011
Summary
This study presents a new method for genetically modifying human embryonic stem cells (hESCs) using lentivirus and fluorescence-activated cell sorting (FACS). This technique enables the creation of homogenous populations of reporter-labeled hESCs for research.
Area of Science:
- Stem cell biology
- Developmental biology
- Genetic engineering
Background:
- Human embryonic stem cells (hESCs) possess pluripotency, making them valuable for studying cell differentiation.
- Efficient genetic modification of hESCs is crucial for tracking cell lineages and understanding developmental mechanisms.
- Existing methods require improvement for reliable reporter gene integration and isolation of modified cells.
Purpose of the Study:
- To develop and describe a robust protocol for the genetic modification of hESCs.
- To enable the generation of homogenous populations of genetically engineered hESCs using reporter constructs.
- To facilitate the identification and isolation of specific cell types derived from hESCs.
Main Methods:
- Transduction of hESCs with a self-inactivating lentivirus carrying a ubiquitous enhanced green fluorescent protein (eGFP) reporter.
- Single-cell isolation and expansion of transduced hESCs.
- Utilizing fluorescence-activated cell sorting (FACS) for the selection of homogenous eGFP-expressing hESC clones.
Main Results:
- Successful transduction of hESCs with the eGFP reporter construct.
- Isolation and expansion of single-cell derived, homogenous populations of genetically modified hESCs.
- Demonstration of FACS as an effective tool for selecting reporter-labeled hESCs.
Conclusions:
- The described lentiviral transduction protocol provides a reliable method for genetic modification of hESCs.
- This approach facilitates the generation of homogenous, reporter-labeled hESC lines for developmental studies.
- The protocol supports the identification and isolation of specific cell types derived from hESCs through reporter expression.
