Citation

Single Cell Analysis of Human Embryonic Stem Cell Differentiation

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Abstract:

The capacity to form nearly all cell types comprising the various tissues in the body has made embryonic stem cells (ESC) a valuable research tool for determining cell lineages. Harnessing the ability to direct the differentiation of human ESCs along specified lineages may provide a means to generate therapeutic cell types for clinical use.

Thus far stem cell culture heterogeneity and the variable response to global inductive cues has been a significant obstacle in obtaining a homogeneous population for directed differentiation protocols. Current literature points to the natural heterogeneity of stem cell populations warranting the necessity to analyze a single cell’s response to an inductive cue. Single cell expression analysis eliminates the variability hindering population studies thereby providing a better understanding of stem cell differentiation.

To examine the heterogeneous response to culture conditions, we have adapted the TaqMan qPCR method of transcript analysis for studying stem cells at a single cell level. We used this assay to profile single human ESCs from undifferentiated colonies as well as after activin-, BMP4-, and serum-induced differentiation. Our results have verified large-scale variability in stem cell populations, and confirmed known genetic relationships in development. Contrary to previous attempts at single cell transcript profiling where data analysis is restricted to qualitative, presence/absence interpretations, careful analysis of the gene expression data revealed that our assay retains a reliably significant quantitative capacity.

Techniques utilized in this study are reproducible and can be used to detect differences between the transcriptomes of individual cells. These methods have comparable sensitivity and accuracy with conventional protocols and are functional in a wide range of cell types. Single cell analysis with our technique will yield valuable insight into the process of lineage commitment and differentiation. It will also provide a platform for the systematic discovery of lineage specific markers useful in refining directed differentiation protocols.
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Association:
Name: Connecticut's Stem Cell Research International Symposium
URL:
http://stemconn.org


Citation:
URL: http://citation.allacademic.com/meta/p319730_index.html
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MLA Citation:

Gibson, Jason., Jakuba, Caroline., Holbrook, Kelly., Boucher, Nathalie., Carter, Mark. and Nelson, Craig. "Single Cell Analysis of Human Embryonic Stem Cell Differentiation" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT, Mar 23, 2009 <Not Available>. 2014-11-29 <http://citation.allacademic.com/meta/p319730_index.html>

APA Citation:

Gibson, J. D., Jakuba, C. M., Holbrook, K. , Boucher, N. , Carter, M. and Nelson, C. E. , 2009-03-23 "Single Cell Analysis of Human Embryonic Stem Cell Differentiation" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT <Not Available>. 2014-11-29 from http://citation.allacademic.com/meta/p319730_index.html

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: The capacity to form nearly all cell types comprising the various tissues in the body has made embryonic stem cells (ESC) a valuable research tool for determining cell lineages. Harnessing the ability to direct the differentiation of human ESCs along specified lineages may provide a means to generate therapeutic cell types for clinical use.

Thus far stem cell culture heterogeneity and the variable response to global inductive cues has been a significant obstacle in obtaining a homogeneous population for directed differentiation protocols. Current literature points to the natural heterogeneity of stem cell populations warranting the necessity to analyze a single cell’s response to an inductive cue. Single cell expression analysis eliminates the variability hindering population studies thereby providing a better understanding of stem cell differentiation.

To examine the heterogeneous response to culture conditions, we have adapted the TaqMan qPCR method of transcript analysis for studying stem cells at a single cell level. We used this assay to profile single human ESCs from undifferentiated colonies as well as after activin-, BMP4-, and serum-induced differentiation. Our results have verified large-scale variability in stem cell populations, and confirmed known genetic relationships in development. Contrary to previous attempts at single cell transcript profiling where data analysis is restricted to qualitative, presence/absence interpretations, careful analysis of the gene expression data revealed that our assay retains a reliably significant quantitative capacity.

Techniques utilized in this study are reproducible and can be used to detect differences between the transcriptomes of individual cells. These methods have comparable sensitivity and accuracy with conventional protocols and are functional in a wide range of cell types. Single cell analysis with our technique will yield valuable insight into the process of lineage commitment and differentiation. It will also provide a platform for the systematic discovery of lineage specific markers useful in refining directed differentiation protocols.


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