Barcoding for clonal tracking of hematopoietic stem cells

by Alexey Bersenev on December 4, 2011 · 0 comments

in hematopoietic, methods

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It is well known that despite phenotypic uniformity, hematopoietic stem cells (HSC) possess great functional heterogeneity. So, individual HSC in heterogeneous population have different functional contribution in blood lineages. Single cell transplantation assay is a golden standard for assessing functional heterogeneity and stemness. Unfortunately, this assay is very difficult to perform technically, time consuming and costly (requires a lot of animals). Researchers were looking for alternative assays, which would allow us to track single stem cells and their clonal contribution in lineage heterogeneity and tissue integrity.

Weissman’s group from Stanford University has developed new technique for identification of single HSC. For the first time they used genetic barcoding for clonal tracking progeny of individual HSC. Let’s look at how was it done:

The researchers began with a FACS-purified population of mouse HSCs and infected them with lentiviruses carrying a library of molecular barcodes. They chose the titer of the infecting virus to optimize the number of cells infected while still ensuring that most cells received a single viral integration. Each virus carried a 33-base-pair (bp) barcode, with a unique 27-bp sequence and a 6-bp ‘identifier’ sequence that was identical for all barcodes in the library. Then the researchers transplanted infected cells into irradiated recipient mice and, several months later, examined the distribution of barcodes in blood cell types of interest.

The authors were able to combine in one assay 3 previously developed and widely used techniques – viral cellular labeling, high-throughput sequencing and DNA barcoding. It still looks a bit complicated with some technical considerations:

The key steps of this clonal tracking technology are (i) ensuring single-cell representations of the barcodes and (ii) handling sequencing errors.

Importantly, barcoding technique can overcome technical limitations and cost of single cell transplantation:

The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice.

In our result, barcodes representing HSC clones demonstrated lineage biases as reported by previous single-cell transplantation experiments. This validates the single-cell precision of our barcode tracking system. Whereas the single-cell transplantation studies used 352 mice, we tracked many more HSC clones using only 7 mice. The barcode tracking system also provided information previously unattainable with conventional single-cell transplantation. For instance, our data revealed two clearly separated HSC populations with distinct lineage biases in each irradiated recipient mouse.

I think, new barcoding method is very promising and worthy of future development. This technique could be applied for clonal tracking of any other type of virus-accessible stem cells.

Nature Methods summary

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