Isolating gene-corrected stem cells without drug selection

Abstract

Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >/=10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a "purity" of >30%, enriched >2,000-fold and with a recovery of approximately 20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >/=3 days.

Fig. 1.

Gene correction by homologous recombination with vectors containing fluorescent protein genes. (A) A green-positive, cyan-negative targeting vector for correcting a gene mutation by homologous recombination with a defective Hprt gene as an example. (Top) The structure of the target gene, which has a deletion of ≈55 kb that removed the promoter and exons 1 and 2, but left exons 3 through 9. (Middle) The gene-correcting vector, which includes a GFP gene and the missing Hprt promoter P and exons 1 and 2 all flanked by the homologous sequences that control the recombination. A CFP gene, located outside the homologous regions, is also part of the targeting vector. (Bottom) The corrected gene with the GFP gene used for positive selection now adjacent to it. The CFP gene used for negative selection is excluded by the homologous recombination. The open boxes and line indicate the correcting DNA. The heavy horizontal lines and filled boxes represent endogenous sequences and exons in the target gene and homologous sequences in the correcting vector. The heavy dashed line indicates bacterial sequences. (B) Mutant stem cells are electroporated with correcting DNA and maintained on feeders for 1-7 days with passages but without drug selection. Cells are dispersed with trypsin and sorted by differences in their forward light scatter (FSC), side scatter (SSC), and pulse width and by their fluorescence after excitation with a 458-nm laser. Cells with green or green plus cyan, or cyan fluorescence are collected in bulk, or singly into the wells of a 384-well plates. Doublets, cells with no fluorescence, or cells fluorescing red are discarded. Colonies derived from sorted cells are counted before and after HAT selection.

Fig. 2.

The time course of events after culture for 1-6 days after electroporation with the green-positive, cyan-negative vector diagramed in Fig. 1 A. The vertical axis shows the percentage of sorted cells fluorescing green. Open bars indicate cells that produce nonfluorescing colonies when expanded. Gray bars indicate cells that produce green-fluorescing HAT-sensitive colonies; these are cells that have incorporated the GFP gene into their genomes nonhomologously. Black bars indicate cells that produce green-fluorescing HAT-resistant colonies; these are the cells in which the mutant gene has been corrected by homologous recombination. The numbers in the boxes show the percentages of sorted cells falling into the three categories indicated by the shading of the bars. The error bars show the standard errors of the results from four experiments. Note that, although a green-positive, cyan-negative vector was used in this experiment, sorting was only for green fluorescence. Note also that the vertical axis of the figure is interrupted and has two scales.

Fig. 3.

Sorting criteria for collecting gene-corrected cells. (A) Green and cyan fluorescence used to collect gene-corrected cells 7 days after electroporation introducing the green-positive, cyan-negative vector diagrammed at the bottom of the panel. The heavy polygon indicates the gating used for collecting green-positive, cyan-negative cells (0.014% of the total input); one in five of these cells produces HAT^R colonies in which the mutant gene is corrected. Less than 1 in 1,000 of green-positive, cyan-positive cells (0.014% of input cells) and none of the green-negative, cyan-positive cells (0.009% of input cells) are HAT^R. Single cells are shown in red. Yellow and blue indicate increasing numbers of cells. (B) Red and cyan fluorescence used to exclude cells with autofluorescence in experiments with the green-positive, cyan-negative vector by rejecting cells within the dashed polygon (0.52% of input cells). These include minor fractions of feeder cells and stem cells that autofluoresce, indicated by square brackets no. 1 and no. 2, respectively. (C) Green and cyan fluorescence used to collect gene-corrected cells 7 days after electroporation introducing the cyan-positive, green-negative vector diagrammed at the bottom of the panel in which the CFP and GFP genes of the earlier vector have been interchanged. The heavy polygon indicates the gating used to collect cyan-positive, green-negative cells (0.01% of input cells); 1 in 3 of these cells produce HAT^R colonies in which the mutant gene has been corrected. Less than 1 in 1,000 of cyan-positive, green-positive cells (0.022% of input cells) and none of the cyan-negative, green-positive cells (0.005% of input cells) are HAT^R. No significant autofluorescence encroaches on the cyan channel, and so the red channel is not needed with this vector, which is the one currently used.