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Posts Tagged ‘skin fibroblast’


Shortened time for cell renewal

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Accelerated Reprogramming and Gene Editing Protocol Can Make Fixed Cells Much Faster

 

Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

Sara E. Howden, John P. Maufort, Bret M. Duffin, Andrew G. Elefanty, Edouard G. Stanley, James A. Thomson
Stem Cell Reports Dec 2015;  5, (6):1109–1118   http://dx.doi.org/10.1016/j.stemcr.2015.10.009
Highlights
  • Episomal reprogramming system is enhanced by expression of miR302/367
  • Gene targeting and reprogramming can be combined in a simple one-step procedure
  • Clonal gene-corrected iPS cell lines can be obtained in as little as 2 weeks

 

Summary

The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

Induced pluripotent stem (iPS) cells, generated by introducing defined factors to reprogram terminally differentiated somatic cells, offer enormous potential for the development of autologous or customized cellular therapies to treat or correct many inherited and acquired diseases (Takahashi et al., 2007, Yu et al., 2007). Complications associated with immunorejection can be avoided through the generation and subsequent disease correction of patient-specific iPS cells, which can be differentiated into relevant cell types for the repopulation and regeneration of a defective tissue or organ. Gene targeting by homologous recombination is the ideal approach for the correction of genetic defects as it enables replacement of the defective allele with a normal functional one without disturbing the remaining genome. The generation of a genetically modified iPS cell line typically involved multiple procedures that required the cells to be in culture for an extensive period, drug selection, and several clonal events (Hockemeyer et al., 2009, Howden et al., 2011, Liu et al., 2011, Zou et al., 2011). In the first step, somatic cells are reprogrammed, and several clones are expanded and characterized. Gene targeting constructs are then introduced, and cells are usually subjected to drug selection to isolate and identify correctly modified iPS cell colonies. Once successfully targeted clones are identified, it is preferable to excise the drug selectable marker, commonly flanked by loxP or FRT sites. Taken together, the multiple steps required for the generation of genetically modified iPS cell lines typically require cells to be in culture for several months, which is not compatible for patients for whom urgent medical intervention is imperative. Furthermore, there is evidence to suggest that increased culture times are associated with undesirable changes in genomic integrity, such as duplications of oncogenic genes (Laurent et al., 2011) and other karyotypic abnormalities (Chen et al., 2008). Here we report that reprogramming and gene targeting can be performed together in a one-step procedure that requires only a single electroporation. Multiple gene-targeted iPS cell clones can be generated from patient cells in as little as 2 weeks, requiring only a single clonal event. The procedure also does not require the use of drug selection and permits the generation of clones that contain “seamless” single base-pair changes, without leaving residual loxP or FRT sites in the host genome.

 

Large image of Figure 1.

Figure 1

Episomal Reprogramming System Is Enhanced with Inclusion of Plasmid Encoding the miR302/367 Cluster

Reprogramming experiments were performed with and without inclusion of the miR302/367 expression plasmid using a normal male fibroblast line. Data represent an average of three independent experiments ± SD.

 

We used an enhanced episomal-based reprogramming system to generate iPS cell lines that would eventually be free of vector sequences. In addition to the seven factors (OCT4, SOX2, NANOG, c-MYC, KLF4, LIN28, and the SV40 Large T-Antigen) encoded by the three oriP-based vectors previously reported to induce pluripotency (Yu et al., 2009), we also forced expression of the micro RNA (miR) 302/367 cluster, which is known to facilitate reprogramming and maintenance of pluripotency (Lin et al., 2008, Miyoshi et al., 2011). The inclusion of an additional episomal vector encoding miR 302/367 resulted in a substantial increase (more than 100-fold) in the total number of iPS cell colonies in human fibroblasts (Figure 1). This plasmid was included in all subsequent reprogramming experiments and was necessary to obtain sufficient iPS cell colony numbers when combining gene targeting and reprogramming in a single step.

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We have also successfully used our one-step protocol to simultaneously reprogram and genetically correct the disease-causing mutation in the patient fibroblasts, an autosomal dominant C > T transition in exon 42 of the PRPF8gene. This was achieved using a plasmid encoding the Cas9 protein fromS. pyogenes (Mali et al., 2013b), a plasmid encoding a PRPF8-specific sgRNA that binds 33 bp upstream of the disease-causing mutation, and a 184-bp single-stranded oligodeoxynucleotide (ssODN) (Figure 3A). The ssODN was engineered to contain four synonymous mutations to minimize the possibility of Cas9 protein re-cutting following homologous recombination and to aid in the identification of clones that had undergone a gene-targeting event (Figure 3A). Approximately 3 weeks post-transfection, we randomly isolated and expanded a total of 72 iPS cell colonies for further analysis. A PCR product encoding the region of interest was amplified from the genomic DNA of all 72 clones using primers flanking the target site, which was subsequently analyzed by Sanger sequencing. Cas9-induced modification of one or both PRPF8 alleles was observed in 22 (31%) of the clones analyzed, most commonly detected as a nonhomologous end joining (NHEJ) event within the intended cut site. Homologous recombination at the target site could be detected in 6 (8%) clones, as evidenced by the loss of the disease-causing mutation or the presence of one or more synonymous mutations carried by the corrective ssODN (Table 1). Genetic correction of the autosomal dominant patient-specific mutation was observed in 2 clones, while targeting of the wild-type allele was observed in 4 clones. We were unable to determine which allele had undergone gene targeting in 1 clone (P.57) due to a 151-bp deletion spanning the site of the mutation. Surprisingly, 1 clone (P.50) appeared to have undergone bi-allelic homologous recombination, as evidenced by correction of the patient-specific mutation and the presence of ssODN-specific synonymous mutations on both alleles (Figures 3B and 3C). However, this clone also contained a 1-bp deletion approximately 50 bp upstream of the intended site of the Cas9-induced double-stranded break. We hypothesize that this is most likely due to homologous recombination with an incorrectly synthesized ssODN rather than an additional mutation caused by NHEJ, which normally occurs at the site of the double-stranded break.

 

Table 1Analysis of Gene-Targeted iPS Cell Clones Derived from Patient with Retinitis Pigmentosa
Clone Modification Observed at PRPF8 Target Site (Exon 42)
P.16 no correction of mutation but presence of SM1 and SM2 on mutant allele; wild-type allele unmodified
P.50 one allele contains SMs 1–3; other allele contains SMs 1–4 and 1-bp deletion ≈30 bp upstream of Cas9 target site
P.57 one allele contains SMs 1–3; other allele contains 151-bp deletion (spanning 124 bp downstream and 7 bp upstream of Cas9 target site) and 105-bp insertion
P.71 correction of mutant allele, but no SMs present; wild-type allele has a 2-bp insertion within Cas9 target site
P.72 wild-type allele contains SMs 1–4; mutant allele has 1-bp deletion
P.73 wild-type allele contains SMs 1–3; mutant allele has 2-bp deletion within Cas9 target site

SM, synonymous mutation.

 

Thumbnail image of Figure 3. Opens large image

http://www.cell.com/cms/attachment/2040890164/2054788036/gr3.jpg

Figure 3

Simultaneous Reprogramming and Genetic Correction of thePRPF8 Gene in Fibroblasts from a Patient with Retinitis Pigmentosa

(A) Schematic diagram of the PRPF8 gene, with mutation in exon 42. The Cas9 target site (red), the patient-specific mutation (blue), and antisense single-stranded DNA template used for gene repair are shown.

(B) Sequencing analysis of exon 42 of the PRPF8 gene in the genomic DNA from uncorrected patient-specific iPS cells. Both wild-type and mutant alleles are shown.

(C) Sequencing analysis of genomic DNA from a single iPS cell clone following successful simultaneous reprogramming and genetic correction of patient-specific fibroblasts. Both alleles appear to have undergone homologous recombination with the corrective ssODN as evidenced by the presence of the ssODN-specific synonymous mutations (SM 1-4) on both alleles. One allele also has a single base-pair deletion, which is most likely caused by an ssODN that was incorrectly synthesized. The location of the patient-specific mutation and synonymous mutations introduced by the repair ssODN are marked by black boxes.

 

Next we attempted to correct the disease-causing mutation in a fibroblast line isolated from an infant with severe combined immunodeficiency (SCID), caused by mutations in the gene encoding adenosine deaminase (ADA). SCID patients could particularly benefit from a one-step protocol that facilitates the expedited generation of gene-corrected iPS cells because without early intervention, such as a bone marrow transplant, patients typically die within the first 1 to 2 years of life. We first attempted to simultaneously reprogram and target DNMT3B in ADA-SCID fibroblasts and identified one EGFP-expressing colony (0.9%) out of a total of 108 iPS cell colonies (Figure S2). PCR analysis confirmed targeting of theDNMT3B locus (see Figure 2E). We next attempted to simultaneously reprogram and correct one of the disease-causing mutations in the ADA-SCID fibroblasts using our one-step protocol. The fibroblasts were derived from a patient who is a compound heterozygote: one allele has a C > T transition in exon 11 of the ADAgene (1,081C > T), and the second allele has an A > G transition in the 3-prime splice site of intron 3, resulting in a deletion of exon 4 from mature mRNA. We chose to correct the C > T transition in exon 11 using an sgRNA specific to the mutant, but not wild-type, exon 11 sequence of the ADA gene (Figure 4A). We hypothesized that this would minimize Cas9 cutting in both alleles, as seen in the majority of the PRPF8 gene-targeted iPS cell lines, where only 1 out of the 6 clones did not have a second allele modified, either by NHEJ or a second homologous recombination event. To facilitate gene correction we used a 175-bp single-stranded corrective ssODN, which was engineered to contain a single synonymous mutation within the Cas9 target site (Figure 4A). A total of 55 colonies were expanded and screened, with Cas9-induced modification of ADAexon 11 observed in 20 (36%) clones, as determined by Sanger sequencing of a 1.4-kb PCR product amplified from genomic DNA using primers flanking the target site. Gene targeting was detected in 3 (5%) clones, as evidenced by the loss of the disease-causing mutation and the presence of the synonymous mutation carried by the corrective ssODN. Genetic correction of the patient-specific mutation in exon 11 was observed in all three clones, without modification of the second allele, indicating that Cas9 preferentially favored the mutant exon 11 sequence (over wild-type). ……

 

Thumbnail image of Figure 4. Opens large image

http://www.cell.com/cms/attachment/2040890164/2054788039/gr4.jpg

Figure 4

Simultaneous Reprogramming and Genetic Correction of ADA-SCID Fibroblasts

(A) Schematic diagram of the ADA gene, with mutation in exon 11. The Cas9 target site (red), the patient-specific mutation (blue), and antisense single-stranded DNA template used for gene repair are shown.

(B) Sequencing analysis of exon 11 of the ADA gene in the genomic DNA of an uncorrected and two gene-corrected iPS cell lines derived from ADA-SCID fibroblasts. One of the gene-corrected lines (clone Bb) was also found to carry a G > A transition approximately 35 bp downstream of the intended DNA double-stranded break, and most likely introduced by an incorrectly synthesized ssODN.

(C) Sequencing analysis of the ADA transcript amplified from the cDNA of an uncorrected and two gene-corrected iPS cell lines. The location of the patient-specific mutation, synonymous mutation, and G > A transition introduced by the repair ssODN are marked by black boxes.

……….

 

We have demonstrated the feasibility of performing reprogramming and gene correction together in a simple one-step procedure that enables the generation of multiple gene-corrected and uncorrected iPS cell lines in as little as 2 weeks, requiring considerably less time and resources compared to conventional multi-step protocols that can take several months to complete. In a therapeutic context this should facilitate transplantation medicine by making gene-corrected cells available to patients in a more timely manner, while potentially minimizing the risks associated with extended cell culture, drug selection, and multiple clonal events. In addition, we anticipate that comparisons between corrected and matched uncorrected control iPS cell lines generated from a single experiment will also be extremely useful for disease modeling and understanding the underlying molecular mechanisms governing disease, because any observed differences between corrected and uncorrected cells can be attributed to the patient-specific mutation rather than differences in genetic background.

However, it is important to note that a number of studies have demonstrated that iPS cell lines derived from skin biopsies typically harbor a unique subset of de novo genetic abnormalities, either in the form of copy-number variation or single base-pair changes (Abyzov et al., 2012, Gore et al., 2011) and that iPS cell lines generated from the same parental line can vary significantly with respect to whole-genome gene expression in the differentiated state (Reinhardt et al., 2013). Nonetheless, it is reasonable to expect that the confounding effects arising from the variations that exist across different iPS cell clones may be minimized by comparing multiple gene-corrected or gene-targeted clones with multiple uncorrected clones. In this regard a consistent difference that is observed exclusively in the corrected versus uncorrected lines can most likely be attributed to the patient-specific mutation rather than variations that may exist from one clone to the next. In the current study we routinely observed targeting efficiencies of > 5%, enabling the generation of multiple gene-targeted and “matched” uncorrected clones from a single experiment.

 

 

 

 

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