It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23–70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1–2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES-like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle. Disclosure of potential conflicts of interest is found at the end of this article.

Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.