Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

Error-free and error-prone DNA repair gene expression data through reprogramming and passage in human iPS cells.

We recently found that DNA repair-related gene expression could be altered by reprogramming as well as the increased expression of genes that accurately convey genomic information, such as homologous recombination (HR) and mismatch repair (MMR), and the decreased expression of error-prone translesion synthesis (TLS) polymerase. Here, we confirmed this change in expression in another cell-line and found that such alteration was maintained by overlapping passages as well as OCT3/4 and NANOG. Our findings suggest that changes in the expression of DNA repair-related genes associated with reprogramming and their maintenance can be novel indicators of the quality control of the cells exhibiting pluripotency.

Increased error-free DNA repair gene expression through reprogramming in human iPS cells.

INTRODUCTION: Many studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied. METHODS: We analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming. RESULTS: Through reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information. CONCLUSIONS: High PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.

Generation of targeted homozygosity in the genome of human induced pluripotent stem cells.

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

Green fluorescent protein as a marker in transgenic mice.

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.

Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.

Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.