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On iPS cells and p53: role of p53–p21 pathway Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by introduction of four genes. Efficiency of this process, however, is low. Here it is reported that up to 10% of transduced mouse embryonic fibroblasts (MEF) lacking p53 became iPS cells, even without the Myc retrovirus. In the p53 -null background, iPS cells can be generated from terminally differentiated T lymphocytes. The authors propose that the p53–p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation. Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the introduction of four genes, but with low efficiency. Here it is reported that 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus, and iPS cells were also generated from terminally differentiated T lymphocytes in the p53 -null background. Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse 1 , 2 , 3 , 4 and in human 5 , 6 , 7 , 8 . The efficiency of this process, however, is low 9 . Pluripotency can be induced without c-Myc, but with even lower efficiency 10 , 11 . A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation 12 , but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53 -null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53–p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.

At present, most cases of unresectable cancer cannot be cured. Genetic mutations, EMT, and cancer stem cells are three major issues linked to poor prognosis in such cases, all connected by inter- and intra-tumor heterogeneity. Issues on inter-/intra-tumor heterogeneity of genetic mutation could be resolved with recent and future technologies of deep sequencers, whereas, regarding such issues as the “same genome, different epigenome/phenotype”, we expect to solve many of these problems in the future through further research in stem cell biology. We herein review and discuss the three major issues in the biology of cancers, especially from the standpoint of stem cell biology.

Induced pluripotent stem (iPS) cells have been generated from mouse and human fibroblasts by the retroviral transduction of four transcription factors. However, the cell origins and molecular mechanisms of iPS cell induction remain elusive. This report describes the generation of iPS cells from adult mouse hepatocytes and gastric epithelial cells. These iPS cell clones appear to be equivalent to embryonic stem cells in gene expression and are competent to generate germline chimeras. Genetic lineage tracings show that liver-derived iPS cells are derived from albumin-expressing cells. No common retroviral integration sites are found among multiple clones. These data suggest that iPS cells are generated by direct reprogramming of lineage-committed somatic cells and that retroviral integration into specific sites is not required.

BackgroundThe esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. MethodsWe established a novel stepwise protocol with transwell culture and an air–liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus.ResultsWe successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation.ConclusionWe revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

The safety of induced pluripotent stem cells seems to depend on how they were generated. Miura et al . examine the effects of the c-myc transgene, tissue of origin and selection method on the tumor-forming propensity of iPS-cell derivatives. We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast–derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.

Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc 1 . Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras 2 , 3 , 4 . Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications 3 . Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc − iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1 , a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1 + cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo , thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney. Stem cells have raised hopes of developing regenerative therapies of renal disease. Here, Osafune et al. provide a protocol for the differentiation of induced human pluripotent stem cells into renal lineages with the capacity to form tubular renal structures in mice.

For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.

Small proline-rich protein 1A (SPRR1A) is recognized as a squamous differentiation marker but is also upregulated in some non-squamous cancers. However, its expression in pancreatic ductal adenocarcinoma (PDAC) has not been investigated. This study elucidated the expression of SPRR1A in PDAC and its effect on the prognosis and malignant behavior of PDAC. We examined the SPRR1A expression by immunohistochemistry in 86 surgical PDAC cases and revealed the relationship between its expression and the prognosis of the PDAC patients. Furthermore, we overexpressed SPRR1A in pancreatic cancer cell lines (PK-1 and Panc-1) and assessed the phenotype and gene expression changes in vitro. Among the 84 cases, excluding 2 with squamous differentiation, 31 (36.9%) had a high SPRR1A expression. The overall survival (median 22.1 months vs. 33.6 months, p = 0.0357) and recurrence-free survival (median 10.7 months vs. 15.5 months, p = 0.0298) were significantly lower in the high-SPRR1A-expression group than in the low-SPRR1A-expression group. A multivariate analysis indicated that a high SPRR1A expression (HR 1.706, 95% CI 1.018 to 2.862, p = 0.0427) and residual tumor status (HR 2.687, 95% CI 1.487 to 4.855, p = 0.00106) were independent prognostic factors. The analysis of TCGA transcriptome data demonstrated that the high-SPRR1A-expression group had a significantly worse prognosis than the low-SPRR1A-expression group, which supported our data. SPRR1A overexpression in PK-1 and Panc-1 did not result in remarkable changes to in vitro phenotypes, such as the cell proliferation, chemo-resistance, EMT, migration or global gene expression. Increased expression of SPRR1A is associated with a poor prognosis in PDAC and may serve as a novel prognostic marker. However, our in vitro study suggests that the SPRR1A expression may be a consequence, not a cause, of the aggressive behavior of PDAC.