Mari Ohnuki

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.

Variation in the safety of induced pluripotent stem cell lines

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.

Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells

Significance In the past few years, findings have been controversial in regard to whether human induced pluripotent stem cells (hiPSCs) are distinct from human embryonic stem cells (hESCs) in their molecular signatures and differentiation properties. In this study, hiPSCs and hESCs have overlapping variations in molecular signatures such as RNA expression and DNA methylation. However, some hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. They need to be identified and eliminated prior to applications in regenerative medicine. We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.

Generation and Characterization of Human Induced Pluripotent Stem Cells

This unit describes how to generate human induced pluripotent stem (iPS) cells and evaluate the qualities of the generated iPS cells. The methods for establishment and maintenance of human iPS cells are similar to those for mouse iPS cells but not identical. In addition, these protocols include excellent procedures for passaging and cryopreservation of human iPS cells established by ES cell researchers, which result in an easy way to culture human iPS cells. Moreover, we include methods for characterizing iPS cells for further research. RT-PCR and immunocytochemistry for detection of pluripotent cell markers, embryoid body differentiation, and teratoma differentiation are used to determine pluripotency in vitro and in vivo, respectively.

Dynamic regulation of human endogenous retroviruses mediates factor-induced reprogramming and differentiation potential

Significance In this study, we found that human endogenous retoriviruses type-H (HERV-Hs) are transiently hyperactivated during reprogramming toward induced pluripotent stem cells (iPSCs) and play important roles in this process. However, when reprogramming is complete and cells acquire full pluripotency, HERV-H activity should decrease to levels comparable with those in embryonic stem cells because failure to resilence this activity leads to the differentiation-defective phenotype in neural lineage. We also found that during reprogramming, reprogramming factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), and Krüppel-like factor 4 (KLF4) (OSK) bind to and activate long-terminal repeats of HERV-Hs. KLF4 possibly precludes Tripartite motif containing 28 and recruits not only OCT3/4 and SOX2, but also E1A binding protein p300 (p300) histone acethyltransferase on HERV-H loci. Therefore, OKSM-induced HERV-H activation constitutes an unanticipated and critical mechanism for iPSC formation. Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s—the long-terminal repeats of HERV type-H (HERV-H)—to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H–driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.

Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm

During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm.

Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.