Mari Ohnuki

If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transducing 4 transcription factors: Oct3/4, Sox2, Klf4, and c-Myc. The investigators now report generating iPS cells from adult human dermal fibroblasts using the same 4 factors. The first step was to optimize retroviral transduction in human fibroblasts as well as conditions of culture. Amphotrophic retrovirus was utilized. Colonies resembling human embryonic stem (hES) cell colonies were observed about 25 days after sampling cells from the facial dermis of a 36-year-old Caucasian female. In general, the human iPS cells expressed hES cell-specific surface antigens rather than stage-specific embryonic antigen. The cells expressed many undifferentiated ES cell-marker genes and they exhibited high telomerase activity. The cells proliferated exponentially for at least 4 months. It proved possible for iPS cells to differentiate into 3 germ layers in vitro. The cells resembled hES cells with regard to morphology, proliferation, gene expression, and epigenetic status of pluripotent cell-specific genes. Among the cell types formed from directed differentiation of human iPS cells were dopaminergic neurons and cardiac myocytes. Injection of human iPS cells subcutaneously into immunodeficient mice was followed after 9 weeks by teratomas containing gut-like epithelial tissues, striated muscle, cartilage, neural tissues, and keratin-containing epidermal tissues. The human iPS clones were not a result of cross-contamination. Although human iPS cells are not identical to hES cells, they should find applications in regenerative medicine once safety issues are overcome.

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.