https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/49782/1/Yamanaka_Cell_131_5.pdf

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

MicroRNAs (miRNAs) are small non-coding RNAs that can modulate mRNA expression. Insights into the roles of miRNAs in development and disease have led to the development of new therapeutic approaches that are based on miRNA mimics or agents that inhibit their functions (antimiRs), and the first such approaches have entered the clinic. This Review discusses the role of different miRNAs in cancer and other diseases, and provides an overview of current miRNA therapeutics in the clinic. In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumour suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. Several miRNA-targeted therapeutics have reached clinical development, including a mimic of the tumour suppressor miRNA miR-34, which reached phase I clinical trials for treating cancer, and antimiRs targeted at miR-122, which reached phase II trials for treating hepatitis. In this article, we describe recent advances in our understanding of miRNAs in cancer and in other diseases and provide an overview of current miRNA therapeutics in the clinic. We also discuss the challenge of identifying the most efficacious therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics.

MicroRNA therapeutics: towards a new era for the management of cancer and other diseases

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

/pdf/highly-efficient-neural-conversion-of-human-es-and-ips-cells-5ewq762oqw.pdf

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.

/pdf/reprogramming-of-human-somatic-cells-to-pluripotency-with-4lycgkg8te.pdf

Reprogramming of human somatic cells to pluripotency with defined factors

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 transd

/pdf/induction-of-pluripotent-stem-cells-from-adult-human-3jc4cxk5x5.pdf

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

Induced pluripotent stem cells (iPSCs) are generated from mouse and human fibroblasts by the introduction of three transcription factors: Oct3/4, Sox2, and Klf4. The proto-oncogene product c-Myc markedly promotes iPSC generation, but also increases tumor formation in iPSC-derived chimeric mice. We report that the promotion of iPSC generation by Myc is independent of its transformation property. We found that another Myc family member, L-Myc, as well as c-Myc mutants (W136E and dN2), all of which have little transformation activity, promoted human iPSC generation more efficiently and specifically compared with WT c-Myc. In mice, L-Myc promoted germline transmission, but not tumor formation, in the iPSC-derived chimeric mice. These data demonstrate that different functional moieties of the Myc proto-oncogene products are involved in the transformation and promotion of directed reprogramming.

/pdf/promotion-of-direct-reprogramming-by-transformation-43ftwvthdg.pdf

Promotion of direct reprogramming by transformation-deficient Myc

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), Kruppel-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.

https://www.pnas.org/content/pnas/111/34/12426.full.pdf

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

https://gladstone.org/u/dsrivastava/Articles/Worringer-CellStemCell-2014.pdf

Megumi Narita

Papers

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

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

Promotion of direct reprogramming by transformation-deficient Myc

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

The let-7/LIN-41 Pathway Regulates Reprogramming to Human Induced Pluripotent Stem Cells by Controlling Expression of Prodifferentiation Genes