Disease-specific induced pluripotent stem cells.

Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.

Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19(Arf) (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf-Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis.

A reprogrammable mouse strain from gene-targeted embryonic stem cells.

The derivation of induced pluripotent stem cells (iPSCs) usually involves the viral introduction of reprogramming factors into somatic cells. Here we used gene targeting to generate a mouse strain with a single copy of an inducible, polycistronic reprogramming cassette, allowing for the induction of pluripotency in various somatic cell types. As these 'reprogrammable mice' can be easily bred, they are a useful tool to study the mechanisms underlying cellular reprogramming.

Sox2 is dispensable for the reprogramming of melanocytes and melanoma cells into induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have been derived at low frequencies from different cell types through ectopic expression of the transcription factors Oct4 and Sox2, combined with either Klf4 and c-Myc or Lin28 and Nanog. In order to generate iPSCs more effectively, it will be crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. Here, we show that both human and mouse melanocytes give rise to iPSCs at higher efficiencies than fibroblasts. Moreover, we demonstrate that a mouse malignant melanoma cell line, which has previously been reprogrammed into embryonic stem cells by nuclear transfer, remains equally amenable to reprogramming into iPSCs by these transcription factors. In contrast to skin fibroblasts, melanocytes and melanoma cells did not require ectopic Sox2 expression for conversion into iPSCs. iPSC lines from melanocytic cells expressed pluripotency markers, formed teratomas and contributed to viable chimeric mice with germ line transmission. Our results identify skin melanocytes as an alternative source for deriving patient-specific iPSCs at increased efficiency and with fewer genetic elements. In addition, our results suggest that cancer cells remain susceptible to transcription factor-mediated reprogramming, which should facilitate the study of epigenetic changes in human cancer.

Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.

Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.

Registered report: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma' by Castellarin and colleagues published in Genome Research in 2012 (Castellarin et al., 2012). The experiment to be replicated is reported in Figure 2. Here, Castellarin and colleagues performed a metagenomic analysis of colorectal carcinoma (CRC) to identify potential associations between inflammatory microorganisms and gastrointestinal cancers. They conducted quantitative real-time PCR on genomic DNA isolated from tumor and matched normal biopsies from a patient cohort and found that the overall abundance of Fusobacterium was 415 times greater in CRC versus adjacent normal tissue. These results confirmed earlier studies and provide evidence for a link between tissue-associated bacteria and tumorigenesis. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF(WT) tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2.

The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.

Registered report: the CD47-signal regulated protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

The Willingham et al., 2012, published in PNAS in 2012. The key experiments being replicated are those reported in Figure 6A-C and Table S4. In these experiments, Willingham et al., 2012 test the safety and efficacy of anti-CD47 antibody treatment in immune competent mice utilizing a syngeneic model of mammary tumor growth in FVB mice. The Reproducibility Project: Cancer Biology is a collaboration between the eLife.

Registered report: tumour vascularization via endothelial differentiation of glioblastoma stem-like cells.

The Nature in 2010 (Ricci-Vitiani et al., 2010). The experiments that will be replicated are those reported in Figure 4B and Supplementary Figure 10B (Ricci-Vitiani et al., 2010), which demonstrate that glioblastoma stem-like cells can derive into endothelial cells, and can be selectively ablated to reduce tumor progression in vivo, and Supplementary Figures S10C and S10D (Ricci-Vitiani et al., 2010), which demonstrate that fully differentiated glioblastoma cells cannot form functionally relevant endothelium. The Reproducibility Project: Cancer Biology is a collaboration between the eLife.

Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination.

Neonatal engraftment by oligodendrocyte progenitor cells (OPCs) permits the myelination of the congenitally dysmyelinated brain. To establish a potential autologous source of these cells, we developed a strategy by which to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs. From three hiPSC lines, as well as from human embryonic stem cells (hESCs), we generated highly enriched OLIG2(+)/PDGFRα(+)/NKX2.2(+)/SOX10(+) human OPCs, which could be further purified using fluorescence-activated cell sorting. hiPSC OPCs efficiently differentiated into both myelinogenic oligodendrocytes and astrocytes, in vitro and in vivo. Neonatally engrafted hiPSC OPCs robustly myelinated the brains of myelin-deficient shiverer mice and substantially increased their survival. The speed and efficiency of myelination by hiPSC OPCs was higher than that previously observed using fetal-tissue-derived OPCs, and no tumors from these grafts were noted as long as 9 months after transplant. These results suggest the potential utility of hiPSC-derived OPCs in treating disorders of myelin loss.

Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc.

Ectopic expression of Oct4, Sox2, cMyc, and Klf4 confers a pluripotent state upon several differentiated cell types, generating induced pluripotent stem cells (iPSCs) [1-8]. iPSC derivation is highly inefficient, and the underlying mechanisms are largely unknown. This low efficiency suggests the existence of additional cooperative factors whose identification is critical for understanding reprogramming. In addition, the therapeutic use of iPSCs relies on the development of efficient nongenetic means of factor delivery, and although a handful of replacement molecules have been identified, their use yields a further reduction to the already low reprogramming efficiency [9-11]. Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use. Here, we demonstrate that inhibition of Tgfbbeta signaling cooperates in the reprogramming of murine fibroblasts by enabling faster, more efficient induction of iPSCs, whereas activation of Tgfbeta signaling blocks reprogramming. In addition to exhibiting a strong cooperative effect, the Tgfbeta receptor inhibitor bypasses the requirement for exogenous cMyc or Sox2, highlighting its dual role as a cooperative and replacement factor. The identification of a highly characterized pathway operating in iPSC induction will open new avenues for mechanistic dissection of the reprogramming process.

Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells.

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy.

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming.

Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.

Guidelines and techniques for the generation of induced pluripotent stem cells.

Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) provides an invaluable resource for regenerative medicine, enabling the generation of patient-specific cells of any lineage without the use of embryonic material. A variety of methods exist for iPSC derivation, all reliant upon manipulation of a select group of transcription factors. We compare the currently reported protocols, identify essential steps common to these methods, and suggest minimal criteria for defining fully reprogrammed cells. In addition, specific procedures aimed to optimize reproducible iPSC derivation are presented, with an emphasis on standardization of certain parameters for accurate comparison between independent experiments.

Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.

Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points, reactivation of endogenous Oct4, Sox2, telomerase, and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells.

A high-efficiency system for the generation and study of human induced pluripotent stem cells.

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained "secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.

Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.

Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.