Vincent Cristofalo “Rising Star”Award: DNA Methylation Landscapes in Aging

The epigenetic code can be thought of as the operating system of the cell. It controls the most basic and critical cellular processes including differentiation, replication, metabolism, and signaling. Yet, with age, the epigenetic landscape is remodeled, bringing about widespread consequences for cellular and tissue identity, integrity, and functioning. But, what if like computer programmers, we could discover how to recode or restore the original program? The revolutionary discoveries by Yamanaka and Takahashi suggests this may be possible. While early experiments showed that Yamanaka factors could be used to convert somatic cells into induced pluripotent stem cells, more recent work by us and others have shown that signatures of epigenetic aging are also wiped clean during this process. What’s more, epigenetic age reversal appears to take place early in the process and thus can be achieved without the cell _needing to dedifferentiate. Building off of this discovery, our lab is combining novel experiments and advanced bioinformatic techniques to decipher the epigenetic code and determine how it is remodeled during aging, development, and reprogramming. In our recent work, we have made advancements in mapping the epigenetic alterations observed in aging and linking them to both cellular processes and disease etiology. We have identified specific age changes in mouse and human cells that reflect mitotic history, cellular senescence, oxidative damage, and mitochondrial dysfunction. We have also demonstrated that these changes inform differences in organismal lifespan and/or disease etiology at the tissue level. Overall, this work has sweeping implications for our basic understanding of epigenetic aging and reprogramming, and will help provide the foundation for potent therapeutics that extend healthspan and lifespan.

Cellular reprogramming and epigenetic rejuvenation

Ageing is an inevitable condition that afflicts all humans. Recent achievements, such as the generation of induced pluripotent stem cells, have delivered preliminary evidence that slowing down and reversing the ageing process might be possible. However, these techniques usually involve complete dedifferentiation, i.e. somatic cell identity is lost as cells are converted to a pluripotent state. Separating the rejuvenative properties of reprogramming from dedifferentiation is a promising prospect, termed epigenetic rejuvenation. Reprogramming-induced rejuvenation strategies currently involve using Yamanaka factors (typically transiently expressed to prevent full dedifferentiation) and are promising candidates to safely reduce biological age. Here, we review the development and potential of reprogramming-induced rejuvenation as an anti-ageing strategy.

Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

Comparative Analysis of Public Data Sets to Identify Stemness Markers That Differentiate Liver Cancer Stem Cells

Cancer stem cells (CSCs) represent an important field in translational medicine for generating novel cancer treatments. To identify important stemness markers in liver CSCs that potentially explain their resistance to treatment, we analyzed 10865 single-cell RNA-seq samples across 42684 coding and non-coding genes. Our results show that CSCs have significantly increased expression of two Yamanaka factors (Oct4, 2.14X and SOX2, 1.13X) and three stemness factors (CD44, 3.25X; KRT7, 2.2X; SOX9, 1.71X), relative to liver progenitor cells. Our study demonstrates the potential power of harnessing shared big data for driving translational medicine for novel hypothesis generation.

JMJD3 acts in tandem with KLF4 to facilitate reprogramming to pluripotency

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3’s catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

Development of a High-Efficacy Reprogramming Method for Generating Human Induced Pluripotent Stem (iPS) Cells from Pathologic and Senescent Somatic Cells

Induced pluripotent stem (iPS) cells are a type of artificial pluripotent stem cell induced by the epigenetic silencing of somatic cells by the Yamanaka factors. Advances in iPS cell reprogramming technology will allow aging or damaged cells to be replaced by a patient’s own rejuvenated cells. However, tissue that is senescent or pathologic has a relatively low reprogramming efficiency as compared with juvenile or robust tissue, resulting in incomplete reprogramming; iPS cells generated from such tissue types do not have sufficient differentiation ability and are therefore difficult to apply clinically. Here, we develop a new reprogramming method and examine it using myofibroblasts, which are pathologic somatic cells, from patient skin tissue and from each of the four heart chambers of a recipient heart in heart transplant surgery. By adjusting the type and amount of vectors containing transcriptional factors for iPS cell reprogramming, as well as adjusting the transfection load and culture medium, the efficiency of iPS cell induction from aged patient skin-derived fibroblasts was increased, and we successfully induced iPS cells from myocardial fibroblasts isolated from the pathologic heart of a heart transplant recipient.

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

Yamanaka reprogramming is revolutionary but inefficient, slow, and stochastic. The underlying molecular events for these mixed outcomes of induction of pluripotent stem cells (iPSC) reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming and are compromised by the fact that only a rare population proceeds towards pluripotency, and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 h, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 h, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted upreprogramming or downreprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.