Methods and Strategies for Procurement, Isolation, Characterization, and Assessment of Senescence of Human Mesenchymal Stem Cells from Adipose Tissue.

Human adipose-derived mesenchymal stem (stromal) cells (hADSC) represent an attractive source of the cells for numerous therapeutic applications in regenerative medicine. These cells are also an efficient model to study biological pathways of stem cell action, tissue injury and disease. Like any other primary somatic cells in culture, industrial-scale expansion of mesenchymal stromal cells (MSC) leads to the replicative exhaustion/senescence as defined by the "Hayflick limit." The senescence is not only greatly effecting in vivo potency of the stem cell cultures but also might be the cause and the source of clinical inconsistency arising from infused cell preparations. In this light, the characterization of hADSC replicative and stressor-induced senescence phenotypes is of great interest.This chapter summarizes some of the essential protocols and assays used at our laboratories and clinic for the human fat procurement, isolation, culture, differentiation, and characterization of mesenchymal stem cells from adipose tissue and the stromal vascular fraction. Additionally, we provide manuals for characterization of hADSC senescence in a culture based on stem cells immunophenotype, proliferation rate, migration potential, and numerous other well-accepted markers of cellular senescence. Such methodological framework will be immensely helpful to design standards and surrogate measures for hADSC-based therapeutic applications.

Piwi Is Required to Limit Exhaustion of Aging Somatic Stem Cells.

Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance.

Adipose stem cells from obese patients show specific differences in the metabolic regulators vitamin D and Gas5.

Adipose tissue is a significant source of mesenchymal stem cells for regenerative therapies; however, caution should be taken as their environmental niche can affect their functional properties. We have previously demonstrated the negative impact of obesity on the function of adipose-derived stem cells (ASCs). Here we have evaluated other possible properties and targets that are altered by obesity such as the recently described long non-coding molecule Gas5, which is involved in glucocorticoid resistance. Using ASCs isolated from obese (oASCs) and control subjects (cASCs), we have analyzed additional metabolic and inflammatory conditions that could be related with their impaired therapeutic potential and consequently their possible usefulness in the clinic.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Mesenchymal Stem Cells from Adipose Tissue in Clinical Applications for Dermatological Indications and Skin Aging.

Operating at multiple levels of control, mesenchymal stem cells from adipose tissue (ADSCs) communicate with organ systems to adjust immune response, provide signals for differentiation, migration, enzymatic reactions, and to equilibrate the regenerative demands of balanced tissue homeostasis. The identification of the mechanisms by which ADSCs accomplish these functions for dermatological rejuvenation and wound healing has great potential to identify novel targets for the treatment of disorders and combat aging. Herein, we review new insights into the role of adipose-derived stem cells in the maintenance of dermal and epidermal homeostasis, and recent advances in clinical applications of ADSCs related to dermatology.

Mesenchymal Stem Cells Secretory Responses: Senescence Messaging Secretome and Immunomodulation Perspective.

Mesenchymal stem/stromal cells (MSC) have been tested in a significant number of clinical trials, where they exhibit regenerative and repair properties directly through their differentiation into the cells of the mesenchymal origin or by modulation of the tissue/organ microenvironment. Despite various clinical effects upon transplantation, the functional properties of these cells in natural settings and their role in tissue regeneration in vivo is not yet fully understood. The omnipresence of MSC throughout vascularized organs equates to a reservoir of potentially therapeutic regenerative depots throughout the body. However, these reservoirs could be subjected to cellular senescence. In this review, we will discuss current progress and challenges in the understanding of different biological pathways leading to senescence. We set out to highlight the seemingly paradoxical property of cellular senescence: its beneficial role in the development and tissue repair and detrimental impact of this process on tissue homeostasis in aging and disease. Taking into account the lessons from the different cell systems, this review elucidates how autocrine and paracrine properties of senescent MSC might impose an additional layer of complexity on the regulation of the immune system in development and disease. New findings that have emerged in the last few years could shed light on sometimes seemingly controversial results obtained from MSC therapeutic applications.

Transcriptional profiling of interleukin-2-primed human adipose derived mesenchymal stem cells revealed dramatic changes in stem cells response imposed by replicative senescence.

Inflammation is a double-edged sword with both detrimental and beneficial consequences. Understanding of the mechanisms of crosstalk between the inflammatory milieu and human adult mesenchymal stem cells is an important basis for clinical efforts. Here, we investigate changes in the transcriptional response of human adipose-derived stem cells to physiologically relevant levels of IL-2 (IL-2 priming) upon replicative senescence. Our data suggest that replicative senescence might dramatically impede human mesenchymal stem cell (MSC) function via global transcriptional deregulation in response to IL-2. We uncovered a novel senescence-associated transcriptional signature in human adipose-derived MSCs hADSCs after exposure to pro-inflammatory environment: significant enhancement of the expression of the genes encoding potent growth factors and cytokines with anti-inflammatory and migration-promoting properties, as well as genes encoding angiogenic and anti-apoptotic promoting factors, all of which could participate in the establishment of a unique microenvironment. We observed transcriptional up-regulation of critical components of the nitric oxide synthase pathway (iNOS) in hADSCs upon replicative senescence suggesting, that senescent stem cells can acquire metastasis-promoting properties via stem cell-mediated immunosuppression. Our study highlights the importance of age as a factor when designing cell-based or pharmacological therapies for older patients and predicts measurable biomarkers characteristic of an environment that is conducive to cancer cells invasiveness and metastasis.

MIR retrotransposon sequences provide insulators to the human genome.

Insulators are regulatory elements that help to organize eukaryotic chromatin via enhancer-blocking and chromatin barrier activity. Although there are several examples of transposable element (TE)-derived insulators, the contribution of TEs to human insulators has not been systematically explored. Mammalian-wide interspersed repeats (MIRs) are a conserved family of TEs that have substantial regulatory capacity and share sequence characteristics with tRNA-related insulators. We sought to evaluate whether MIRs can serve as insulators in the human genome. We applied a bioinformatic screen using genome sequence and functional genomic data from CD4(+) T cells to identify a set of 1,178 predicted MIR insulators genome-wide. These predicted MIR insulators were computationally tested to serve as chromatin barriers and regulators of gene expression in CD4(+) T cells. The activity of predicted MIR insulators was experimentally validated using in vitro and in vivo enhancer-blocking assays. MIR insulators are enriched around genes of the T-cell receptor pathway and reside at T-cell-specific boundaries of repressive and active chromatin. A total of 58% of the MIR insulators predicted here show evidence of T-cell-specific chromatin barrier and gene regulatory activity. MIR insulators appear to be CCCTC-binding factor (CTCF) independent and show a distinct local chromatin environment with marked peaks for RNA Pol III and a number of histone modifications, suggesting that MIR insulators recruit transcriptional complexes and chromatin modifying enzymes in situ to help establish chromatin and regulatory domains in the human genome. The provisioning of insulators by MIRs across the human genome suggests a specific mechanism by which TE sequences can be used to modulate gene regulatory networks.

Mammalian-wide interspersed repeat (MIR)-derived enhancers and the regulation of human gene expression.

BACKGROUND: Mammalian-wide interspersed repeats (MIRs) are the most ancient family of transposable elements (TEs) in the human genome. The deep conservation of MIRs initially suggested the possibility that they had been exapted to play functional roles for their host genomes. MIRs also happen to be the only TEs whose presence in-and-around human genes is positively correlated to tissue-specific gene expression. Similar associations of enhancer prevalence within genes and tissue-specific expression, along with MIRs' previous implication as providing regulatory sequences, suggested a possible link between MIRs and enhancers. RESULTS: To test the possibility that MIRs contribute functional enhancers to the human genome, we evaluated the relationship between MIRs and human tissue-specific enhancers in terms of genomic location, chromatin environment, regulatory function, and mechanistic attributes. This analysis revealed MIRs to be highly concentrated in enhancers of the K562 and HeLa human cell-types. Significantly more enhancers were found to be linked to MIRs than would be expected by chance, and putative MIR-derived enhancers are characterized by a chromatin environment highly similar to that of canonical enhancers. MIR-derived enhancers show strong associations with gene expression levels, tissue-specific gene expression and tissue-specific cellular functions, including a number of biological processes related to erythropoiesis. MIR-derived enhancers were found to be a rich source of transcription factor binding sites, underscoring one possible mechanistic route for the element sequences co-option as enhancers. There is also tentative evidence to suggest that MIR-enhancer function is related to the transcriptional activity of non-coding RNAs. CONCLUSIONS: Taken together, these data reveal enhancers to be an important cis-regulatory platform from which MIRs can exercise a regulatory function in the human genome and help to resolve a long-standing conundrum as to the reason for MIRs' deep evolutionary conservation.

Compound cis-regulatory elements with both boundary and enhancer sequences in the human genome.

MOTIVATION: It has been suggested that presumably distinct classes of genomic regulatory elements may actually share common sets of features and mechanisms. However, there has been no genome-wide assessment of the prevalence of this phenomenon. RESULTS: To evaluate this possibility, we performed a bioinformatic screen for the existence of compound regulatory elements in the human genome. We identified numerous such colocated boundary and enhancer elements from human CD4(+) T cells. We report evidence that such compound regulatory elements possess unique chromatin features and facilitate cell type-specific functions related to inflammation and immune response in CD4(+) T cells.

Epigenetics components of aging in the central nervous system.

This review highlights recent discoveries that have shaped the emerging viewpoints in the field of epigenetic influences in the central nervous system (CNS), focusing on the following questions: (i) How is the CNS shaped during development when precursor cells transition into morphologically and molecularly distinct cell types, and is this event driven by epigenetic alterations?; ii) How do epigenetic pathways control CNS function?; (iii) What happens to "epigenetic memory" during aging processes, and do these alterations cause CNS dysfunction?; (iv) Can one restore normal CNS function by manipulating the epigenome using pharmacologic agents, and will this ameliorate aging-related neurodegeneration? These and other still unanswered questions remain critical to understanding the impact of multifaceted epigenetic machinery on the age-related dysfunction of CNS.

BroadPeak: a novel algorithm for identifying broad peaks in diffuse ChIP-seq datasets.

SUMMARY: Although some histone modification chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) signals show abrupt peaks across narrow and specific genomic locations, others have diffuse distributions along chromosomes, and their large contiguous enrichment landscapes are better modeled as broad peaks. Here, we present BroadPeak, an algorithm for the identification of such broad peaks from diffuse ChIP-seq datasets. We show that BroadPeak is a linear time algorithm that requires only two parameters, and we validate its performance on real and simulated histone modification ChIP-seq datasets. BroadPeak calls peaks that are highly coincident with both the underlying ChIP-seq tag count distributions and relevant biological features, such as the gene bodies of actively transcribed genes, and it shows superior overall recall and precision of known broad peaks from simulated datasets. AVAILABILITY: The source code and documentations are available at http://jordan.biology.gatech.edu/page/software/broadpeak/.

Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells.

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.

Chromatin signature discovery via histone modification profile alignments.

We report on the development of an unsupervised algorithm for the genome-wide discovery and analysis of chromatin signatures. Our Chromatin-profile Alignment followed by Tree-clustering algorithm (ChAT) employs dynamic programming of combinatorial histone modification profiles to identify locally similar chromatin sub-regions and provides complementary utility with respect to existing methods. We applied ChAT to genomic maps of 39 histone modifications in human CD4(+) T cells to identify both known and novel chromatin signatures. ChAT was able to detect chromatin signatures previously associated with transcription start sites and enhancers as well as novel signatures associated with a variety of regulatory elements. Promoter-associated signatures discovered with ChAT indicate that complex chromatin signatures, made up of numerous co-located histone modifications, facilitate cell-type specific gene expression. The discovery of novel L1 retrotransposon-associated bivalent chromatin signatures suggests that these elements influence the mono-allelic expression of human genes by shaping the chromatin environment of imprinted genomic regions. Analysis of long gene-associated chromatin signatures point to a role for the H4K20me1 and H3K79me3 histone modifications in transcriptional pause release. The novel chromatin signatures and functional associations uncovered by ChAT underscore the ability of the algorithm to yield novel insight on chromatin-based regulatory mechanisms.

Epigenetics: judge, jury and executioner of stem cell fate.

Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This "epigenetic landscape" governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases.

Do human transposable element small RNAs serve primarily as genome defenders or genome regulators?

It is currently thought that small RNA (sRNA) based repression mechanisms are primarily employed to mitigate the mutagenic threat posed by the activity of transposable elements (TEs). This can be achieved by the sRNA guided processing of TE transcripts via Dicer-dependent (e.g., siRNA) or Dicer-independent (e.g., piRNA) mechanisms. For example, potentially active human L1 elements are silenced by mRNA cleavage induced by element encoded siRNAs, leading to a negative correlation between element mRNA and siRNA levels. On the other hand, there is emerging evidence that TE derived sRNAs can also be used to regulate the host genome. Here, we evaluated these two hypotheses for human TEs by comparing the levels of TE derived mRNA and TE sRNA across six tissues. The genome defense hypothesis predicts a negative correlation between TE mRNA and TE sRNA levels, whereas the genome regulatory hypothesis predicts a positive correlation. On average, TE mRNA and TE sRNA levels are positively correlated across human tissues. These correlations are higher than seen for human genes or for randomly permuted control data sets. Overall, Alu subfamilies show the highest positive correlations of element mRNA and sRNA levels across tissues, although a few of the youngest, and potentially most active, Alu subfamilies do show negative correlations. Thus, Alu derived sRNAs may be related to both genome regulation and genome defense. These results are inconsistent with a simple model whereby TE derived sRNAs reduce levels of standing TE mRNA via transcript cleavage, and suggest that human cells efficiently process TE transcripts into sRNA based on the available message levels. This may point to a widespread role for processed TE transcripts in genome regulation or to alternative roles of TE-to-sRNA processing including the mitigation of TE transcript cytotoxicity.

Protein interactions with piALU RNA indicates putative participation of retroRNA in the cell cycle, DNA repair and chromatin assembly.

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.

On the presence and role of human gene-body DNA methylation.

DNA methylation of promoter sequences is a repressive epigenetic mark that down-regulates gene expression. However, DNA methylation is more prevalent within gene-bodies than seen for promoters, and gene-body methylation has been observed to be positively correlated with gene expression levels. This paradox remains unexplained, and accordingly the role of DNA methylation in gene-bodies is poorly understood. We addressed the presence and role of human gene-body DNA methylation using a meta-analysis of human genome-wide methylation, expression and chromatin data sets. Methylation is associated with transcribed regions as genic sequences have higher levels of methylation than intergenic or promoter sequences. We also find that the relationship between gene-body DNA methylation and expression levels is non-monotonic and bell-shaped. Mid-level expressed genes have the highest levels of gene-body methylation, whereas the most lowly and highly expressed sets of genes both have low levels of methylation. While gene-body methylation can be seen to efficiently repress the initiation of intragenic transcription, the vast majority of methylated sites within genes are not associated with intragenic promoters. In fact, highly expressed genes initiate the most intragenic transcription inconsistent with the previously held notion that gene-body methylation serves to repress spurious intragenic transcription to allow for efficient transcriptional elongation. These observations lead us to propose a model to explain the presence of human gene-body methylation. This model holds that the repression of intragenic transcription by gene-body methylation is largely epiphenomenal, and suggests that gene-body methylation levels are predominantly shaped via the accessibility of the DNA to methylating enzyme complexes.

Genome-wide prediction and analysis of human chromatin boundary elements.

Boundary elements partition eukaryotic chromatin into active and repressive domains, and can also block regulatory interactions between domains. Boundary elements act via diverse mechanisms making accurate feature-based computational predictions difficult. Therefore, we developed an unbiased algorithm that predicts the locations of human boundary elements based on the genomic distributions of chromatin and transcriptional states, as opposed to any intrinsic characteristics that they may possess. Application of our algorithm to ChIP-seq data for histone modifications and RNA Pol II-binding data in human CD4(+) T cells resulted in the prediction of 2542 putative chromatin boundary elements genome wide. Predicted boundary elements display two distinct features: first, position-specific open chromatin and histone acetylation that is coincident with the recruitment of sequence-specific DNA-binding factors such as CTCF, EVI1 and YYI, and second, a directional and gradual increase in histone lysine methylation across predicted boundaries coincident with a gain of expression of non-coding RNAs, including examples of boundaries encoded by tRNA and other non-coding RNA genes. Accordingly, a number of the predicted human boundaries may function via the synergistic action of sequence-specific recruitment of transcription factors leading to non-coding RNA transcriptional interference and the blocking of facultative heterochromatin propagation by transcription-associated chromatin remodeling complexes.

Adult stem cells: simply a tool for regenerative medicine or an additional piece in the puzzle of human aging?

Adult stem cells have taken center stage in current research related to regenerative medicine and pharmacogenomic studies seeking new therapeutic interventions. As we learn more about these cells, it is becoming apparent that the next big leap in our understanding of adult stem cell biology and adult stem cell aging will depend on the integration of approaches from various disciplines. Major advances and technological breakthroughs at the crossroad of fields such as biomaterials, genomics, epigenomics, and proteomics will enable the design of better tools to model human diseases, and warrant safe usage of adult stem cells in the clinic.