Genomic Variability within an Organism Exposes Its Cell Lineage Tree

What is the lineage relation among the cells of an organism? The answer is sought by developmental biology, immunology, stem cell research, brain research, and cancer research, yet complete cell lineage trees have been reconstructed only for simple organisms such as Caenorhabditis elegans. We discovered that somatic mutations accumulated during normal development of a higher organism implicitly encode its entire cell lineage tree with very high precision. Our mathematical analysis of known mutation rates in microsatellites (MSs) shows that the entire cell lineage tree of a human embryo, or a mouse, in which no cell is a descendent of more than 40 divisions, can be reconstructed from information on somatic MS mutations alone with no errors, with probability greater than 99.95%. Analyzing all ~1.5 million MSs of each cell of an organism may not be practical at present, but we also show that in a genetically unstable organism, analyzing only a few hundred MSs may suffice to reconstruct portions of its cell lineage tree. We demonstrate the utility of the approach by reconstructing cell lineage trees from DNA samples of a human cell line displaying MS instability. Our discovery and its associated procedure, which we have automated, may point the way to a future “Human Cell Lineage Project” that would aim to resolve fundamental open questions in biology and medicine by reconstructing ever larger portions of the human cell lineage tree

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations

Methods for lineage tracing on the organism-wide level

Determining the lineage origin of cell types is a major goal in developmental biology. Furthermore, lineage tracing is a powerful approach for understanding the origin of developmental defects as well as the origin of diseases such as cancer. There is now a variety of complementary approaches for identifying lineage relationships, ranging from direct observation of cell divisions by light microscopy to genetic labeling of cells using inducible recombinases and fluorescent reporters. A recent development, and the main topic of this review article, is the use of high-throughput sequencing data for lineage analysis. This emerging approach holds the promise of increased multiplexing capacity, allowing lineage analysis of large cell numbers up to the organism-wide level combined with simultaneous transcription profiling by single cell RNA sequencing

Estimating Cell Depth from Somatic Mutations

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions

Lack of increases in methylation at three CpG-rich genomic loci in non-mitotic adult tissues during aging

<p>Abstract</p> <p>Background</p> <p>Cell division occurs during normal human development and aging. Despite the likely importance of cell division to human pathology, it has been difficult to infer somatic cell mitotic ages (total numbers of divisions since the zygote) because direct counting of lifetime numbers of divisions is currently impractical. Here we attempt to infer relative mitotic ages with a molecular clock hypothesis. Somatic genomes may record their mitotic ages because greater numbers of replication errors should accumulate after greater numbers of divisions. Mitotic ages will vary between cell types if they divide at different times and rates.</p> <p>Methods</p> <p>Age-related increases in DNA methylation at specific CpG sites (termed "epigenetic molecular clocks") have been previously observed in mitotic human epithelium like the intestines and endometrium. These CpG rich sequences or "tags" start unmethylated and potentially changes in methylation during development and aging represent replication errors. To help distinguish between mitotic versus time-associated changes, DNA methylation tag patterns at 8–20 CpGs within three different genes, two on autosomes and one on the X-chromosome were measured by bisulfite sequencing from heart, brain, kidney and liver of autopsies from 21 individuals of different ages.</p> <p>Results</p> <p>Levels of DNA methylation were significantly greater in adult compared to fetal or newborn tissues for two of the three examined tags. Consistent with the relative absence of cell division in these adult tissues, there were no significant increases in tag methylation after infancy.</p> <p>Conclusion</p> <p>Many somatic methylation changes at certain CpG rich regions or tags appear to represent replication errors because this methylation increases with chronological age in mitotic epithelium but not in non-mitotic organs. Tag methylation accumulates differently in different tissues, consistent with their expected genealogies and mitotic ages. Although further studies are necessary, these results suggest numbers of divisions and ancestry are at least partially recorded by epigenetic replication errors within somatic cell genomes.</p

Muscle-Bound Primordial Stem Cells Give Rise to Myofiber-Associated Myogenic and Non-Myogenic Progenitors

Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density

Cell Lineage Analysis of the Mammalian Female Germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development

Origins of lymphatic and distant metastases in human colorectal cancer

The spread of cancer cells from primary tumors to regional lymph nodes is often associated with reduced survival. One prevailing model to explain this association posits that fatal, distant metastases are seeded by lymph node metastases. This view provides a mechanistic basis for the TNM staging system and is the rationale for surgical resection of tumor-draining lymph nodes. Here we examine the evolutionary relationship between primary tumor, lymph node, and distant metastases in human colorectal cancer. Studying 213 archival biopsy samples from 17 patients, we used somatic variants in hypermutable DNA regions to reconstruct high-confidence phylogenetic trees. We found that in 65% of cases, lymphatic and distant metastases arose from independent subclones in the primary tumor, whereas in 35% of cases they shared common subclonal origin. Therefore, two different lineage relationships between lymphatic and distant metastases exist in colorectal cancer

Reconstruction of Cell Lineage Trees in Mice

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance

Variations in brain DNA

It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain) of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.This study was supported by grants from the BBVA Foundation and MICINN-MINECO.Peer reviewedPeer Reviewe