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Globally, the human population is aging, with an increased proportion of people in "old age" (over 60 years). This trend leads to a growing demand in aging research, stimulating studies in animal models such as mice, fish, and invertebrates. Recently, we published a research summary on the aging of hematopoietic stem cells (HSCs) in C57BL/6 mice based on 12 gene expression datasets. Here, I discuss in greater detail the added value of taking an integrated view, rather than considering each publication separately, to determine genes involved in aging. Considerable variation exists between lists of differentially expressed (DE) genes in HSCs, comparing young and old mice. This variation can result from factors such as inconsistent definitions of "young" and "old", technical variations and variations between laboratory mouse strains. We previously demonstrated that the variation between gene lists could be circumvented by forming a unified list of DE genes-the "aging list"-with citation indexes attached. The most frequently detected DE genes [approximately 200 most cited, which we named the "aging signature" (AS)] were highly consistent across publications. Gene Ontology classification of the AS list identified additional sources of variation between studies: one comes from the specifics of how the data are collected and analyzed; another comes from inconsistencies between how we define the gene categories. As discussed, overcoming these variations is the next challenge toward an integral approach to our systematic knowledge of the aging process., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Platelets and erythrocytes constitute over 95% of all hematopoietic stem cell output. However, the clonal dynamics of HSC contribution to these lineages remains largely unexplored., Results: We use lentiviral genetic labeling of mouse hematopoietic stem cells to quantify output from all lineages, nucleate, and anucleate, simultaneously linking these with stem and progenitor cell transcriptomic phenotypes using single-cell RNA-sequencing. We observe dynamic shifts of clonal behaviors through time in same-animal peripheral blood and demonstrate that acute platelet depletion shifts the output of multipotent hematopoietic stem cells to the exclusive production of platelets. Additionally, we observe the emergence of new myeloid-biased clones, which support short- and long-term production of blood cells., Conclusions: Our approach enables kinetic studies of multi-lineage output in the peripheral blood and transcriptional heterogeneity of individual hematopoietic stem cells. Our results give a unique insight into hematopoietic stem cell reactivation upon platelet depletion and of clonal dynamics in both steady state and under stress., (© 2023. The Author(s).)

Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)-the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate "young-like" HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs., (© 2022. The Author(s).)

Clonal Hematopoiesis (CH) is a common, age-related phenomenon of growing scientific interest, due to its association with hematologic malignancy, cardiovascular disease and decreased overall survival. CH is commonly attributed to the preferential outgrowth of a mutant hematopoietic stem cell (HSC) with enhanced fitness, resulting in clonal imbalance. In-depth understanding of the relation between HSC clonal dynamics, CH and hematologic malignancy requires integration of fundamental lineage tracing studies with clinical data. However, this is hampered by lack of a uniform definition of CH and by inconsistency in the analytical methods used for its quantification. Here, we propose a conceptual and analytical framework for the definition and measurement of CH. First, we transformed the conceptual definition of CH into the CH index, which provides a quantitative measure of clone numbers and sizes. Next, we generated a set of synthetic data, based on the beta-distribution, to simulate clonal populations with different degrees of imbalance. Using these clonal distributions and the CH index as a reference, we tested several established indices of clonal diversity and (in-)equality for their ability to detect and quantify CH. We found that the CH index was distinct from any of the other tested indices. Nonetheless, the diversity indices (Shannon, Simpson) more closely resembled the CH index than the inequality indices (Gini, Pielou). Notably, whereas the inequality indices mainly responded to changes in clone sizes, the CH index and the tested diversity indices also responded to changes in the number of clones in a sample. Accordingly, these simulations indicate that CH can result not only by skewing clonal abundancies, but also by variation in their overall numbers. Altogether, our model-based approach illustrates how a formalized definition and quantification of CH can provide insights into its pathogenesis. In the future, use of the CH index or Shannon index to quantify clonal diversity in fundamental as well as clinical clone-tracing studies will promote cross-disciplinary discussion and progress in the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bystrykh and Belderbos.)

Cellular barcoding is a relatively simple method that allows quantitative assessment of the clonal dynamics of normal, nonmalignant hematopoietic stem cells and of leukemia. Cellular barcodes are (semi-)random synthetic DNA sequences of a fixed length, which are used to uniquely mark and track cells over time. A successful barcoding experiment consists of several essential steps, including library production, transfection, transduction, barcode retrieval, and barcode data analysis. Key challenges are to obtain sufficient number of barcoded cells to conduct experiments and reliable barcode data analysis. This is especially relevant for experiments using primary leukemia cells (which are of limited availability and difficult to transduce), when studying low levels of chimerism, or when the barcoded cell population is sorted in different smaller subpopulations (e.g., lineage contribution of normal hematopoietic stem cells in murine xenografts). In these settings, retrieving accurate barcode data from low input material using standard PCR amplification techniques might be challenging and more sophisticated approaches are required. In this chapter we describe the procedures to transfect and transduce patient-derived leukemia cells, to retrieve barcoded data from both high and low input material, and to filter barcode data from sequencing noise prior to quantitative clonal analysis.

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-resistant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rγ -/- (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patient-derived xenografts had significantly fewer clones compared with placebo-treated mice (100 ± 10 vs. 160 ± 15 clones, p = 0.0005), while clonal complexity in vincristine- and dexamethasone-treated xenografts was unaffected (115 ± 33 and 150 ± 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapy-mediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Umbilical cord blood (UCB) provides an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Administration of sufficient donor HSCs is critical to restore recipient hematopoiesis and to maintain long-term polyclonal blood formation. However, due to lack of unique markers, the frequency of HSCs among UCB CD34 + cells is the subject of ongoing debate, urging for reproducible strategies for their counting. Here, we used cellular barcoding to determine the frequency and clonal dynamics of human UCB HSCs and to determine how data analysis methods affect these parameters. We transplanted lentivirally barcoded CD34 + cells from 20 UCB donors into Nod/Scid/IL2Ry -/- (NSG) mice (n = 30). Twelve recipients (of 8 UCB donors) engrafted with >1% GFP + cells, allowing for clonal analysis by multiplexed barcode deep sequencing. Using multiple definitions of clonal diversity and strategies for data filtering, we demonstrate that differences in data analysis can change clonal counts by several orders of magnitude and propose methods to improve their consistency. Using these methods, we show that the frequency of NSG-repopulating cells was low (median ∼1 HSC/10 4 CD34 + UCB cells) and could vary up to 10-fold between donors. Clonal patterns in blood became increasingly consistent over time, likely reflecting initial output of transient progenitors, followed by long-term HSCs with stable hierarchies. The majority of long-term clones displayed multilineage output, yet clones with lymphoid- or myeloid-biased output were also observed. Altogether, this study uncovers substantial interdonor and analysis-induced variability in the frequency of UCB CD34 + clones that contribute to post-transplant hematopoiesis. As clone tracing is increasingly relevant, we urge for universal and transparent methods to count HSC clones during normal aging and upon transplantation., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Expansion of hematopoietic stem cells (HSCs) is a 'holy grail' of regenerative medicine, as successful stem cell transplantations depend on the number and quality of infused HSCs. Although many attempts have been pursued to either chemically or genetically increase HSC numbers, neither clonal analysis of these expanded cells nor their ability to support mature blood lineages has been demonstrated. Here we show that miR-125a, at the single cell level, can expand murine long-term repopulating HSCs. In addition, miR-125a increases clone longevity, clone size and clonal contribution to hematopoiesis. Unexpectedly, we found that miR-125a expanded HSCs clones were highly homogenously distributed across multiple anatomical sites. Interestingly, these miR-125a overexpressing cells had enhanced mobility and were more frequently detected in the spleen. Our study reveals a novel, cell-intrinsically controlled mechanism by which HSC migration is regulated.

In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7-binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genetic and phenotypic heterogeneity of human leukemia is thought to drive leukemia progression through a Darwinian process of selection and evolution of increasingly malignant clones. However, the lack of markers that uniquely identify individual leukemia clones precludes high-resolution tracing of their clonal dynamics. Here, we use cellular barcoding to analyze the clonal behavior of patient-derived leukemia-propagating cells (LPCs) in murine xenografts. Using a leukemic cell line and diagnostic bone marrow cells from 6 patients with B-progenitor cell acute lymphoblastic leukemia, we demonstrate that patient-derived xenografts were highly polyclonal, consisting of tens to hundreds of LPC clones. The number of clones was stable within xenografts but strongly reduced upon serial transplantation. In contrast to primary recipients, in which clonal composition was highly diverse, clonal composition in serial xenografts was highly similar between recipients of the same donor and reflected donor clonality, supporting a deterministic, clone-size-based model for clonal selection. Quantitative analysis of clonal abundance in several anatomic sites identified 2 types of anatomic asymmetry. First, clones were asymmetrically distributed between different bones. Second, clonal composition in the skeleton significantly differed from extramedullary sites, showing similar numbers but different clone sizes. Altogether, this study shows that cellular barcoding and xenotransplantation providea useful model to study the behavior of patient-derived LPC clones, which provides insights relevant for experimental studies on cancer stem cells and for clinical protocols for the diagnosis and treatment of leukemia., (© 2017 by The American Society of Hematology.)

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cellular barcoding is a recently rediscovered tool to trace the clonal output of individual cells with genetically distinct and heritable DNA sequences. Each year a few dozens of papers are published using the cellular barcoding technique. Those publications largely focus on mutually related issues, namely: counting cells capable of clonal proliferation and expansion, monitoring clonal dynamics in time, tracing the origin of differentiated cells, characterizing the differentiation potential of stem cells and similar topics. Apart from their biological content, claims and conclusions, these studies show remarkable diversity in technical aspects of the barcoding method and sometimes in major conclusions. Although a diversity of approaches is quite usual in data analysis, deviant handling of barcode data might directly affect experimental results and their biological interpretation. Here, we will describe typical challenges and caveats in cellular barcoding publications available so far.

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.

Accurate monitoring of tumor dynamics and leukemic stem cell (LSC) heterogeneity is important for the development of personalized cancer therapies. In this study, we experimentally induced distinct types of leukemia in mice by enforced expression of Cbx7. Simultaneous cellular barcoding allowed for thorough analysis of leukemias at the clonal level and revealed high and unpredictable tumor complexity. Multiple LSC clones with distinct leukemic properties coexisted. Some of these clones remained dormant but bore leukemic potential, as they progressed to full-blown leukemia after challenge. LSC clones could retain multilineage differentiation capacities, where one clone induced phenotypically distinct leukemias. Beyond a detailed insight into CBX7-driven leukemic biology, our model is of general relevance for the understanding of tumor dynamics and clonal evolution., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

miRNAs have been implicated in all stages of hematopoiesis including maintenance of self-renewal of hematopoietic stem cells (HSCs) and differentiation into mature blood cells. Regulation by miRNAs is markedly intertwined with transcription factors. In this review, we highlight miRNAs shown to be important for HSC maintenance and lineage differentiation with focus on their interaction with transcription factors. We also pay attention to the diverse modes of miRNA regulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype., (Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Background: DNA barcodes are short unique sequences used to label DNA or RNA-derived samples in multiplexed deep sequencing experiments. During the demultiplexing step, barcodes must be detected and their position identified. In some cases (e.g., with PacBio SMRT), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements., Results: In our analysis, barcode sequences showed high rates of coincidental similarities with the Mus musculus reference DNA. This problem became more acute when the length of the barcode sequence decreased and the number of barcodes in the set increased. The method presented in this paper controls the tail area-based false discovery rate to distinguish between barcoded and unbarcoded reads. This method helps to establish the highest acceptable minimal distance between reads and barcode sequences. In a proof of concept experiment we correctly detected barcodes in 83% of the reads with a precision of 89%. Sensitivity improved to 99% at 99% precision when the adjacent primer sequence was incorporated in the analysis. The analysis was further improved using a paired end strategy. Following an analysis of the data for sequence variants induced in the Atp1a1 gene of C57BL/6 murine melanocytes by ultraviolet light and conferring resistance to ouabain, we found no evidence of cross-contamination of DNA material between samples., Conclusion: Our method offers a proper quantitative treatment of the problem of detecting barcoded reads in a noisy sequencing environment. It is based on the false discovery rate statistics that allows a proper trade-off between sensitivity and precision to be chosen.

Clonal tracking of hematopoietic stem and progenitor cells (HSPCs) has proven valuable for studying their behavior in murine recipients. Now in Cell Stem Cell, Kim et al. (2014) and Wu et al. (2014) extend these analyses to nonhuman primates, providing insights into dynamics of HSPC expansion and lineage commitment following autologous transplantation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make-up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long-term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols.

Cellular barcoding is a relatively recent technique aimed at clonal analysis of a proliferating cell population of any kind. The method was shown to be particularly successful in monitoring clonal contributions of hematopoietic stem cells (HSCs). An essential step of the method is retroviral or lentiviral labeling of the hematopoietic cells. The unique feature of the method is the generation of a vector library containing specific artificial DNA tags, generally known as barcodes. The library must satisfy multiple essential requirements. Importantly, considering the number of possible variations within the barcode sequence, the actual size of the barcoded vector library, and the number of clonogenic (stem) cells in the given experiment should be in ratios far from saturation. Excessive bias in barcodes frequencies must be avoided, and the library size must be assessed prior to the sequencing analysis. The final sequencing results must undergo statistical filtering. If all requirements are met, the method ensures profound sensitivity and accuracy for monitoring of the clonal fluctuations in a wide range of biological experiments.

Background: High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag or barcode that is attached to the sequencing or amplification primer and hence appears at the beginning of the sequence in every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and Levenshtein codes., Result: Levenshtein codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this paper we demonstrate the decreased error correction capability of Levenshtein codes in a DNA context and suggest an adaptation of Levenshtein codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaption we take the DNA context into account and redefine the word length whenever an insertion or deletion is revealed. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors., Conclusion: We present an adaptation of Levenshtein codes to DNA contexts capable of correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of recovering the new length of the corrupted codeword and of correcting on average more random mutations than traditional Levenshtein or Hamming codes.As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.

The number of hematopoietic stem cells (HSCs) that contributes to blood formation and the dynamics of their clonal contribution is a matter of ongoing discussion. Here, we use cellular barcoding combined with multiplex high-throughput sequencing to provide a quantitative and sensitive analysis of clonal behavior of hundreds of young and old HSCs. The majority of transplanted clones steadily contributes to hematopoiesis in the long-term, although clonal output in granulocytes, T cells, and B cells is substantially different. Contributions of individual clones to blood are dynamically changing; most of the clones either expand or decline with time. Finally, we demonstrate that the pool of old HSCs is composed of multiple small clones, whereas the young HSC pool is dominated by fewer, but larger, clones.

Background: A fragmentation of genomic DNA by restriction digestion is a popular step in many applications. Usually attention is paid to the expected average size of the DNA fragments. Another important parameter, randomness of restriction, is regularly implied but rarely verified. This parameter is crucial to the expectation, that either all fragments made by restriction will be suitable for the method of choice, or only a fraction of those will be effectively used by the method. If only a fraction of the fragments are used, we often should know whether the used fragments are representative of the whole genome. With a modern knowledge of mouse, human and many other genomes, frequencies and distributions of restriction sites and sizes of corresponding DNA fragments can be analyzed in silico. In this manuscript, the mouse genome was systematically scanned for frequencies of complementary 4-base long palindromes., Findings and Conclusions: The study revealed substantial heterogeneity in distribution of those sites genome-wide. Only few palindromes showed close to random pattern of distribution. Overall, the distribution of frequencies for most palindromes is much wider than expected by random occurrence. In practical terms, accessibility of genome upon restriction can be improved by a selective combination of restrictases using a few combinatorial rules. It is recommended to mix at least 3 restrictases, their recognition sequences (palindrome) should be the least similar to each other. Principles of the optimization and optimal combinations of restrictases are provided.

Histone deacetylase inhibitors (HDIs) have been shown to enhance hematopoietic stem and progenitor cell activity and improve stem cell outcomes after ex vivo culture. Identification of gene targets of HDIs is required to understand the full potential of these compounds and can allow for improved stem cell culturing protocols. The molecular process that underlies the biological effects of valproic acid (VPA), a widely used HDI, on hematopoietic stem/progenitor cells was investigated by studying the early-response genes of VPA. These genes were linked to VPA-induced enhancement of cell function as measured by in vitro assays. Genome-wide gene expression studies revealed over-representation of genes involved in glutathione metabolism, receptor and signal transducer activity, and changes in the hematopoietic stem/progenitor cells surface profile after short, 24-hour VPA treatment. Sca-1, a well-known and widely used stem cell surface marker, was identified as a prominent VPA target. We showed that multiple HDIs induce Sca-1 expression on hematopoietic cells. VPA strongly preserved Sca-1 expression on Lin(-)Sca1(+)ckit(+) cells, but also reactivated Sca-1 on committed progenitor cells that were Sca-1(neg), thereby reverting them to the Lin(-)Sca1(+)ckit(+) phenotype. We demonstrated that reacquired Sca-1 expression coincided with induced self-renewal capacity as measured by in vitro replating assays, while Sca-1 itself was not required for the biological effects of VPA as demonstrated using Sca-1-deficient progenitor cells. In conclusion, our results show that VPA modulates several genes involved in multiple signal transduction pathways, of which Sca-1 was shown to mark cells with increased self-renewal capacity in response to HDIs., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The number of stem cells contributing to hematopoiesis has been a matter of debate. Many studies use retroviral tagging of stem cells to measure clonal contribution. Here we argue that methodological factors can impact such clonal analyses. Whereas early studies had low resolution, leading to underestimation, recent methods may result in an overestimation of stem-cell counts. We discuss how restriction enzyme choice, PCR bias, high-throughput sequencing depth and tagging method could affect the conclusions of clonal studies.

Hematopoietic stem/progenitor cell (HSPC) traits differ between genetically distinct mouse strains. For example, DBA/2 mice have a higher HSPC frequency compared with C57BL/6 mice. We performed a genetic screen for micro-RNAs that are differentially expressed between LSK, LS(-)K(+), erythroid and myeloid cells isolated from C57BL/6 and DBA/2 mice. This analysis identified 131 micro-RNAs that were differentially expressed between cell types and 15 that were differentially expressed between mouse strains. Of special interest was an evolutionary conserved miR cluster located on chromosome 17 consisting of miR-99b, let-7e, and miR-125a. All cluster members were most highly expressed in LSKs and down-regulated upon differentiation. In addition, these microRNAs were higher expressed in DBA/2 cells compared with C57BL/6 cells, and thus correlated with HSPC frequency. To functionally characterize these microRNAs, we overexpressed the entire miR-cluster 99b/let-7e/125a and miR-125a alone in BM cells from C57BL/6 mice. Overexpression of the miR-cluster or miR-125a dramatically increased day-35 CAFC activity and caused severe hematopoietic phenotypes upon transplantation. We showed that a single member of the miR-cluster, namely miR-125a, is responsible for the majority of the observed miR-cluster overexpression effects. Finally, we performed genome-wide gene expression arrays and identified candidate target genes through which miR-125a may modulate HSPC fate.

The diversity and scope of multiplex parallel sequencing applications is steadily increasing. Critically, multiplex parallel sequencing applications methods rely on the use of barcoded primers for sample identification, and the quality of the barcodes directly impacts the quality of the resulting sequence data. Inspection of the recent publications reveals a surprisingly variable quality of the barcodes employed. Some barcodes are made in a semi empirical fashion, without quantitative consideration of error correction or minimal distance properties. After systematic comparison of published barcode sets, including commercially distributed barcoded primers from Illumina and Epicentre, methods for improved, Hamming code-based sequences are suggested and illustrated. Hamming barcodes can be employed for DNA tag designs in many different ways while preserving minimal distance and error-correcting properties. In addition, Hamming barcodes remain flexible with regard to essential biological parameters such as sequence redundancy and GC content. Wider adoption of improved Hamming barcodes is encouraged in multiplex parallel sequencing applications.

Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.

Genetical genomics is a strategy for mapping gene expression variation to expression quantitative trait loci (eQTLs). We performed a genetical genomics experiment in four functionally distinct but developmentally closely related hematopoietic cell populations isolated from the BXD panel of recombinant inbred mouse strains. This analysis allowed us to analyze eQTL robustness/sensitivity across different cellular differentiation states. Although we identified a large number (365) of "static" eQTLs that were consistently active in all four cell types, we found a much larger number (1,283) of "dynamic" eQTLs showing cell-type-dependence. Of these, 140, 45, 531, and 295 were preferentially active in stem, progenitor, erythroid, and myeloid cells, respectively. A detailed investigation of those dynamic eQTLs showed that in many cases the eQTL specificity was associated with expression changes in the target gene. We found no evidence for target genes that were regulated by distinct eQTLs in different cell types, suggesting that large-scale changes within functional regulatory networks are uncommon. Our results demonstrate that heritable differences in gene expression are highly sensitive to the developmental stage of the cell population under study. Therefore, future genetical genomics studies should aim at studying multiple well-defined and highly purified cell types in order to construct as comprehensive a picture of the changing functional regulatory relationships as possible., Competing Interests: The authors have declared that no competing interests exist.

Background: The Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases., Results: Applying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data., Conclusion: The accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used.

Several studies have suggested that the cyclin-dependent kinase (CDK) inhibitor p21 plays a crucial role in regulating hematopoietic stem and progenitor pool size. To allow assessment of long-term stem cell functioning in vivo, we have backcrossed a p21 null allele to C57BL/6 (B6) mice, the most commonly used mouse strain in hematopoietic stem cell research. In various in vitro assays, the homozygous deletion of the p21 allele did not affect the number of hematopoietic cells in B6 mice. Furthermore, the competitive repopulation ability was not different between p21-deficient and wild-type stem cells from both young and aged (20-month-old) mice. These results show that p21 is not essential for regulation of stem cell number in steady state. When proliferative stress was applied on p21-deficient stem cells by serial transplantation of 1,500 Lin(-)Sca-1(+)c-kit(+) (LSK) cells, again no detrimental effect was observed on cobblestone area-forming cell (CAFC) frequency and competitive repopulating ability. However, when bone marrow cells from mice that received 2 Gy of irradiation were transplanted, p21 deficiency resulted in a more than fourfold reduction in competitive repopulation index. Finally, we did not find major differences in cell cycle status and global gene expression patterns between LSK cells from p21-deficient and wild-type mice. Our findings indicate that the background of mice used for studying the function of a gene by genetic modification may determine the outcome. Cumulatively, our data fail to support the notion that p21 is essential for stem cell function during steady-state hematopoiesis, but may be relatively more important under conditions of cellular stress.

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.

Short-oligonucleotide arrays typically contain multiple probes per gene. In genetical genomics applications a statistical model for the individual probe signals can help in separating "true" differential mRNA expression from "ghost" effects caused by polymorphisms, misdesigned probes, and batch effects. It can also help in detecting alternative splicing, start, or termination.

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.

Hematopoietic stem cells from different strains of mice vary widely with respect to their cell cycle activity. In the present study we used complementary genetic and genomic approaches to identify molecular pathways affecting this complex trait. We identified a major quantitative trait locus (QTL) associated with variation in cell proliferation in C57BL/6 and DBA/2 mice to a 10 centimorgan (cM) region on chromosome 11. A congenic mouse model confirmed that a genomic interval on chromosome 11 in isolation confers the proliferation phenotype. To detect candidate genes we performed subtractive hybridizations and gene arrays using cDNA from highly enriched stem cells from parental strains. Intriguingly, a disproportionate number of differentially expressed genes mapped to chromosome 11 and, more specifically, these transcripts occurred in 3 distinct clusters. The largest cluster colocalized exactly with the cell cycling QTL. Such clustering suggested the involvement of genetic variation that affects higher-order chromosomal organization. This hypothesis was reinforced by the fact that differentially expressed genes mapped to recombination "coldspots," as a consequence of which clustered genes are collectively inherited. These findings suggest the functional interdependence of these closely linked genes. Our data are consistent with the hypothesis that this isolated cell cycle QTL does not result from a mutation in a single gene but rather is a consequence of variable expression of a collection of highly linked genes.

Tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) of Amycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD-ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MNO), a decameric protein with 50-kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (> 640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazolium-dye-linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5'-deazaflavin and at least one other low-molecular-mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD-ADH complex is carried out by component 1 (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5'-deazaflavin was present in component 3. Other gram-positive bacteria having a TD-ADH complex also produced 5'-deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. methanolica, and probably also by other gram-positive bacteria containing MNO or TD-ADH, proceeds via TD-ADH. The likeliness of 5'-deazaflavin participation in this process is discussed.

Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50,000-Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

Cell-free extracts of methanol-grown Amycolatopsis methanolica contain dye-linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate-ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were found for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. (1987) Eur. J. Biochem. 166, 575-579] which was at that time considered to be an aldehyde dehydrogenase. Formate-ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and CO2 or a factor-formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Recent advances in biotechnology of methanol-utilizing yeasts are briefly summarized. The emphasis is given to production of some fine and commercial chemicals such as formaldehyde, formate, hydrogen peroxide, dihydroxyacetone, ATP, FAD as well as proteins, specifically alcohol oxidase. The advantages of mutants and recombinants derived from methylotrophic yeasts for efficient production of various useful materials are demonstrated.