Donor-to-Donor Heterogeneity in the Clonal Dynamics of Transplanted Human Cord Blood Stem Cells in Murine Xenografts.

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/104 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.

Clonal selection and asymmetric distribution of human leukemia in murine xenografts revealed by cellular barcoding.

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.

Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus.

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.

Counting stem cells: methodological constraints.

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.

Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding.

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.

microRNAs in hematopoiesis.

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.

Enhancing the detection of barcoded reads in high throughput DNA sequencing data by controlling the false discovery rate.

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.

Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells.

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.

Why an integrated view of gene expression studies on hematopoiesis in mouse aging is better than the sum of their parts.

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.

Levenshtein error-correcting barcodes for multiplexed DNA sequencing.

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.

Panhematopoietic RNA barcoding enables kinetic measurements of nucleate and anucleate lineages and the activation of myeloid clones following acute platelet depletion.

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.

Cellular barcoding tool for clonal analysis in the hematopoietic system.

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.

Expression quantitative trait loci are highly sensitive to cellular differentiation state.

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.

Measures of Clonal Hematopoiesis: Are We Missing Something?

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.

Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity.

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.

Clonal Analysis of Patient-Derived Samples Using Cellular Barcodes.

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.

Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes.

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.

Correction: Quantitative distribution of patient-derived leukemia clones in murine xenografts revealed by cellular barcodes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1.

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.

MiR-125a enhances self-renewal, lifespan, and migration of murine hematopoietic stem and progenitor cell clones.

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.