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.

Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood.

Maternal inflammation during pregnancy can have detrimental effects on embryonic development that persist during adulthood. However, the underlying mechanisms and insights in the responsible cell types are still largely unknown. Here we report the effect of maternal inflammation on fetal microglia, the innate immune cells of the central nervous system (CNS). In mice, a challenge with LPS during late gestation stages (days 15-16-17) induced a pro-inflammatory response in fetal microglia. Adult whole brain microglia of mice that were exposed to LPS during embryonic development displayed a persistent reduction in pro-inflammatory activation in response to a re-challenge with LPS. In contrast, hippocampal microglia of these mice displayed an increased inflammatory response to an LPS re-challenge. In addition, a reduced expression of brain-derived neurotrophic factor (BDNF) was observed in hippocampal microglia of LPS-offspring. Microglia-derived BDNF has been shown to be important for learning and memory processes. In line with these observations, behavioral- and learning tasks with mice that were exposed to maternal inflammation revealed reduced home cage activity, reduced anxiety and reduced learning performance in a T-maze. These data show that exposure to maternal inflammation during late gestation results in long term changes in microglia responsiveness during adulthood, which is different in nature in hippocampus compared to total brain microglia.

Sox2 Sustains Recruitment of Oligodendrocyte Progenitor Cells following CNS Demyelination and Primes Them for Differentiation during Remyelination.

The Sox family of transcription factors have been widely studied in the context of oligodendrocyte development. However, comparatively little is known about the role of Sox2, especially during CNS remyelination. Here we show that the expression of Sox2 occurs in oligodendrocyte progenitor cells (OPCs) in rodent models during myelination and in activated adult OPCs responding to demyelination, and is also detected in multiple sclerosis lesions. In normal adult white matter of both mice and rats, it is neither expressed by adult OPCs nor by oligodendrocytes (although it is expressed by a subpopulation of adult astrocytes). Overexpression of Sox2 in rat OPCs in vitro maintains the cells in a proliferative state and inhibits differentiation, while Sox2 knockout results in decreased OPC proliferation and survival, suggesting that Sox2 contributes to the expansion of OPCs during the recruitment phase of remyelination. Loss of function in cultured mouse OPCs also results in an impaired ability to undergo normal differentiation in response to differentiation signals, suggesting that Sox2 expression in activated OPCs also primes these cells to eventually undergo differentiation. In vivo studies on remyelination following experimental toxin-induced demyelination in mice with inducible loss of Sox2 revealed impaired remyelination, which was largely due to a profound attenuation of OPC recruitment and likely also due to impaired differentiation. Our results reveal a key role of Sox2 expression in OPCs responding to demyelination, enabling them to effectively contribute to remyelination. SIGNIFICANCE STATEMENT: Understanding the mechanisms of CNS remyelination is central to developing effective means by which this process can be therapeutically enhanced in chronic demyelinating diseases such as multiple sclerosis. In this study, we describe the role of Sox2, a transcription factor widely implicated in stem cell biology, in CNS myelination and remyelination. We show how Sox2 is expressed in oligodendrocyte progenitor cells (OPCs) preparing to undergo differentiation, allowing them to undergo proliferation and priming them for subsequent differentiation. Although Sox2 is unlikely to be a direct therapeutic target, these data nevertheless provide more information on how OPC differentiation is controlled and therefore enriches our understanding of this important CNS regenerative process.

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.