The developmental stage of the medulloblastoma cell-of-origin restricts Sonic hedgehog pathway usage and drug sensitivity.

Sonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.

A comprehensive transcriptome signature of murine hematopoietic stem cell aging.

We surveyed 16 published and unpublished data sets to determine whether a consistent pattern of transcriptional deregulation in aging murine hematopoietic stem cells (HSC) exists. Despite substantial heterogeneity between individual studies, we uncovered a core and robust HSC aging signature. We detected increased transcriptional activation in aged HSCs, further confirmed by chromatin accessibility analysis. Unexpectedly, using 2 independent computational approaches, we established that deregulated aging genes consist largely of membrane-associated transcripts, including many cell surface molecules previously not associated with HSC biology. We show that Selp (P-selectin), the most consistent deregulated gene, is not merely a marker for aged HSCs but is associated with HSC functional decline. Additionally, single-cell transcriptomics analysis revealed increased heterogeneity of the aged HSC pool. We identify the presence of transcriptionally "young-like" HSCs in aged bone marrow. We share our results as an online resource and demonstrate its utility by confirming that exposure to sympathomimetics or deletion of Dnmt3a/b molecularly resembles HSC rejuvenation or aging, respectively.

Mouse parotid salivary gland organoids for the in vitro study of stem cell radiation response.

OBJECTIVE: Hyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro. MATERIALS AND METHODS: Single cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response. RESULTS: Single cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland. CONCLUSIONS: Murine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.

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.

Aging of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) ensure a balanced production of all blood cells throughout life. As they age, HSCs gradually lose their self-renewal and regenerative potential, whereas the occurrence of cellular derailment strongly increases. Here we review our current understanding of the molecular mechanisms that contribute to HSC aging. We argue that most of the causes that underlie HSC aging result from cell-intrinsic pathways, and reflect on which aspects of the aging process may be reversible. Because many hematological pathologies are strongly age-associated, strategies to intervene in aspects of the stem cell aging process may have significant clinical relevance.

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.

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.

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.

Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands.

Adult stem cells are often touted as therapeutic agents in the regenerative medicine field, however data detailing both the engraftment and functional capabilities of solid tissue derived human adult epithelial stem cells is scarce. Here we show the isolation of adult human salivary gland (SG) stem/progenitor cells and demonstrate at the single cell level in vitro self-renewal and differentiation into multilineage organoids. We also show in vivo functionality, long-term engraftment, and functional restoration in a xenotransplantation model. Indeed, transplanted human salisphere-derived cells restored saliva production and greatly improved the regenerative potential of irradiated SGs. Further selection for c-Kit expression enriched for cells with enhanced regenerative potencies. Interestingly, interaction of transplanted cells with the recipient SG may also be involved in functional recovery. Thus, we show for the first time that salispheres cultured from human SGs contain stem/progenitor cells capable of self-renewal and differentiation and rescue of saliva production. Our study underpins the therapeutic promise of salisphere cell therapy for the treatment of xerostomia.

Epigenetic perturbations in aging stem cells.

Stem cells maintain homeostasis in all regenerating tissues during the lifespan of an organism. Thus, age-related functional decline of such tissues is likely to be at least partially explained by molecular events occurring in the stem cell compartment. Some of these events involve epigenetic changes, which may dictate how an aging genome can lead to differential gene expression programs. Recent technological advances have made it now possible to assess the genome-wide distribution of an ever-increasing number of epigenetic marks. As a result, the hypothesis that there may be a causal role for an altered epigenome contributing to the functional decline of cells, tissues, and organs in aging organisms can now be explored. In this paper, we review recent developments in the field of epigenetic regulation of stem cells, and how this may contribute to aging.

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.

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.

The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation.

Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small molecules can serve as tools to manipulate cell fate decisions. Here, we tested 2 small molecules, valproic acid (VPA) and lithium (Li), to inhibit differentiation. HSPCs exposed to VPA and Li during differentiation-inducing culture preserved an immature cell phenotype, provided radioprotection to lethally irradiated recipients, and enhanced in vivo repopulating potential. Anti-differentiation effects of VPA and Li were observed also at the level of committed progenitors, where VPA re-activated replating activity of common myeloid progenitor and granulocyte macrophage progenitor cells. Furthermore, VPA and Li synergistically preserved expression of stem cell-related genes and repressed genes involved in differentiation. Target genes were collectively co-regulated during normal hematopoietic differentiation. In addition, transcription factor networks were identified as possible primary regulators. Our results show that the combination of VPA and Li potently delays differentiation at the biologic and molecular levels and provide evidence to suggest that combinatorial screening of chemical compounds may uncover possible additive/synergistic effects to modulate stem cell fate decisions.

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'.

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

How CBX proteins regulate normal and leukemic blood cells.

Hematopoietic stem cell (HSC) fate decisions are dictated by epigenetic landscapes. The Polycomb Repressive Complex 1 (PRC1) represses genes that induce differentiation, thereby maintaining HSC self-renewal. Depending on which chromobox (CBX) protein (CBX2, CBX4, CBX6, CBX7, or CBX8) is part of the PRC1 complex, HSC fate decisions differ. Here, we review how this occurs. We describe how CBX proteins dictate age-related changes in HSCs and stimulate oncogenic HSC fate decisions, either as canonical PRC1 members or by alternative interactions, including non-epigenetic regulation. CBX2, CBX7, and CBX8 enhance leukemia progression. To target, reprogram, and kill leukemic cells, we suggest and describe multiple therapeutic strategies to interfere with the epigenetic functions of oncogenic CBX proteins. Future studies should clarify to what extent the non-epigenetic function of cytoplasmic CBX proteins is important for normal, aged, and leukemic blood cells.

CD61 identifies a superior population of aged murine HSCs and is required to preserve quiescence and self-renewal.

ABSTRACT: Aging leads to a decline in function of hematopoietic stem cells (HSCs) and increases susceptibility to hematological disease. We found CD61 to be highly expressed in aged murine HSCs. Here, we investigate the role of CD61 in identifying distinct subpopulations of aged HSCs and assess how expression of CD61 affects stem cell function. We show that HSCs with high expression of CD61 are functionality superior and retain self-renewal capacity in serial transplantations. In primary transplantations, aged CD61High HSCs function similarly to young HSCs. CD61High HSCs are more quiescent than their CD61Low counterparts. We also show that in aged bone marrow, CD61High and CD61Low HSCs are transcriptomically distinct populations. Collectively, our research identifies CD61 as a key player in maintaining stem cell quiescence, ensuring the preservation of their functional integrity and potential during aging. Moreover, CD61 emerges as a marker to prospectively isolate a superior, highly dormant population of young and aged HSCs, making it a valuable tool both in fundamental and clinical research.

Shared Ageing Research Models (ShARM): a new facility to support ageing research.

In order to manage the rise in life expectancy and the concomitant increased occurrence of age-related diseases, research into ageing has become a strategic priority. Mouse models are commonly utilised as they share high homology with humans and show many similar signs and diseases of ageing. However, the time and cost needed to rear aged cohorts can limit research opportunities. Sharing of resources can provide an ethically and economically superior framework to overcome some of these issues but requires dedicated infrastructure. Shared Ageing Research Models (ShARM) ( www.ShARMUK.org ) is a new, not-for-profit organisation funded by Wellcome Trust, open to all investigators. It collects, stores and distributes flash frozen tissues from aged murine models through its biorepository and provides a database of live ageing mouse colonies available in the UK and abroad. It also has an online environment (MICEspace) for collation and analysis of data from communal models and discussion boards on subjects such as the welfare of ageing animals and common endpoints for intervention studies. Since launching in July 2012, thanks to the generosity of researchers in UK and Europe, ShARM has collected more than 2,500 tissues and has in excess of 2,000 mice registered in live ageing colonies. By providing the appropriate support, ShARM has been able to bring together the knowledge and experience of investigators in the UK and Europe to maximise research outputs with little additional cost and minimising animal use in order to facilitate progress in ageing research.