Adam Wilkinson

All Publications

• Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature Wilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R. V., Yamamoto, R., Loh, K. M., Nakamura, Y., Watanabe, M., Nakauchi, H., Yamazaki, S. 2019

  Abstract

  Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

  View details for DOI 10.1038/s41586-019-1244-x

  View details for PubMedID 31142833

• Stabilizing hematopoietic stem cells in vitro. Current opinion in genetics & development Wilkinson, A. C., Nakauchi, H. 2020; 64: 1–5

  Abstract

  Hematopoietic stem cells (HSCs) can regenerate all lineages of the adult blood and immune systems long-term following transplantation via a combination of self-renewal and multipotent differentiation. HSCs are therefore an important cell type in both basic research and in the clinic, where HSC transplantation is a curative therapy for a range of diseases. However, as a rare bone marrow cell population, the characterization and collection of HSCs can often be challenging. This has led to a large search for in vitro culture conditions that support the growth of functional HSCs and the in vitro stabilization of the HSC state represents a major goal in the field. Here, we review recent progress towards stabilizing HSCs in vitro.

  View details for DOI 10.1016/j.gde.2020.05.035

  View details for PubMedID 32570191

• In vivo and ex vivo haematopoietic stem cell expansion. Current opinion in hematology Yamamoto, R., Wilkinson, A. C., Nakauchi, H. 2020

  Abstract

  PURPOSE OF REVIEW: Haematopoietic stem cells (HSCs) are characterized by two key features: self-renewal ability and multilineage differentiation potential. Through these cellular activities, HSCs sustain blood and immune system homeostasis throughout life and can also reconstitute the entire haematopoietic system within a bone marrow ablated recipient. This approach of HSC transplantation is used clinically as a curative treatment option for numerous haematological diseases, both malignant and nonmalignant.RECENT FINDINGS: Elucidation of the mechanism of HSC expansion represents a major focus within haematology. Here, we review the recent progress towards understanding HSC expansion in vivo and ex vivo, including a discussion of recent clonal transplantation assays and the development of novel ex vivo culture systems.SUMMARY: Recent findings provide exciting promise for improving the safety and efficacy of current HSC-based therapies as well as for the development of new therapeutic paradigms.

  View details for DOI 10.1097/MOH.0000000000000593

  View details for PubMedID 32452877

• Hope for hematological diseases. Science (New York, N.Y.) Wilkinson, A. C. 2020; 367 (6483): 1206

  View details for DOI 10.1126/science.aba6108

  View details for PubMedID 32165580

• Long-term ex vivo expansion of mouse hematopoietic stem cells. Nature protocols Wilkinson, A. C., Ishida, R., Nakauchi, H., Yamazaki, S. 2020

  Abstract

  Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

  View details for DOI 10.1038/s41596-019-0263-2

  View details for PubMedID 31915389

• Lineage commitment of hematopoietic stem cells and progenitors: insights from recent single cell and lineage tracing technologies. Experimental hematology Loughran, S., Haas, S., Wilkinson, A. C., Klein, A., Brand, M. 2020

  Abstract

  Blood production is essential to maintain human health, and even small perturbations in hematopoiesis can cause disease. Hematopoiesis has therefore been the focus of much research for many years. Experiments determining the lineage potentials of hematopoietic stem and progenitor cells (HSPCs) in vitro and after transplantation revealed a hierarchy of progenitor cell states, where differentiating cells undergo lineage commitment - a series of irreversible changes that progressively restrict their potential. New technologies have recently been developed that allow for a more detailed analysis of the molecular states and fates of differentiating HSPCs. Proteomic and lineage-tracing approaches, alongside single cell transcriptomic analyses have recently helped to reveal the biological complexity underlying lineage commitment during hematopoiesis. Recent insights from these new technologies were presented by Drs. Marjorie Brand and Allon Klein in the Summer 2019 ISEH Webinar, and are discussed in this Perspective.

  View details for DOI 10.1016/j.exphem.2020.07.002

  View details for PubMedID 32653531

• Haematopoietic stem cell self-renewal in vivo and ex vivo. Nature reviews. Genetics Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. 2020

  Abstract

  The self-renewal capacity of multipotent haematopoietic stem cells (HSCs) supports blood system homeostasis throughout life and underlies the curative capacity of clinical HSC transplantation therapies. However, despite extensive characterization of the HSC state in the adult bone marrow and embryonic fetal liver, the mechanism of HSC self-renewal has remained elusive. This Review presents our current understanding of HSC self-renewal in vivo and ex vivo, and discusses important advances in ex vivo HSC expansion that are providing new biological insights and offering new therapeutic opportunities.

  View details for DOI 10.1038/s41576-020-0241-0

  View details for PubMedID 32467607

• Single-cell lineage tracing approaches in hematology research - technical considerations. Experimental hematology Carrelha, J., Lin, D. S., Rodriguez-Fraticelli, A. E., Luis, T. C., Wilkinson, A. C., Cabezas-Wallscheid, N., Tremblay, C. S., Haas, S. 2020

  Abstract

  The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding the hematopoietic system activity in health and disease, and have made a major impact in how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single cell transplantation, lentiviral barcoding, and sleeping beauty barcoding. This Perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

  View details for DOI 10.1016/j.exphem.2020.07.007

  View details for PubMedID 32735908

• Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion. Experimental hematology Nishimura, T., Hsu, I., Martinez-Krams, D. C., Nakauchi, Y., Majeti, R., Yamazaki, S., Nakauchi, H., Wilkinson, A. C. 2019

  Abstract

  Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

  View details for DOI 10.1016/j.exphem.2019.11.007

  View details for PubMedID 31874780

• Expanded Potential Stem Cell Media as a tool to study human developmental hematopoiesis in vitro. Experimental hematology Wilkinson, A. C., Ryan, D. J., Kucinski, I., Wang, W., Yang, J., Nestorowa, S., Diamanti, E., Tsang, J. C., Wang, J., Campos, L. S., Yang, F., Fu, B., Wilson, N., Liu, P., Gottgens, B. 2019

  Abstract

  Pluripotent stem cell (PSC) differentiation in vitro represents a powerful and tractable model to study mammalian development, and an unlimited source of cells for regenerative medicine. Within hematology, in vitro PSC hematopoiesis affords novel insights into blood formation and represents an exciting potential approach to generate hematopoietic and immune cell types for transplantation and transfusion. Most studies to date have focused on in vitro hematopoiesis from mouse PSCs and human PSCs. However, differences in mouse and human PSC culture protocols have complicated the translation of discoveries between these systems. We recently developed a novel chemical media formulation, Expanded Potential Stem Cell Medium (EPSCM), that maintains mouse PSCs in a unique cellular state that affords extraembryonic differentiation capacity. Herein, we demonstrate that EPSCM can be directly used to stably maintain human PSCs. We further show that human PSCs maintained in EPSCM can spontaneously form embryoid bodies and undergo in vitro hematopoiesis using a simple differentiation protocol, similar to mouse PSC differentiation. EPSCM-maintained human PSCs generated at least two hematopoietic cell populations, which displayed distinct transcriptional profiles by RNA-sequencing analysis. EPSCM also supports gene targeting using homologous recombination, affording generation of a SPI1 (PU.1) reporter PSC line to study and track in vitro hematopoiesis. EPSCM therefore provides a useful tool not only to study pluripotency but also hematopoietic cell specification and developmental-lineage commitment.

  View details for DOI 10.1016/j.exphem.2019.07.003

  View details for PubMedID 31326613

• Biological implications of clonal hematopoiesis. Experimental hematology Luis, T. C., Wilkinson, A. C., Beerman, I., Jaiswal, S., Shlush, L. I. 2019

  Abstract

  Adult hematological malignancies, such as acute myeloid leukemia, are thought to arise through the gradual acquisition of oncogenic mutations within long-lived hematopoietic stem cells (HSCs). Genomic analysis of peripheral blood DNA has recently identified leukemia-associated genetic mutations within otherwise healthy individuals, an observation that is strongly associated with age. These genetic mutations are often found at high frequency, suggesting dominance of a mutant HSC clone. Expansion of clones carrying other mutations not associated with leukemia or larger chromosomal deletions were also observed. This clinical observation has been termed clonal hematopoiesis, a condition associated with increased the risk of both hematological malignancy and cardiovascular disease. Here, we discuss the identification of clonal hematopoiesis and its implications on human health, based on the May 2019 International Society for Experimental Hematology New Investigator Committee Webinar.

  View details for DOI 10.1016/j.exphem.2019.08.004

  View details for PubMedID 31472170

• Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity EXPERIMENTAL HEMATOLOGY Wilkinson, A. C., Morita, M., Nakauchi, H., Yamazaki, S. 2018; 63: 12–16

  View details for DOI 10.1016/j,exphem.2018.04.004

  View details for Web of Science ID 000439763500002

• Changing concepts in hematopoietic stem cells. Science (New York, N.Y.) Yamamoto, R., Wilkinson, A. C., Nakauchi, H. 2018; 362 (6417): 895–96

  View details for PubMedID 30467158

• Rapid Mast Cell Generation from Gata2 Reporter Pluripotent Stem Cells. Stem cell reports Kauts, M. L., De Leo, B., Rodríguez-Seoane, C., Ronn, R., Glykofrydis, F., Maglitto, A., Kaimakis, P., Basi, M., Taylor, H., Forrester, L., Wilkinson, A. C., Göttgens, B., Saunders, P., Dzierzak, E. 2018; 11 (4): 1009–20

  Abstract

  Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.

  View details for DOI 10.1016/j.stemcr.2018.08.007

  View details for PubMedID 30197119

  View details for PubMedCentralID PMC6178197

• Branched-chain amino acid metabolism in cancer. Current opinion in clinical nutrition and metabolic care Ananieva, E. A., Wilkinson, A. C. 2018; 21 (1): 64–70

  Abstract

  The current review aims to provide an update on the recent biomedical interest in oncogenic branched-chain amino acid (BCAA) metabolism, and discusses the advantages of using BCAAs and expression of BCAA-related enzymes in the treatment and diagnosis of cancers.An accumulating body of evidence demonstrates that BCAAs are essential nutrients for cancer growth and are used by tumors in various biosynthetic pathways and as a source of energy. In addition, BCAA metabolic enzymes, such as the cytosolic branched-chain aminotransferase 1 (BCAT1) and mitochondrial branched-chain aminotransferase 2, have emerged as useful prognostic cancer markers. BCAT1 expression commonly correlates with more aggressive cancer growth and progression, and has attracted substantial scientific attention in the past few years. These studies have found the consequences of BCAT1 disruption to be heterogeneous; not all cancers share the same requirements for BCAA metabolites and the function of BCAT1 appears to vary between cancer types.Both oncogenic mutations and cancer tissue-of-origin influence BCAA metabolism and expression of BCAA-associated metabolic enzymes. These new discoveries need to be taken into consideration during the development of new cancer therapies that target BCAA metabolism.

  View details for PubMedID 29211698

  View details for PubMedCentralID PMC5732628

• The hematopoietic stem cell diet. International journal of hematology Wilkinson, A. C., Yamazaki, S. 2018; 107 (6): 634–41

  Abstract

  Hematopoietic stem cells (HSCs) are responsible for sustaining life-long blood formation or hematopoiesis and are also used clinically in a form of bone marrow transplantation, a curative cellular therapy for a range of hematological diseases. HSCs are maintained throughout adult life by a complex biological niche or microenvironment, which is thought to be composed of a range of cellular, molecular, and metabolic components. The metabolic components of the HSC niche have become of increasing interest over the past few years. It is now well-recognized that metabolic activity is intimately linked to HSC function, and dysregulation of these metabolic pathways result in hematological pathologies such as leukemia. Here, we review the recent progress in this field including our current understanding of the "dietary" requirements of HSCs and how nutrition influences HSC activity. These recent findings have suggested promising new metabolic approaches to improve clinical HSC transplantation and leukemia therapies.

  View details for PubMedID 29605874

• Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment. Cell stem cell Yamamoto, R., Wilkinson, A. C., Ooehara, J., Lan, X., Lai, C. Y., Nakauchi, Y., Pritchard, J. K., Nakauchi, H. 2018; 22 (4): 600–607.e4

  Abstract

  Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs.

  View details for PubMedID 29625072

• An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors. Stem cell reports Ieyasu, A., Ishida, R., Kimura, T., Morita, M., Wilkinson, A. C., Sudo, K., Nishimura, T., Ohehara, J., Tajima, Y., Lai, C., Otsu, M., Nakamura, Y., Ema, H., Nakauchi, H., Yamazaki, S. 2017

  Abstract

  Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

  View details for DOI 10.1016/j.stemcr.2017.01.015

  View details for PubMedID 28238792

• Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method SCIENTIFIC REPORTS Tajima, Y., Ito, K., Umino, A., Wilkinson, A. C., Nakauchi, H., Yamazaki, S. 2017; 7

  Abstract

  The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation.

  View details for DOI 10.1038/srep40684

  View details for Web of Science ID 000392272500001

  View details for PubMedID 28098173

  View details for PubMedCentralID PMC5241640

• Establishment of mouse expanded potential stem cells. Nature Yang, J., Ryan, D. J., Wang, W., Tsang, J. C., Lan, G., Masaki, H., Gao, X., Antunes, L., Yu, Y., Zhu, Z., Wang, J., Kolodziejczyk, A. A., Campos, L. S., Wang, C., Yang, F., Zhong, Z., Fu, B., Eckersley-Maslin, M. A., Woods, M., Tanaka, Y., Chen, X., Wilkinson, A. C., Bussell, J., White, J., Ramirez-Solis, R., Reik, W., Göttgens, B., Teichmann, S. A., Tam, P. P., Nakauchi, H., Zou, X., Lu, L., Liu, P. 2017; 550 (7676): 393–97

  Abstract

  Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

  View details for PubMedID 29019987

• Technical Considerations for the Use of CRISPR/Cas9 in Hematology Research. Experimental hematology Gundry, M. C., Dever, D. P., Yudovich, D., Bauer, D. E., Haas, S., Wilkinson, A. C., Singbrant, S. 2017

  Abstract

  The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has recently been "repurposed" to efficiently mutate mammalian genomes in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of "correcting" dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic.

  View details for PubMedID 28757433

• Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity. Cell systems Wilkinson, A. C., Nakauchi, H., Göttgens, B. 2017; 5 (4): 319–31

  Abstract

  Transcription factor (TF) networks are a key determinant of cell fate decisions in mammalian development and adult tissue homeostasis and are frequently corrupted in disease. However, our inability to experimentally resolve and interrogate the complexity of mammalian TF networks has hampered the progress in this field. Recent technological advances, in particular large-scale genome-wide approaches, single-cell methodologies, live-cell imaging, and genome editing, are emerging as important technologies in TF network biology. Several recent studies even suggest a need to re-evaluate established models of mammalian TF networks. Here, we provide a brief overview of current and emerging methods to define mammalian TF networks. We also discuss how these emerging technologies facilitate new ways to interrogate complex TF networks, consider the current open questions in the field, and comment on potential future directions and biomedical applications.

  View details for PubMedID 29073372

• In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells by Gfi1b, c-Fos, and Gata2 Overexpression within Teratoma. Stem cell reports Tsukada, M., Ota, Y., Wilkinson, A. C., Becker, H. J., Osato, M., Nakauchi, H., Yamazaki, S. 2017; 9 (4): 1024–33

  Abstract

  Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) could potentially provide unlimited HSCs for clinical transplantation, a curative treatment for numerous blood diseases. However, to date, bona fide HSC generation has been largely unsuccessful in vitro. We have previously described proof of concept for in vivo HSC generation from PSCs via teratoma formation. However, our first-generation system was complex and the output low. Here, we further optimize this technology and demonstrate the following: (1) simplified HSC generation using transcription factor overexpression; (2) improved HSC output using c-Kit-deficient host mice, and (3) that teratomas can be transplanted and cryopreserved. We demonstrate that overexpression of Gfi1b, c-Fos, and Gata2, previously reported to transdifferentiate fibroblasts into hematopoietic progenitors in vitro, can induce long-term HSC formation in vivo. Our in vivo system provides a useful platform to investigate new strategies and re-evaluate existing strategies to generate HSCs and study HSC development.

  View details for PubMedID 28943250

  View details for PubMedCentralID PMC5639260

• Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation SCIENCE Taya, Y., Ota, Y., Wilkinson, A. C., Kanazawa, A., Watarai, H., Kasai, M., Nakauchi, H., Yamazaki, S. 2016; 354 (6316): 1152-1155

  Abstract

  A specialized bone marrow microenvironment (niche) regulates hematopoietic stem cell (HSC) self-renewal and commitment. For successful donor-HSC engraftment, the niche must be emptied via myeloablative irradiation or chemotherapy. However, myeloablation can cause severe complications and even mortality. Here we report that the essential amino acid valine is indispensable for the proliferation and maintenance of HSCs. Both mouse and human HSCs failed to proliferate when cultured in valine-depleted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction emptied the mouse bone marrow niche and afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical role for valine in HSC maintenance and suggest that dietary valine restriction may reduce iatrogenic complications in HSC transplantation.

  View details for DOI 10.1126/science.aag3145

  View details for Web of Science ID 000388916400043

  View details for PubMedID 27934766

• Integrated genome-scale analysis of the transcriptional regulatory landscape in a blood stem/progenitor cell model. Blood Wilson, N. K., Schoenfelder, S., Hannah, R., Sánchez Castillo, M., Schütte, J., Ladopoulos, V., Mitchelmore, J., Goode, D. K., Calero-Nieto, F. J., Moignard, V., Wilkinson, A. C., Jimenez-Madrid, I., Kinston, S., Spivakov, M., Fraser, P., Göttgens, B. 2016; 127 (13): e12-23

  Abstract

  Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.

  View details for DOI 10.1182/blood-2015-10-677393

  View details for PubMedID 26809507

• Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium CELL REPORTS Watson, J. K., Rulands, S., Wilkinson, A. C., Wuidart, A., Ousset, M., Van Keymeulen, A., Goettgens, B., Blanpain, C., Simons, B. D., Rawlins, E. L. 2015; 12 (1): 90-101

  Abstract

  Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.

  View details for DOI 10.1016/j.celrep.2015.06.011

  View details for Web of Science ID 000357673300011

  View details for PubMedID 26119728

• Decoding the regulatory network of early blood development from single-cell gene expression measurements. Nature biotechnology Moignard, V., Woodhouse, S., Haghverdi, L., Lilly, A. J., Tanaka, Y., Wilkinson, A. C., Buettner, F., Macaulay, I. C., Jawaid, W., Diamanti, E., Nishikawa, S., Piterman, N., Kouskoff, V., Theis, F. J., Fisher, J., Göttgens, B. 2015; 33 (3): 269-276

  Abstract

  Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

  View details for DOI 10.1038/nbt.3154

  View details for PubMedID 25664528

• CODEX: a next-generation sequencing experiment database for the haematopoietic and embryonic stem cell communities. Nucleic acids research Sánchez-Castillo, M., Ruau, D., Wilkinson, A. C., Ng, F. S., Hannah, R., Diamanti, E., Lombard, P., Wilson, N. K., Gottgens, B. 2015; 43 (Database issue): D1117-23

  Abstract

  CODEX (http://codex.stemcells.cam.ac.uk/) is a user-friendly database for the direct access and interrogation of publicly available next-generation sequencing (NGS) data, specifically aimed at experimental biologists. In an era of multi-centre genomic dataset generation, CODEX provides a single database where these samples are collected, uniformly processed and vetted. The main drive of CODEX is to provide the wider scientific community with instant access to high-quality NGS data, which, irrespective of the publishing laboratory, is directly comparable. CODEX allows users to immediately visualize or download processed datasets, or compare user-generated data against the database's cumulative knowledge-base. CODEX contains four types of NGS experiments: transcription factor chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), histone modification ChIP-Seq, DNase-Seq and RNA-Seq. These are largely encompassed within two specialized repositories, HAEMCODE and ESCODE, which are focused on haematopoiesis and embryonic stem cell samples, respectively. To date, CODEX contains over 1000 samples, including 221 unique TFs and 93 unique cell types. CODEX therefore provides one of the most complete resources of publicly available NGS data for the direct interrogation of transcriptional programmes that regulate cellular identity and fate in the context of mammalian development, homeostasis and disease.

  View details for DOI 10.1093/nar/gku895

  View details for PubMedID 25270877

• Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification. Development Wilkinson, A. C., Kawata, V. K., Schütte, J., Gao, X., Antoniou, S., Baumann, C., Woodhouse, S., Hannah, R., Tanaka, Y., Swiers, G., Moignard, V., Fisher, J., Hidetoshi, S., Tijssen, M. R., de Bruijn, M. F., Liu, P., Göttgens, B. 2014; 141 (20): 4018-4030

  Abstract

  Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis.

  View details for DOI 10.1242/dev.115709

  View details for PubMedID 25252941

• Transcriptional regulation of haematopoietic stem cells. Advances in experimental medicine and biology Wilkinson, A. C., Göttgens, B. 2013; 786: 187-212

  Abstract

  Haematopoietic stem cells (HSCs) are a rare cell population found in the bone marrow of adult mammals and are responsible for maintaining the entire haematopoietic system. Definitive HSCs are produced from mesoderm during embryonic development, from embryonic day 10 in the mouse. HSCs seed the foetal liver before migrating to the bone marrow around the time of birth. In the adult, HSCs are largely quiescent but have the ability to divide to self-renew and expand, or to proliferate and differentiate into any mature haematopoietic cell type. Both the specification of HSCs during development and their cellular choices once formed are tightly controlled at the level of transcription. Numerous transcriptional regulators of HSC specification, expansion, homeostasis and differentiation have been identified, primarily from analysis of mouse gene knockout experiments and transplantation assays. These include transcription factors, epigenetic modifiers and signalling pathway effectors. This chapter reviews the current knowledge of these HSC transcriptional regulators, predominantly focusing on the transcriptional regulation of mouse HSCs, although transcriptional regulation of human HSCs is also mentioned where relevant. Due to the breadth and maturity of this field, we have prioritised recently identified examples of HSC transcriptional regulators. We go on to highlight additional layers of control that regulate expression and activity of HSC transcriptional regulators and discuss how chromosomal translocations that result in fusion proteins of these HSC transcriptional regulators commonly drive leukaemias through transcriptional dysregulation.

  View details for DOI 10.1007/978-94-007-6621-1_11

  View details for PubMedID 23696358

• RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction CELL REPORTS Wilkinson, A. C., Ballabio, E., Geng, H., North, P., Tapia, M., Kerry, J., Biswas, D., Roeder, R. G., Allis, C. D., Melnick, A., de Bruijn, M. F., Milne, T. A. 2013; 3 (1): 116-127

  Abstract

  The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

  View details for DOI 10.1016/j.celrep.2012.12.016

  View details for Web of Science ID 000321891400014

  View details for PubMedID 23352661

• Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses. Biology open Wilkinson, A. C., Goode, D. K., Cheng, Y., Dickel, D. E., Foster, S., Sendall, T., Tijssen, M. R., Sanchez, M., Pennacchio, L. A., Kirkpatrick, A. M., Göttgens, B. 2013; 2 (11): 1229-1238

  Abstract

  Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2(-/-) Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability.

  View details for DOI 10.1242/bio.20136296

  View details for PubMedID 24244860

• Rfam: updates to the RNA families database NUCLEIC ACIDS RESEARCH Gardner, P. P., Daub, J., Tate, J. G., Nawrocki, E. P., Kolbe, D. L., Lindgreen, S., Wilkinson, A. C., Finn, R. D., Griffiths-Jones, S., Eddy, S. R., Bateman, A. 2009; 37: D136-D140

  Abstract

  Rfam is a collection of RNA sequence families, represented by multiple sequence alignments and covariance models (CMs). The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, using sensitive BLAST filters in combination with CMs. A minority of families with a very broad taxonomic range (e.g. tRNA and rRNA) provide the majority of the sequence annotations, whilst the majority of Rfam families (e.g. snoRNAs and miRNAs) have a limited taxonomic range and provide a limited number of annotations. Recent improvements to the website, methodologies and data used by Rfam are discussed. Rfam is freely available on the Web at http://rfam.sanger.ac.uk/and http://rfam.janelia.org/.

  View details for DOI 10.1093/nar/gkn766

  View details for Web of Science ID 000261906200024

  View details for PubMedID 18953034