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Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR–Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.Haematopoietic stem and progenitor cell (HSPC) gene therapy using lentiviral or gammaretroviral vectors has now been approved for clinical use. In this Review, Ferrari, Thrasher and Aiuti discuss the history of HSPC gene therapy, the clinical promise of gene-editing HPSCs and the use of HSPC gene therapy to treat specific diseases.

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.Wilkinson and colleagues discuss haematopoietic stem cell (HSC) self-renewal in mice and humans. Experimental techniques for assaying HSC self-renewal are addressed, along with biological mechanisms regulating HSC self-renewal in vivo and ex vivo, and the therapeutic implications of this understanding.

This protocol describes the long-term culture of liver and pancreas 3D organoids from human and mouse, and differentiation of liver organoids in vitro and in vivo . Methodology for genetic manipulation of these self-renewing organoids is also detailed. Adult somatic tissues have proven difficult to expand in vitro , largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro . Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro , which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

Key Points Stem cells perpetuate themselves through self-renewal and they replenish mature cells to maintain tissue homeostasis throughout the lifespan of an organism. Stem cell function is precisely regulated by various intrinsic mechanisms, in coordination with extrinsic stimuli; recent studies have revealed critical roles for stem cell metabolism in maintaining stem cell self-renewal. Stem cells reside within specialized niches (for example, a hypoxic niche), where specific local conditions have a role in maintaining a quiescent state that is essential for preserving the self-renewal capacity of stem cells. Many types of stem cells heavily rely on anaerobic glycolysis, rather than mitochondrial oxidative phosphorylation, to produce the low levels of intracellular reactive oxygen species, which inhibit stem cell ageing. The balance between stem cell quiescence and proliferation is regulated by the nutrient-sensitive PI3K–AKT–mTOR and AMPK pathways, and Gln metabolism. Recent studies have uncovered a crucial role for fatty acid metabolism in the self-renewal of haematopoietic stem cells (HSCs) through the control it exerts over stem cell fate decisions. Studies of mouse models and advances in metabolomic analysis, particularly of haematopoietic stem cells, have revealed how metabolic cues from anaerobic glycolysis, bioenergetic signalling, the AKT–mTOR pathway, and Gln and fatty acid metabolism, affect the balance between stem cell self-renewal and differentiation. Understanding how metabolic pathways regulate fate decisions may be beneficial therapeutically. A distinctive feature of stem cells is their capacity to self-renew to maintain pluripotency. Studies of genetically-engineered mouse models and recent advances in metabolomic analysis, particularly in haematopoietic stem cells, have deepened our understanding of the contribution made by metabolic cues to the regulation of stem cell self-renewal. Many types of stem cells heavily rely on anaerobic glycolysis, and stem cell function is also regulated by bioenergetic signalling, the AKT–mTOR pathway, Gln metabolism and fatty acid metabolism. As maintenance of a stem cell pool requires a finely-tuned balance between self-renewal and differentiation, investigations into the molecular mechanisms and metabolic pathways underlying these decisions hold great therapeutic promise.

Tumor organoids derived from the most common subtypes of primary liver cancer recapitulate the histologic and molecular features of the tissues of origin, even after long-term culture. These in vitro models, as well as those for colorectal cancer reported in Crespo et al. in a previous issue, are amenable for drug screening and allow the identification of therapeutic approaches with potential for cancer treatment. Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo . PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches. Haematopoietic stem cells rely on glycolysis for their energy demands but whether this affects their fate is unknown. Here, the authors show that forcing the cells to rely on glycolysis is important for self-renewal and that this involves a reduction in mitochondrial mass.

The maintenance and proliferation of hematopoietic stem cells after injury is regulated by signals from the bone marrow stem cell niche. The bone marrow niche has mystified scientists for many years, leading to widespread investigation to shed light into its molecular and cellular composition. Considerable efforts have been devoted toward uncovering the regulatory mechanisms of hematopoietic stem cell (HSC) niche maintenance. Recent advances in imaging and genetic manipulation of mouse models have allowed the identification of distinct vascular niches that have been shown to orchestrate the balance between quiescence, proliferation and regeneration of the bone marrow after injury. Here we highlight the recently discovered intrinsic mechanisms, microenvironmental interactions and communication with surrounding cells involved in HSC regulation, during homeostasis and in regeneration after injury and discuss their implications for regenerative therapy.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation 1 , which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias 2 . Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche 3 – 5 , stable ex vivo HSC expansion has previously been unattainable 6 , 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 cultures 8 ; 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 1 month, 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 50 cells 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. An albumin-free culture system for the long-term ex vivo expansion of mouse haematopoietic stem cells produces 236- to 899-fold expansion, and generates cultures that robustly engraft in recipient mice without toxic pre-conditioning.

Analysis of transplantation of single haematopoietic stem cells in mice defines stable lineage-restricted fates in long-term self-renewing multipotent stem cells, including a class of multipotent stem cells that exclusively replenishes the megakaryocyte/platelet lineage. The many fates of stem cells in the blood line Many blood disorders can be treated with haematopoietic (blood-generating) stem cell (HSC) transplants, but such treatment does not always lead to efficient replenishment of all blood lineages. Through single-cell transplantation of HSCs in mice, Sten Eirik Jacobsen and colleagues define lineage-restricted fates of long-term self-renewing cells. They identify a class of HSC that effectively replenishes the megakaryocyte and platelet lineages over other lineages, and other HSCs that are more able to participate in megakaryocyte, erythroid and myeloid lineages despite being able to sustain lymphoid potential. Genetic lineage tracing also shows that platelet-biased HSCs are able to support unperturbed adult haematopoiesis. Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells 1 , securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations 2 , 3 , 4 . HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality 5 , 6 . Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs 7 , 8 , 9 , 10 , 11 , a finding that is supported by studies of unperturbed haematopoiesis 2 , 3 , 4 , 12 and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs 7 , 8 , 9 , 10 , 13 . Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency 11 , 14 . Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.

Key Points T lymphocytes transition through progressive stages of differentiation that are characterized by a stepwise loss of functional and therapeutic potential. Subsets of mature T cells exhibit the stem cell-like attributes of self-renewal, multipotency and the ability to undergo asymmetric division. Evolutionarily conserved pathways regulating stemness are active in T cells, including T memory stem cells, T helper 17 cells and interleukin-17 (IL-17)-producing CD8 + T cells. Pharmacological and genetic induction of stem cell pathways can be used to generate tumour-specific T cells with stem cell-like properties. Reprogramming terminally differentiated tumour-reactive T cells to display naive or stem cell-like functionalities might be obtained through the expression of transcription factors or microRNAs that are associated with naive or T memory stem cells. Stem cell-like T cells possess enhanced capacities to engraft, persist and mediate prolonged immune attack against tumour masses that are sustained by long-lived cancer stem cells. Treating cancer patients with T cell-based therapies has shown some some promise in the clinic, but not all patients respond. There could be many reasons for this, some of which might be addressed by using the best possible antitumour T cell. What are the biological properties of such a cell and can we generate one? Stem cells are defined by the ability to self-renew and to generate differentiated progeny, qualities that are maintained by evolutionarily conserved pathways that can lead to cancer when deregulated. There is now evidence that these stem cell-like attributes and signalling pathways are also shared among subsets of mature memory T lymphocytes. We discuss how using stem cell-like T cells can overcome the limitations of current adoptive T cell therapies, including inefficient T cell engraftment, persistence and ability to mediate prolonged immune attack. Conferring stemness to antitumour T cells might unleash the full potential of cellular therapies.