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While the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Following damage, interleukin-1β (IL-1β) signalling-dependent modulation of Jag1 and Jag2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives the reprogramming and acquisition of differentiation plasticity. We identify the transcription factor Fosl2 (also known as Fra2) for secretory cell fate conversion to alveolar type 2 cells that retain the distinct genetic and epigenetic signatures of secretory lineages. We also reveal that human secretory cells positive for KDR (also known as FLK-1) display a conserved capacity to generate alveolar type 2 cells via Notch inhibition. Our results demonstrate the functional role of an IL-1β–Notch–Fosl2 axis in the fate decision of secretory cells during injury repair, proposing a potential therapeutic target for human lung alveolar regeneration. Using human airway organoids and mouse models, Choi et al. show that an IL-1β–Notch–Fosl2 signalling axis regulates the conversion of secretory cells into alveolar type 2 cells after injury.

Objective: Neonatal stroke affects 1 in 2800 live births and is a major cause of neurological injury. The Sonic Hedgehog (Shh) signaling pathway is critical for central nervous system (CNS) development and has neuroprotective and reparative effects in different CNS injury models. Previous studies have demonstrated beneficial effects of small molecule Shh-Smoothened-agonist (SAG) against neonatal cerebellar injury and it improves Down syndrome-related brain structural deficits in mice. Here, we investigated SAG neuroprotection in rat models of neonatal ischemia-reperfusion (stroke) and adult focal white matter injury. Methods: We used transient middle cerebral artery occlusion at P10 and ethidium bromide injection in adult rats to induce damage. Following surgery and SAG or vehicle treatment we analyzed tissue loss, cell proliferation and fate, and behavioral outcome. Results: We report that a single dose of SAG administered following neonatal stroke preserved brain volume, reduced inflammation, enhanced oligodendrocyte progenitor cell (OPC) and EC proliferation, and resulted in long-term cognitive improvement. Single-dose SAG also promoted proliferation of OPCs following focal demyelination in the adult rat. Conclusion: These findings indicate benefit of one-time SAG treatment post-insult in reducing brain injury and improving behavioral outcome after experimental neonatal stroke.

The brain is responsive to an ever-changing environment, enabling the organism to learn and change behaviour accordingly. Efforts to understand the underpinnings of this plasticity have almost exclusively focussed on the functional and underlying structural changes that neurons undergo at neurochemical synapses. What has received comparatively little attention is the involvement of activity-dependent myelination in such plasticity and the functional output of circuits controlling behaviour. The traditionally held view of myelin as a passive insulator of axons is changing to one of lifelong changes in myelin, modulated by neuronal activity and experience. Here we review the nascent evidence of the functional role of myelin plasticity in strengthening circuit functions that underlie learning and behaviour

Like many adult stem cell populations, the capacity of oligodendrocyte progenitor cells (OPCs) to proliferate and differentiate is substantially impaired with aging. Previous work has shown that tissue-wide transient expression of the pluripotency factors Oct4, Sox2, Klf4 and c-Myc extends lifespan and enhances somatic cell function. Here we show that just one of these factors, c-Myc, is sufficient to determine the age state of OPC: c-Myc expression in aged OPCs drives their functional rejuvenation, while its inhibition in neonatal OPCs induces an aged-like phenotype, as determined by in vitro assays and transcriptome analysis. Increasing c-Myc expression in aged OPCs in vivo restores their proliferation and differentiation capacity, thereby enhancing regeneration in an aged central nervous system environment. Our results directly link Myc to cellular activity and cell age state, with implications for understanding regeneration in the context of aging, and provide important insights into the biology of stem cell aging. Transient expression of the pluripotency factors Oct4, Sox2, Klf4 and c-Myc can mitigate the effects of stem cell aging on tissue health. Neumann and colleagues show that Myc expression alone converts aged oligodendrocyte progenitor cells into neonatal-like cells, and is sufficient to enhance central nervous system regeneration in an otherwise aged environment.

In 2018, Shipp and colleagues from Harvard proposed a new genetic classification system for diffuse large B cell lymphoma (DLBCL)(1). At the same time, the Staudt laboratory at the National Cancer Institute (NCI) also proposed a genetic classification(2), refined in a subsequent publication(3), with subgroups that could be clearly matched to those of the Harvard study. More recently, a UK Haematological Malignancy Research Network (HMRN) sequencing study of almost a thousand patients independently reached strikingly similar conclusions over the molecular subclassification of DLBCL(4). These studies, and the degree of consensus between them, are important for those of us who treat DLBCL. They provide a potential handle with which to rationalise the biological heterogeneity that has so far blighted our ability to deploy biologically targeted agents successfully in the first-line treatment of DLBCL. From a biological perspective these studies redefine entirely how we view the classification and pathogenesis of DLBCL. However, from the perspective of a clinician faced with an individual patient with DLBCL the implications are much less clear. Indeed, significant challenges must be overcome before these complex classifications systems can be incorporated into standard clinical practice or prospective therapeutic trials.

Genome editing technologies such as zinc finger nucleases, TALENs and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality-controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class I null platelets using genome editing technologies for β2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoiesis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality-controlled platelet units.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene-networks involved in the exit from naïve pluripotency remains fragmentary. Here we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems-biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naive to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve-associated genes tightly connected to the in vivo epiblast state-transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funneling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signaling- pathways direct this pivotal mammalian cell-state transition.

For Parkinson's disease (PD) the main driver of patients’ well-being and clinical trials is the course disease takes over time (progression and prognosis), not susceptibility. The pace of deterioration varies dramatically between patients for reasons that are poorly understood. Most previous genome-wide analyses were static and could not resolve the critical time dimension. To characterize how genetic variation influences the progression of PD over time to dementia, which is a major determinant for quality of life, we performed a longitudinal genome-wide survival study (GWSS). 11.2 million deeply imputed variants in 3,821 PD patients who were prospectively tracked with 36,123 visits over a median of 6.7 years from disease onset (inter-quartile range, 4.2 years) were analyzed. Progression to dementia and decline in Mini Mental State Exam scores was determined using cox proportional hazards and mixed random and fixed effects models. The Regulating Synaptic Membrane Exocytosis 2 (RIMS2) locus achieved genome-wide significance in the discovery population and was validated in the replication population. The RIMS2 variant conferred a hazard ratio (HR) of 4.77 (95% confidence interval (CI) 3.01 – 7.56) with P = 2.78 × 10-11. Two other loci reached meta-analysis P values, CamPaIGN: Roger Barker, Tom Foltynie, Caroline Williams-Gray, Trevor Robbins, Carol Brayne, Sarah Mason, Sophie Winder-Rhodes contributed to the design and running of the trial as well as the recruitment and assessment of patients and DNA sample collection and preparation. Ruwani Wijeyekoon contributed to DNA sample preparation. The CamPaIGN study was sponsored by the University of Cambridge UK and has received funding from the Wellcome Trust, the Medical Research Council, the Patrick Berthoud Trust, and the NIHR Cambridge Biomedical Research Centre.

The last few decades have produced a large number of proof-of-concept studies in regenerative medicine. However, the route to clinical adoption is fraught with technical and translational obstacles that frequently consign promising academic solutions to the so-called “valley of death.” This review is intended to serve as a blueprint for translational regenerative medicine: we suggest principles to help guide cell and material selection, present key in vivo imaging modalities and argue that the host immune response should be considered throughout therapeutic development. Finally, we suggest a pathway to navigate the often complex regulatory and manufacturing landscape of translational regenerative medicine., J.P.K.A. acknowledges support from an Arthritis Research U.K. Foundation Fellowship (21138) and the Medical Research Council (MRC) (MR/S00551X/1). T.J.K. acknowledges the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Individual European Fellowship (Grant Agreement No. 746980). A.C.R. acknowledges support from the Wellcome Trust Institutional Translational Partnership Award (208858/Z/17/Z). P.S.P. acknowledges funding from the UK Regenerative Medicine Platform (MR/K026739/1), the MRC (MR/R026416/1), and the EPSRC (EP/R511638/1). C.M.M. was supported by the MRC-funded UK Regenerative Medicine Platform Immunomodulation Hub award (MR/L022699/1). W.L.K. is supported by the MRC/UKRI Innovate Fellowship (MR/S005528/1). V.P. acknowledges funding from the UK Regenerative Medicine Platform (MR/R015724/1). R.O.C.O acknowledges support from the UK Regenerative Medicine Platform “Acellular / Smart Materials – 3D Architecture” (MR/R015651/1), the Rosetrees Trust, Wessex Medical Research and Biotechnology and Biological Sciences Research Council (BBSRC) (BB/P017711/1). D.J.S. acknowledges support from a British Heart Foundation (BHF) Intermediate Basic Science Research Fellowship (FS/15/33/31608), the BHF Centre for Regenerative Medicine (RM/17/1/33377), and an MRC Project Grant (MR/R026416/1). F.M.W. acknowledges support from the UK Regenerative Medicine Platform (MR/LO22699/1, MR/R015635/1, MR/K026666/1). S.J.F. acknowledges support from the UK Regenerative Medicine Platform Engineered Cell Environment Hub and the MRC. R.A.B. acknowledges MRC-WT funding of the Cambridge Stem Cell Institute and MRC funding of the UK Regenerative Medicine Platform Pluripotent Stem and Engineered Cell hub (MR/R015724/1) along with funding from the Rosetrees Trust (A1519 M654) and Cure Parkinson's Trust. R.A.B. is also supported by NIHR funding of Biomedical Research Centre Cambridge (146281). R.A.B. is an NIHR Senior Investigator (NF-SI-0616-10011) and a 23 P.I. in the MRC/WT Stem Cell Institute (203151/Z/16/Z). M.M.S. acknowledges support from the grant from the UK Regenerative Medicine Platform “Acellular / Smart Materials – 3D Architecture” (MR/R015651/1), the European Research Council (ERC) Seventh Framework Programme Consolidator grant “Naturale CG” (616417), the Rosetrees Trust and the Wellcome Trust Senior Investigator Award (098411/Z/12/Z). The authors would also like to thank David Pan (MRC) for his input into this manuscript during the course of Phases 1 and 2 of the UK Regenerative Medicine Platform.

Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythaemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the haematopoietic stem/progenitor cell (HSPC) landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking HSCs with megakaryocytes and prospectively identify a novel intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Finally, relative to wild-type HSCs, mutant CALR HSCs show increases in JAK-STAT signalling, the unfolded protein response, cell cycle, and a previously undescribed upregulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis., Work in the Göttgens lab is supported by the Medical Research Council (MR/M008975/1), Wellcome (206328/Z/17/Z), Blood Cancer UK (18002), and Cancer Research UK (RG83389, jointly with A.R.G). Work in the Green lab is supported by Wellcome (RG74909), WBH Foundation (RG91681), and Cancer Research UK (RG83389, jointly with B.G.).

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSCs by activating stress myelopoiesis, such roles in LSCs are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator that drives myeloid differentiation and activates inflammatory programs in both HSCs and LSCs. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across cohorts of patients with AML, each with distinct phenotypic and clinical properties. S1PR3 was high in LSCs and blasts of mature myeloid samples with linkages to chemosensitivity, whereas S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset. Significance: S1PR3 is a novel regulator of myeloid fate in normal hematopoiesis that is heterogeneously expressed in AML. S1PR3 marks a subset of less primitive AML cases with a distinct inflammatory signature and therefore has clinical implications as both a therapeutic target and a biomarker to distinguish primitive from mature AML. See related commentary by Yang et al., p. 3. This article is highlighted in the In This Issue feature, p. 1

Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy invariably infiltrating the bone marrow. Although treatment options for patients with advanced disease have significantly improved in the past years, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary bone marrow failure. The underlying mechanisms impairing normal hematopoiesis in patients with CLL are poorly defined., We would like to express our deepest gratitude to patients who donated blood for this research. Samples were obtained with assistance from the Cambridge Blood and Stem Cell Biobank, funded by the Cambridge Cancer Centre and Cambridge Stem Cell Institute. This work was funded by the Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA and Royal Society. Research in I.R. and E.L. laboratories is supported by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute.

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.

Huntington’s disease (HD) is a devastating, autosomal-dominant neurodegenerative disease, for which there are currently no disease-modifying therapies. Clinical trials to replace the damaged striatal medium spiny neurons (MSN) have been attempted in the past two decades but have met with only limited success. In this study, we investigated whether a clonal, conditionally immortalized neural stem cell line (CTX0E03), which has already shown safety and signals of efficacy in chronic ischemic stroke patients, could rescue deficits seen in an animal model of HD. After CTX0E03 transplantation into the quinolinic acid (QA)-lesioned rat model of HD, behavioral changes were measured using the rotarod, stepping and staircase tests. In vivo differentiation and neuronal connections of the transplanted CTX0E03 cells was evaluated with immunohistochemical staining and retrograde tracing with Fluoro-Gold. We found that transplantation of CTX0E03 gave rise to a significant behavioral improvement compared with the sham- or fibroblast-transplanted group. Transplanted CTX0E03 formed medium spiny neurons (MSNs) (DARPP-32) and GABAergic neurons (GABA, GAD65/67) with BDNF expression in the striatum, while cortically transplanted cells formed Tbr1-positive neurons. Using a retrograde-label we also found stable engraftment and connection of the transplanted cells with host brain tissues. CTX0E03 transplantation also reduced glial scar formation and inflammation, as well as increasing endogenous neurogenesis and angiogenesis. Overall, our results demonstrate that CTX0E03, a clinical-grade neural stem cell line, is effective for pre-clinical test in HD, and, therefore, will be useful for clinical development in the treatment of HD patients., This research was supported by the National Research Foundation of Korea (NRF14 2017M3A9B4061407), Republic of Korea and by the internal funding of ReNeuron, United Kingdom and iPS Bio, Inc., Republic of Korea.

The murine developing epicardium heterogeneously expresses the transcription factors TCF21 and WT1. Here, we show that this cell heterogeneity is conserved in human epicardium, regulated by BNC1 and associated with cell fate and function. Single cell RNAseq of epicardium derived from human pluripotent stem cells (hPSC-epi) revealed that distinct epicardial sub-populations are defined by high levels of expression for the transcription factors BNC1 or TCF21. WT1+ cells are included in the BNC1+ population, which was confirmed in human foetal hearts. THY1 emerged as a membrane marker of the TCF21 population. We show that THY1+ cells can differentiate into cardiac fibroblast (CF) and smooth muscle cells (SMC), while THY1-cells were predominantly restricted to SMC. Knocking down BNC1 during the establishment of the epicardial populations resulted in a homogeneous, predominantly, TCF21high population. Network inference methods using transcriptomic data from the different cell lineages derived from the hPSC-epi, delivered a core transcriptional network organized around WT1, TCF21 and BNC1. This study is a step towards engineering sub-populations of epicardial cells with selective biological activities and unveils a list of epicardial regulators.

In November 2018, the US Food & Drug Administration (FDA) issued a press release that stated: “The potential health benefits of regenerative medicine have spurred major progress in stem-cell biology over the past several decades. But we continue to see bad actors exploit the scientific promise of this field to mislead vulnerable patients into believing they’re being given safe, effective treatments; when instead these stem cell producers are leveraging the field’s hype to push unapproved, unproven, illegal, and potentially unsafe products.", None

Summary Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+). Furthermore, we show that, in adenomatous crypts (APC−/−), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics., Graphical Abstract, Highlights • Somatic mtDNA mutations for quantitative lineage tracing in human intestine • Human intestinal stem cells evolve according to a neutral drift process • Loss of APC causes an increase in the stem cell loss/replacement rate • Intestinal crypts divide once every 30–40 years in the normal human colon, Baker et al. examine the in vivo stem cell biology of human colonic crypts. They reveal that each crypt contains a small number of functional stem cells and that stem cell division is predominantly symmetric and also quantify perturbation of stem cell architecture within adenomas. Additionally, they measure the division rate of human colonic crypts as once every 30–40 years in the healthy colon and demonstrate that the crypt division rate is increased 10-fold within small adenomas.

In cycling tissues that exhibit high turnover, tissue maintenance and repair are coordinated by stem cells. But, how frequently stem cells are replaced following differentiation, aging or injury remains unclear. By drawing together the results of recent lineage-tracing studies, we propose that tissue stem cells are routinely lost and replaced in a stochastic manner. We show that stem cell replacement leads to neutral competition between clones, resulting in two characteristic and recurring patterns of clone fate dynamics, which provide a unifying framework for interpreting clone fate data and for measuring rates of stem cell loss and replacement in vivo. Thus, we challenge the concept of the stem cell as an immortal, slow-cycling, asymmetrically dividing cell., A.M.K. is supported by the Engineering and Physical Sciences Research Council fellowship.

This study demonstrates that EZH2 has stage-specific and diametrically opposite roles during the induction and maintenance stages of AML. However, different transcriptional programs are affected at each stage, identifying mutant EZH2 as a prognostic marker and paradoxically wild-type EZH2 as a potential therapeutic target

Xenopus embryos provide a rapid and accessible in vivo model, expressing a plethora of endogenous kinase and phosphatase enzymes that control protein phosphorylation, often affecting physiological function. Traditionally, detection of protein phosphorylation has been achieved by radioisotope phosphate labelling of proteins, sometimes with in vitro assays using recombinant proteins, or by site-specific phospho-antibodies. However, target phospho-sites and kinases responsible are often unknown, and use of radioactive isotopes is not always desirable. Therefore, as a first step to determining functional significance of potential phosphorylation, it is useful to show that a protein can be phosphorylated in vivo in Xenopus eggs and/or embryos. This protocol describes a non-radioactive method to visualise protein phosphorylation by exposing the protein to the egg/embryo kinase environment and observing a difference in protein migration on SDS-PAGE and western blot with and without treatment with lamda phosphatase enzyme. Subsequent investigation of the ability of site-specific phospho-mutant proteins to recapitulate the effect of phosphatase treatment can be used to explore the identity of the phosphorylated sites. Moreover, detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of post-translational modifications., Manuscript amended to include: L.H. was funded by a Medical Research Council Studentship. Research in AP’s laboratory is supported by Medical Research Council grants MR/K018329/1, MR/L021129/1, and core support from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. LH is supported by a Peterhouse Fellowship.

Stem cell fate decisions are driven by a broad array of signals, both chemical and mechanical. Although much progress has been made in our understanding of the impact of chemical signals on cell fate choice, much less is known about the role and influence of mechanical signalling, particularly in embryonic stem (ES) cells. Many studies use substrates with different stiffness to study mechanical signalling, but changing substrate stiffness can induce secondary effects which are difficult to disentangle from the direct effects of forces/mechanical signals. To probe the direct impact of mechanical stress on cells, we developed an adaptable cell substrate stretcher to exert specific, reproducible forces on cells. Using this device to test the response of ES cells to tensile strain, we found that cells experienced a transient influx of calcium followed by an upregulation of the so-called immediate and early genes. On longer time scales, however, ES cells in ground state conditions were largely insensitive to mechanical stress. Nonetheless, as ES cells exited the ground state, their susceptibility to mechanical signals increased, resulting in broad transcriptional changes. Our findings suggest that exit from ground state of pluripotency is unaffected by mechanical signals, but that these signals could become important during the next stage of lineage specification. A better understanding of this process could improve our understanding of cell fate choice in early development and improve protocols for differentiation guided by mechanical cues.

Oligodendrocyte progenitor cells (OPCs), which differentiate into myelinating oligodendrocytes during central nervous system (CNS) development, are the main proliferative cells in the adult brain. OPCs are conventionally considered a homogeneous population, particularly with respect to their electrophysiological properties, but this has been debated. We show, by using single-cell electrophysiological recordings, that OPCs start out as a homogeneous population, but become functionally heterogeneous, varying both within and between brain regions and with age. These electrophysiological changes in OPCs correlate with the differentiation potential of OPCs; thus, they may underlie the differentiational differences in OPCs between regions and likewise differentiation failure with age.

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.

Proneural basic-helix-loop-helix (bHLH) proteins, such as Neurogenin2 (Ngn2) and Ascl1, are critical regulators at the onset of neuronal differentiation. Endogenously they have largely complementary expression patterns, and have conserved roles in the specification of distinct neuronal subtypes. In Xenopus embryos, xNgn2 is the master regulator of primary neurogenesis forming sensory, inter- and motor neurons within the neural plate, while xAscl1 is the master regulator of autonomic neurogenesis, forming noradrenergic neurons in the antero-ventral region of the embryo. Here we characterise neuronal subtype identity of neurons induced by xNgn2 in the ectoderm of whole Xenopus embryos in comparison with xAscl1, and in ectodermal “animal cap” explants. We find that the transcriptional cascades mediating primary and autonomic neuron formation are distinct, and while xNgn2 and xAscl1 can upregulate genes associated with a non-endogenous cascade, this expression is spatially restricted within the embryo. xNgn2 is more potent than xAscl1 at inducing primary neurogenesis as assayed by neural-β-tubulin. In ectoderm of the intact embryo, these induced primary neurons have sensory characteristics with no upregulation of motor neuron markers. In contrast, xNgn2 is able to up-regulate both sensory and motor neuron markers in naïve ectoderm of animal cap explants, suggesting a non-permissive environment for motor identity in the patterned ectoderm of the whole embryo., This work is supported by the Wellcome Trust [203151] (AP). LH has been supported by an MRC Doctoral Training Award and Peterhouse Research Fellowship.

This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance., CPS is funded through a Children’s Liver Disease Foundation (CLDF) studentship. DAL is funded by the Medical Research Council, Wellcome Trust, GlaxoSmithKline, the Rosetrees Trust, EPSRC and UCLH NIHR Biomedical Research Centre. LV, PM, MCB, NH are funded by ERC Relieve-IMDs and ERC advanced grant New-Chol, Cambridge University Hospitals National Institute for Health Research Biomedical Research Center, and the core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. STR is funded by an MRC Clinician Scientist Fellowship award.

Lineage segregation in the mouse embryo is a finely controlled process dependent upon coordination of signalling pathways and transcriptional responses. Here we employ a conditional deletion system to investigate embryonic patterning and lineage specification in response to loss of Oct4. We first observe ectopic expression of Nanog in Oct4-negative postimplantation epiblast cells. The expression domains of lineage markers are subsequently disrupted. Definitive endoderm expands at the expense of mesoderm; the anterior/posterior axis is positioned more distally and an ectopic posterior-like domain appears anteriorly, suggesting a role for Oct4 in maintaining the embryonic axis. Although primitive streak forms in the presumptive proximal-posterior region, epithelial-to mesenchymal transition is impeded by an increase of E-cadherin, leading to complete tissue disorganisation and failure to generate germ layers. In explant and in vitro differentiation assays, Oct4 mutants also show upregulation of E-cadherin and Foxa2, suggesting a cell-autonomous phenotype. We confirm requirement for Oct4 in self-renewal of postimplantation epiblast ex vivo. Our results indicate a role for Oct4 in orchestrating multiple fates and enabling expansion, correct patterning and lineage choice in the post-implantation epiblast., This work was supported by the Wellcome Trust, Biotechnology and Biological Sciences Research Council, Medical Research Council and the University of Cambridge. The Cambridge Stem Cell Institute is supported by core funding from the Wellcome Trust and Medical Research Council.

Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates resulting in alterations in autophagy. However, the consequences of impaired autophagy on neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation, as well as post-mortem material from patients with Huntington’s disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation, but also directly contributes to global alterations of intracellular post-transcriptional networks., The work was supported by grants from the Swedish Research Council (# K2014-62X-22527-01-3 and K2014-62X-20404-08-5), the Swedish Foundation for Strategic Research (# FFL12-0074), the Swedish Brain Foundation (# FO2014-0106); the Swedish excellence project Basal Ganglia Disorders Linnaeus Consortium (Bagadilico), and the Swedish Government Initiative for Strategic Research Areas (MultiPark & StemTherapy).

Oligodendrocyte Progenitor Cells (OPCs) first appear at mid embryogenic stages during development of the mammalian CNS and a mitotically active population of them remains present even into late adulthood. During the life-time of the organism they initially proliferate and migrate in order to populate the whole nervous tissue, then they massively generate oligodendrocytesand finally they switch to a less mitotically active phase generating new oligodendrocytes at a slow rate in the adult brain; importantly, they can regenerate acutely or chronically destroyed myelin. All the above depend on the capacity of OPCs to regulate their cell cycle within different contexts. In this review we describe the development of OPCs, their differential mitotic behavior in various conditions (embryo, disease, ageing), we discuss what is known about the mechanisms that control their cell cycle and wehighlightfew interesting and still open questions.

Activation of retinoid X receptor γ and vitamin D receptor (VDR) heterodimer via vitamin D enhances oligodendrocyte progenitor differentiation, whereas blocking VDR impairs differentiation and myelination ex vivo., The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines.

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra‐ and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

Apathy is one of the most common psychiatric symptoms experienced by patients with Huntington’s disease (HD). It appears early, progresses with the disease course and has been shown to contribute significantly to caregiver burden. However, what is understood by apathy in HD is not clearly defined nor the underlying mechanisms responsible for it. In this review, we discuss the concept of apathy in the context of HD and propose that a consensus regarding its conceptualisation and subsequently its diagnostic criteria would significantly benefit the field. In order to undertake this work, we began by reviewing the existing literature on the definition and assessment of apathy in HD, its underlying neurobiological basis and its relationship to other related features such as abulia, anhedonia and alexithymia. In the context of HD, apathy could be described by a loss of or diminished motivation, emotion and goal-directed behaviour that is not best explained by motor or social constraints of the disease. However, there is an urgent need to better understand the characteristics of apathy specifically in HD, how they evolve across the disease course, and how they relate to central dopaminergic pathways. Only by undertaking such work can we hope to better understand this early and disabling aspect of HD.

Pluripotent stem cells (PSCs) have the potential to revolutionize biomedical science; however, while it is simple to reproducibly obtain comparable, stable cell lines in mouse, those produced from human material typically show significant variability both within and between cell lines. This is likely due to differences in the cell identity of conventional mouse and human PSCs. It is hoped that recently identified conditions to reprogram human cells to a naïve-like state will produce better PSCs resulting in reproducible experimental outcomes and more consistent differentiation protocols. In this review we discuss the latest literature on the discovery of human naïve-like stem cells and examine how similar they are to both mouse naïve cells and the preimplantation human epiblast.

Background: Recently, the development of Parkinson’s disease (PD) has been linked to a number of genetic risk factors, of which the most common is glucocerebrosidase (GBA) mutations. Methods: We investigated PD and Gaucher Disease (GD) patient derived skin fibroblasts using biochemistry assays. Results: PD patient derived skin fibroblasts have normal glucocerebrosidase (GCase) activity, whilst patients with PD and GBA mutations have a selective deficit in GCase enzyme activity and impaired autophagic flux. Conclusions: This data suggests that only PD patients with a GBA mutation have altered GCase activity and autophagy, which may explain their more rapid clinical progression.

Oesophageal cancer remains one of the least explored malignancies. However, in recent years its increasing incidence and poor prognosis have stimulated interest from the cancer community to understand the pathways to the initiation and progression of the disease. Critical understanding of the molecular processes controlling changes in stem cell fate and the cross-talk with their adjacent stromal neighbours will provide essential knowledge on the mechanisms that go awry in oesophageal carcinogenesis. Advances in linage tracing techniques have represented a powerful technique to start understanding changes in oesophageal cell behaviour in response to mutations and mutagens that favour tumour development. Environmental cues also constitute an important factor in the aetiology of oesophageal cancer. The oesophageal epithelium is a tissue exposed to harsh environmental conditions that not only damage the DNA of epithelial cells but also result in an active stromal reaction, promoting tumour progression. Ultimately, cancer represents a complex interplay between malignant cells and their microenvironment. Indeed, increasing evidence suggests that the accumulation of somatic mutations is not the sole cause of cancer. Instead, non-cell autonomous components, such as the stroma, can significantly contribute from the earliest stages of tumour formation. The realisation that stromal cells play an important role in cancer has transformed this cellular compartment into an attractive and emerging research field. It is becoming increasingly apparent that the tumour microenvironment provides unique opportunities to identify early diagnostic and prognostic markers, as well as potential therapeutic strategies that may synergise with those targeting tumour cells. This chapter compiles recent observations on oesophageal epithelial stem cell biology, and how environmental and micro-environmental changes may lead to oesophageal disease and cancer.

Cancers evolve as a consequence of multiple somatic lesions, with competition between subclones and sequential subclonal evolution. Some driver mutations arise either early or late in the evolution of different individual tumours, suggesting that the final malignant properties of a subclone reflect the sum of mutations acquired rather than the order in which they arose. However, very little is known about the cellular consequences of altering the order in which mutations are acquired. Recent studies of human myeloproliferative neoplasms demonstrates that the order in which individual mutations are acquired has a dramatic impact on the cell biological and molecular properties of tumor initiating cells. Differences in clinical presentation, complications, and response to targeted therapy were all observed and implicate mutation order as an important player in cancer biology. These observations represent the first demonstration that the order of mutation acquisition influences stem and progenitor cell behaviour and clonal evolution in any cancer. Thus far, the impact of different mutation orders has only been studied in hematological malignancies, and analogous studies of solid cancers are now required.

Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.

Remyelination is the regenerative response to demyelination. Although the oligodendrocyte progenitor is established as the major source of remyelinating cells, there is no conclusive evidence on whether mature, differentiated oligodendrocytes can also contribute to remyelination. Using two different inducible myelin-CreER mouse strains in which mature oligodendrocytes were prelabeled by the expression of membrane-bound Green fluorescent protein, we found that after focal spinal cord demyelination, the surrounding surviving labeled oligodendrocytes did not proliferate but remained at a consistent density. Furthermore, existing (prelabeled) oligodendrocytes showed no evidence of incorporation or migration into the lesioned area, or of process extension from the peripheral margins into the lesion. Thus, mature oligodendrocytes do not normally contribute to remyelination and are therefore not a promising target for regenerative therapy.

Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. © 2016 by John Wiley & Sons, Inc.

Enhancing central nervous system (CNS) myelin regeneration is recognized as an important strategy to ameliorate the devastating consequences of demyelinating diseases such as multiple sclerosis. Previous findings have indicated that myelin proteins, which accumulate following demyelination, inhibit remyelination by blocking the differentiation of rat oligodendrocyte progenitor cells (OPCs) via modulation of PKCα. We therefore screened drugs for their potential to overcome this differentiation block. From our screening, tamoxifen emerges as a potent inducer of OPC differentiation in vitro. We show that the effects of tamoxifen rely on modulation of the estrogen receptors ERα, ERβ, and GPR30. Furthermore, we demonstrate that administration of tamoxifen to demyelinated rats in vivo accelerates remyelination. Tamoxifen is a well-established drug and is thus a promising candidate for a drug to regenerate myelin, as it will not require extensive safety testing. In addition, Tamoxifen plays an important role in biomedical research as an activator of inducible genetic models. Our results highlight the importance of appropriate controls when using such models.

The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile “cell-in-a-test-tube” system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis., Research in AP’s laboratory is supported by Medical Research Council grants MR/K018329/1 and MR/L021129/1, and core support from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. GM was supported by Michael Levin at Tufts University through grants from the G. Harold and Leila Y. Mathers Charitable Foundation and the Physical Science Oncology Center supported by Award Number U54CA143876 from the National Cancer Institute.

Myeloproliferative neoplasms (MPNs) are a set of chronic hematopoietic neoplasms with overlapping clinical and molecular features. Recent years have witnessed considerable advances in our understanding of their pathogenetic basis. Due to their protracted clinical course, the evolution to advanced hematological malignancies, and the accessibility of neoplastic tissue, the study of MPNs has provided a window into the earliest stages of tumorigenesis. With the discovery of mutations in CALR, the majority of MPN patients now bear an identifiable marker of clonal disease; however, the mechanism by which mutated CALR perturbs megakaryopoiesis is currently unresolved. We are beginning to understand better the role of JAK2V617F homozygosity, the function of comutations in epigenetic regulators and spliceosome components, and how these mutations cooperate with JAK2V617F to modulate MPN phenotype.

Extracellular vesicles (EVs) contain many proteins, both cytosolic and surface bound. The current model for EV biogenesis dictates that cytosolic proteins remain in the lumen and cell surface proteins reside on the outside of vesicles. This is consistent with the traditional protein trafficking pathway, where proteins destined for the plasma membrane contain a signal sequence targeting them to the secretory pathway. According to this ‘classical’ pathway for membrane and secretory protein trafficking, proteins lacking a signal sequence should not reside at the cell surface. It has been shown that transmembrane proteins are retained in the membrane of EVs and RNAs reside in the lumen of EVs. However, there is little known about the packaging and location of other proteins enriched in EVs. Annexin A2 is a cytosolic protein abundant in EVs. We show for the first time that Annexin A2 is expressed not only in the lumen of EVs as predicted but also on the surface of EVs. This raises fundamental questions regarding Annexin A2 transport to the outer leaflet of the EV membrane as it lacks a signal peptide for secretion.

Funder: Wellcome Trust, Hematopoiesis is one of the best studied adult stem-cell systems, with a differentiation hierarchy progressing from immature hematopoietic stem cells to over 10 distinct mature cell types. Recent technological breakthroughs now make it possible to define transcriptional profiles in thousands of individual cells. Facilitated by the wealth of prior data on cell purification and analysis strategies, hematopoiesis has been one of the earliest experimental systems to which many of these new single-cell sequencing technologies have been applied. In this review, the authors focus on recent studies, which have shed light on heterogeneity within individual populations as well as the relationships between populations, and also attempt to characterize the differences between normal and disease/perturbed states.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

Induced pluripotent stem cells (iPSCs) have the potential to generate patient-specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late-outgrowth endothelial progenitor cells (L-EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L-EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof-of-principle for clinical applications we generated EPC-iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L-EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L-EPC reprogramming allowed for the reliable generation of iPSCs in a 96-well format, which is compatible with high-throughput platforms. Array comparative genome hybridization analysis of L-EPCs versus donor-matched circulating monocytes demonstrated that L-EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC-iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L-EPC line. This work identifies L-EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.

Technologies allowing for the derivation of patient-specific neurons from somatic cells are emerging as powerfulin vitrotools to investigate the intrinsic cellular pathological behaviours of the diseases that affect these patients. While the use of patient-derived neurons to model Parkinson’s disease (PD) has only just begun, these approaches have allowed us to begin investigating disease pathogenesis in a unique way. In this paper, we discuss the advances made in the field of cellular reprogramming to model PD and discuss the pros and cons associated with the use of such cells.

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.

Glioblastoma (GBM) is the most aggressive primary brain tumor and thrives in a microenvironment of relative immunosuppression. The poor clinical outcome of these malignant tumors requires the development of novel treatment options/therapeutic regimens. Accordingly, numerous immunotherapies for GBM are currently being tested in ongoing clinical trials. Herein we have examined the ability of the FDA approved drug topotecan to suppress programmed death-ligand 1 (PD-L1) expression. Our results suggest a role for topotecan as an adjuvant therapy in treatment regimens targeting certain GBM patient subpopulations in whom the expression of PD-L1 has been confirmed., This research was supported by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke/National Institutes of Health (NINDS/NIH).