The suspected link between infection by Zika virus (ZIKV), a re-emerging flavivirus, and microcephaly is an urgent global health concern. The direct target cells of ZIKV in the developing human fetus are not clear. Here we show that a strain of the ZIKV, MR766, serially passaged in monkey and mosquito cells efficiently infects human neural progenitor cells (hNPCs) derived from induced pluripotent stem cells. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection increases cell death and dysregulates cell-cycle progression, resulting in attenuated hNPC growth. Global gene expression analysis of infected hNPCs reveals transcriptional dysregulation, notably of cell-cycle-related pathways. Our results identify hNPCs as a direct ZIKV target. In addition, we establish a tractable experimental model system to investigate the impact and mechanism of ZIKV on human brain development and provide a platform to screen therapeutic compounds. The suspected link between ZIKV infection and microcephaly is an urgent global health concern. Tang et al. report that ZIKV virus directly infects human cortical neural progenitor cells with high efficiency, resulting in stunted growth of this cell population and transcriptional dysregulation.
Genetic analyses have shaped much of our understanding of cancer. However, it is becoming increasingly clear that cancer cells display features of normal tissue organization, where cancer stem cells (CSCs) can drive tumor growth. Although often considered as mutually exclusive models to describe tumor heterogeneity, we propose that the genetic and CSC models of cancer can be harmonized by considering the role of genetic diversity and nongenetic influences in contributing to tumor heterogeneity. We offer an approach to integrating CSCs and cancer genetic data that will guide the field in interpreting past observations and designing future studies. This Review proposes that the genetic and CSC models of cancer can be harmonized by considering the role of genetic diversity and nongenetic influences in contributing to tumor heterogeneity.
Clinical investigations using stem cell products in regenerative medicine are addressing a wide spectrum of conditions using a variety of stem cell types. To date, there have been few reports of safety issues arising from autologous or allogeneic transplants. Many cells administered show transient presence for a few days with trophic influences on immune or inflammatory responses. Limbal stem cells have been registered as a product for eye burns in Europe and mesenchymal stem cells have been approved for pediatric graft versus host disease in Canada and New Zealand. Many other applications are progressing in trials, some with early benefits to patients. Clinical investigations using stem cell products are addressing a wide spectrum of conditions using many different stem cell types. Trounson and McDonald review the clinical trials in which data have now been published and highlight areas where progress is being made as well as failures and areas of concern.
Adult hippocampal neurogenesis declines in aging rodents and primates. Aging humans are thought to exhibit waning neurogenesis and exercise-induced angiogenesis, with a resulting volumetric decrease in the neurogenic hippocampal dentate gyrus (DG) region, although concurrent changes in these parameters are not well studied. Here we assessed whole autopsy hippocampi from healthy human individuals ranging from 14 to 79 years of age. We found similar numbers of intermediate neural progenitors and thousands of immature neurons in the DG, comparable numbers of glia and mature granule neurons, and equivalent DG volume across ages. Nevertheless, older individuals have less angiogenesis and neuroplasticity and a smaller quiescent progenitor pool in anterior-mid DG, with no changes in posterior DG. Thus, healthy older subjects without cognitive impairment, neuropsychiatric disease, or treatment display preserved neurogenesis. It is possible that ongoing hippocampal neurogenesis sustains human-specific cognitive function throughout life and that declines may be linked to compromised cognitive-emotional resilience. Boldrini et al. find persistent adult neurogenesis in humans into the eighth decade of life, despite declines in quiescent stem cell pools, angiogenesis, and neuroplasticity. Over a 65-year age span, proliferating neural progenitors, immature and mature granule neurons, glia, and dentate gryus volume were unchanged.
The link between Zika virus (ZIKV) infection and microcephaly has raised urgent global alarm. The historical African ZIKV MR766 was recently shown to infect cultured human neural precursor cells (NPCs), but unlike the contemporary ZIKV strains, it is not believed to cause microcephaly. Here we investigated whether the Asian ZIKV strain SZ01 could infect NPCs in vivo and affect brain development. We found that SZ01 replicates efficiently in embryonic mouse brain by directly targeting different neuronal linages. ZIKV infection leads to cell-cycle arrest, apoptosis, and inhibition of NPC differentiation, resulting in cortical thinning and microcephaly. Global gene expression analysis of infected brains reveals upregulation of candidate flavirus entry receptors and dysregulation of genes associated with immune response, apoptosis, and microcephaly. Our model provides evidence for a direct link between Zika virus infection and microcephaly, with potential for further exploration of the underlying mechanisms and management of ZIKV-related pathological effects during brain development. The suspected link between Zika virus (ZIKV) infection and microcephaly has raised urgent global alarm. However, there is so far no direct evidence for ZIKV infection impacting brain development. In this study, Li, Xu, and colleagues show that ZIKV replicates efficiently in the mouse embryonic brain by mainly targeting neural progenitor cells. They also show that infected brains are smaller with enlarged ventricles and a thinner cortex, consistent with a microcephalic phenotype.
Cancer stem cells (CSCs) are tumor cells that have the principal properties of self-renewal, clonal tumor initiation capacity, and clonal long-term repopulation potential. CSCs reside in niches, which are anatomically distinct regions within the tumor microenvironment. These niches maintain the principle properties of CSCs, preserve their phenotypic plasticity, protect them from the immune system, and facilitate their metastatic potential. In this perspective, we focus on the CSC niche and discuss its contribution to tumor initiation and progression. Since CSCs survive many commonly employed cancer therapies, we examine the prospects of targeting the niche components as preferable therapeutic targets. In this perspective, Plaks et al. focus on the CSC niche and discuss its contribution to tumor initiation and progression. Since CSCs survive many commonly employed cancer therapies, they also examine the prospects of targeting the niche components as preferable therapeutic targets.
Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly. Dang et al. show that Zika virus (ZIKV) attenuates growth in cerebral organoids from human embryonic stem cells by targeting neural progenitors. ZIKV activates the TLR3-mediated innate immune response, leading to dysregulation of a network of genes involved in neurogenesis, axon guidance, apoptosis, and differentiation.
Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect. Correction of a disease-causing CFTR mutation in cultured intestinal stem cells from cystic fibrosis patients is demonstrated using the CRISPR/Cas9 system.
The recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina. We also show that AXL expression in radial glia is conserved in developing mouse and ferret cortex and in human stem cell-derived cerebral organoids, highlighting multiple experimental systems that could be applied to study mechanisms of ZIKV infectivity and effects on brain development. The recent outbreak of Zika virus and the association with fetal abnormalities including microcephaly represents a global health emergency. Kriegstein and colleagues survey the expression of candidate Zika virus entry proteins to suggest that high AXL expression in neural stem cells may render this population selectively vulnerable to viral infection.
In addition to their stem/progenitor properties, mesenchymal stromal cells (MSCs) possess broad immunoregulatory properties that are being investigated for potential clinical application in treating immune-based disorders. An informed view of the scope of this clinical potential will require a clear understanding of the dynamic interplay between MSCs and the innate and adaptive immune systems. In this Review, we outline current insights into the ways in which MSCs sense and control inflammation, highlighting the central role of macrophage polarization. We also draw attention to functional differences seen between vivo and in vitro contexts and between species. Finally, we discuss progress toward clinical application of MSCs, focusing on GvHD as a case study. This Review discusses the role of MSCs in sensing and controlling inflammation, highlighting the central role of macrophage polarization, differences based on species and physiological contexts, and potential clinical applications.
Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR , 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR cells were -GFP , -DsRed , and GFP , markers which also highly enriched CFU-Fs, but negative for -CreER and -CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR cells were quiescent, but they proliferated after injury. Therefore, LepR cells are the major source of bone and adipocytes in adult bone marrow. Zhou et al. reveal that cells positive for Leptin receptor (LepR) are the main source of mesenchymal stromal cells (MSCs), bone-forming progenitors, and adipocytes in adult mouse bone marrow.
Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells. Through sequential chemical screening, Theunissen et al. identify a combination of kinase inhibitors that induces and maintains defining features of naive pluripotency in human embryonic stem cells.
Cell therapy can improve cardiac function in animals and humans after injury, but the mechanism is unclear. We performed cell therapy experiments in genetically engineered mice that permanently express green fluorescent protein (GFP) only in cardiomyocytes after a pulse of 4-OH-tamoxifen. Myocardial infarction diluted the GFP cardiomyocyte pool, indicating refreshment by non-GFP progenitors. Cell therapy with bone marrow-derived c-kit cells, but not mesenchymal stem cells, further diluted the GFP pool, consistent with c-kit cell-mediated augmentation of cardiomyocyte progenitor activity. This effect could not be explained by transdifferentiation to cardiomyocytes by exogenously delivered c-kit cells or by cell fusion. Therapy with c-kit cells but not mesenchymal stem cells improved cardiac function. These findings suggest that stimulation of endogenous cardiogenic progenitor activity is a critical mechanism of cardiac cell therapy. ► c-kit cell therapy stimulates endogenous cardiomyocyte progenitors ► Nkx2.5- and Gata4-expressing progenitors increase in the infarct border zone ► c-kit cell therapy leads to improved cardiac function ► No evidence for cardiomyocyte transdifferentiation by bone marrow progenitors
N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 30 untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.
Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1 cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-β (PDGFRβ) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1 cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1 MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury. The cellular origins of fibrosis in different tissues are unclear. Kramann et al. show that Gli1 marks a network of perivascular mesenchymal-stem-cell-like cells that generate myofibroblasts and play a central role in organ fibrosis after injury, and ablating these cells ameliorates fibrosis and rescues organ function.
N6-methyl-adenosine (m 6 A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m 6 A by mapping the m 6 A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m 6 A modification, including transcripts encoding core pluripotency transcription factors. m 6 A is enriched over 3′ untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m 6 A methylases, led to m 6 A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m 6 A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.
The cancer stem cell (CSC) model has been established as a cellular mechanism that contributes to phenotypic and functional heterogeneity in diverse cancer types. Recent observations, however, have highlighted many complexities and challenges: the CSC phenotype can vary substantially between patients, tumors may harbor multiple phenotypically or genetically distinct CSCs, metastatic CSCs can evolve from primary CSCs, and tumor cells may undergo reversible phenotypic changes. Although the CSC concept will have clinical relevance in specific cases, accumulating evidence suggests that it will be imperative to target all CSC subsets within the tumor to prevent relapse.
Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes. In vivo molecular dynamics of adult stem cells have been elusive. Shin et al. used single-cell RNA-seq and a novel bioinformatic approach named Waterfall to reconstruct somatic stem cell dynamics with unprecedented temporal resolution. The genome-wide molecular transitions they identified suggest commonalities among different somatic stem cell systems.
Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. ► Modified mRNAs can express reprogramming proteins and evade antiviral response ► Highly efficient derivation of human iPSCs without genomic integration ► RNA-derived iPSCs faithfully recapitulate the properties of human ESCs ► Efficient directed differentiation of iPSCs to differentiated myotubes
Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications. Recent work suggested that off-target mutagenesis is a problem for genome-editing technology, but Veres et al. show that, at least for human pluripotent stem cells, the frequency of such mutations appears to be low.