- Email: [email protected]
Organoids Develop Motor Skills: 3D Human Neuromuscular Junctions
Cell Stem Cell
Previews Organoids Develop Motor Skills: 3D Human Neuromuscular Junctions Justin K. Ichida1,2,3,* and Chien-Ping Ko4 1Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 2Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, CA 90033, USA 3Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 4Section of Neurobiology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2020.01.003
Stem cell technology enables the production of three-dimensional organ-like structures, but engineering multi-tissue anatomy has proven difficult. In this issue of Cell Stem Cell, Martins et al. (2020) show that generating a common progenitor cell for posterior spinal cord and muscle enables the formation of functional neuromuscular junctions in single organoids. Recent advances in pluripotent stem cell technology allow the in vitro generation of three-dimensional organoids that contain the multitude of cell types observed in vivo and self-organize into appropriate substructures (McCauley and Wells, 2017). Within the nervous system, cerebral organoids can recapitulate cortical and subcortical structures, providing new insights into human brain development and disease (Bershteyn et al., 2017). However, reconstructing the neurological circuits that span multiple central nervous system (CNS) regions or connect CNS and non-CNS tissues is a nascent area of study. While fusion of organoids differentiated in separate compartments enables synapse formation between different regions of the nervous system (Marton and Pașca, 2019), it remains unclear whether this strategy is reproducible enough to enable developmental and disease studies. In this issue of Cell Stem Cell, Martins et al. (2020) leverage neuromesodermal progenitor cells, which give rise to both the posterior nervous system and surrounding muscle (Tzouanacou et al., 2009), to generate organoids that contain spinal cord neurons, Schwann cells, and muscle. The resulting organoids self-organize into spatially separated spinal cord and muscle regions, reproducibly form functional neuromuscular junctions (NMJs) decorated with terminal Schwann cells, exhibit central pattern generator-like circuits, and accurately model the NMJ dysfunction observed in myasthenia gravis (MG).
NMJ dysfunction underlies severe neuromuscular diseases including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and MG. Development of patient-specific in vitro NMJ models would provide new insights into disease mechanisms and catalyze the advancement of new therapies for these diseases. Contractile myotubes form in 3D culture (Afshar Bakooshli et al., 2019), suggesting that organoids might enable longer-term culture of muscle. However, differentiating neural and nonneural organoids separately and later fusing them could present reproducibility and scalability challenges. To circumvent this, Martins et al. (2020) devised an elegant strategy based on the fact that in mammals, the posterior spinal cord and at least some of the surrounding muscle originate from a common bipotent progenitor known as the neuromesodermal progenitor cell (Henrique et al., 2015; Tzouanacou et al., 2009). Previous studies showed that WNT and FGF signaling could induce the formation of neuromesodermal progenitors from pluripotent stem cells (Gouti et al., 2014; Henrique et al., 2015), and Martins et al. (2020) used this strategy in 2D human pluripotent stem cell cultures to generate neuromesodermal progenitors (Figure 1). They formed 3D aggregates of the neuromesodermal progenitors and promoted their expansion with FGF, HGF, and IGF. After 5 days, the neuromesodermal progenitors proliferated and differentiated into cells restricted to neural and mesodermal fates which separated
spatially in the neuromuscular organoids (NMOs) (Figure 1). Over the next 50– 100 days, the organoids reached 5–6 mm in diameter and displayed an elongated morphology with neural tissue on one end and muscle cells on the other. More than 80% of the organoids from 4 pluripotent stem cell lines possessed this unique morphology, demonstrating the reproducibility of this technique. Single-cell RNA sequencing at early time points confirmed the posterior identity of the NMOs and the generation of both neural and mesodermal lineages. Single-cell RNA-seq analysis of days 5 and 50 showed that the neural lineages gave rise to neuronal spinal cord motor neurons and interneurons, an early derivative of the neuroectodermal branch resulted in neural crest and Schwann cell production, and the mesodermal progenitors differentiated into skeletal muscle, epithelial cells, and cartilage. RNA sequencing confirmed the reproducibility of forming these different cell types in similar quantities across multiple differentiations. The segregation of neural and muscle compartments was maintained at day 50 when anatomical and functional NMJs became apparent. Fluorescence light microscopy showed clusters of acetylcholine receptors (AChRs) seen at the site of neurites contacting fast-twitch skeletal muscle fibers. In addition, terminal Schwann cells at NMJs, as well as glial cells (GFAP+) and myelination (MBP+), were detected in the NMOs. Furthermore, proliferating muscle progenitors and
Cell Stem Cell 26, February 6, 2020 ª 2020 Elsevier Inc. 131
Cell Stem Cell
Previews Day -3
Day 0 CHIR bFGF
Pluripotent Stem Cells
Day 1
Day 5
bFGF HGF IGF
Neuromesodermal Progenitor Cells
Day 50
Functional NMJs and Muscle Contraction
Neural Muscle
Aggregates
Neural/ Mesodermal Organoids
Neuromuscular Organoids
Myasthenia Gravis Model
Neuronal Circuits
Figure 1. Overview of Neuromuscular Organoid Production and Functional Characterization Stem cell-derived neuromesodermal progenitors differentiated into neural and muscle tissue that developed synchronized neuronal firing and functional neuromuscular junctions capable of modeling myasthenia gravis.
nents. It is also not clear whether the decline of NMJs from day 50 to day 100 seen in NMOs could be due to motor neuron death. Nevertheless, future studies can optimize this NMO model and it seems likely one could integrate other cell types that play key roles in neurodegenerative diseases such as astrocytes and microglia, as well as to include different spinal cord regions and muscle fiber types to address selective vulnerability seen in ALS and SMA. This important study by Martins et al. (2020) will undoubtedly lead to breakthroughs in our understanding of NMJ development and disease mechanisms. ACKNOWLEDGMENTS
satellite-like cells were seen under the basal lamina. Electron microscopy confirmed the tripartite NMJs showing the three key cellular elements: (1) the presynaptic nerve terminals containing synaptic vesicles and active zones, (2) terminal Schwann cells, and (3) the postsynaptic muscle fibers with junctional folds and organized sarcomeres, which are all normal features of mammalian NMJs (Li et al., 2018). Importantly, these NMJs were functional as shown by the fact that signs of muscle contraction, which could be inhibited with curare (AChR blocker), appeared around 50 days in NMOs. Another important finding is the generation of central pattern generator-like circuit in NMOs. Using a multi-electrode array system, Martins et al. (2020) have demonstrated spontaneous calcium oscillations and electrical activities, some of them with synchronous firing seen at day 50. Acute glutamate treatment in day 30 and day 50 organoids significantly increased frequency of spontaneous activity, which was inhibited by glutamate receptor inhibitors (APV and CNQX). Likewise, acetylcholine treatment also increased significantly the electrical activity, consistent with the appearance of AChR clusters at the NMJ at this stage. Furthermore, Martins et al. (2020) showed the presence of interneurons, and treatments with NMDA/5HT, which evoke the rhythmic activity of the locomotor central pattern generators, increased local firings and synchronous network activity from day 30 to day 50 NMOs. 132 Cell Stem Cell 26, February 6, 2020
To test whether the NMOs could model NMJ disorders, Martins et al. (2020) investigated MG, which is caused by autoantibodies primarily against AChRs at the NMJ (Verschuuren et al., 2016). NMOs at day 50 treated with autoantibodies from MG patients showed a significant reduction in the number of AChR clusters, muscle contraction rate, and muscle contraction amplitude as compared with control patients. It would be interesting to use similar approaches to model Lambert-Eaton myasthenic syndrome, which is caused by autoantibodies attacking voltage-gated calcium channels resulting in less ACh release at the NMJ (Verschuuren et al., 2016). When employed using patient-specific iPSCs, the 3D neuromuscular organoids generated by Martins et al. (2020) will be valuable for studying pathogenic mechanisms underlying ALS and SMA. Studies of these diseases have suffered from the lack of human-based models that recapitulate NMJ biology and include critical components such as terminal Schwann cells. The scalable nature of NMOs will provide an ideal platform for drug screening and developing therapies for NMJ diseases. Martins et al. (2020) showed that NMJs form at similar rates in organoids from different batches and lines, which will facilitate translational studies. It is uncertain whether the proportion of NMJs that contain myelinated axons and terminal Schwann cells is consistent between batches and high enough to permit the study of these important NMJ compo-
We are grateful for funding from NIH grant R01NS094721 and the Spinal Muscular Atrophy Foundation to C.-P.K. We also acknowledge funding from NIH grants R01NS097850 and R01DC015530, the New York Stem Cell Foundation, the Tau Consortium, and the Merkin Family Foundation to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator and the Richard N. Merkin Assistant Professor of Stem Cell Biology and Regenerative Medicine at USC. DECLARATION OF INTERESTS C.-P.K. is a Cure SMA Translational Advisory Council member and J.K.I. is a co-founder of AcuraStem, Inc. REFERENCES Afshar Bakooshli, M., Lippmann, E.S., Mulcahy, B., Iyer, N., Nguyen, C.T., Tung, K., Stewart, B.A., van den Dorpel, H., Fuehrmann, T., Shoichet, M., et al. (2019). A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction. eLife 8, 8. Bershteyn, M., Nowakowski, T.J., Pollen, A.A., Di Lullo, E., Nene, A., Wynshaw-Boris, A., and Kriegstein, A.R. (2017). Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia. Cell Stem Cell 20, 435–449.e4. Gouti, M., Tsakiridis, A., Wymeersch, F.J., Huang, Y., Kleinjung, J., Wilson, V., and Briscoe, J. (2014). In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biol. 12, e1001937. Henrique, D., Abranches, E., Verrier, L., and Storey, K.G. (2015). Neuromesodermal progenitors and the making of the spinal cord. Development 142, 2864–2875. Li, L., Xiong, W.C., and Mei, L. (2018). Neuromuscular Junction Formation, Aging, and Disorders. Annu. Rev. Physiol. 80, 159–188. Martins, J.-M.F., Fischer, C., Urzi, A., Vidal, R., Kunz, S., Ruffault, P.-L., Kabuss, L., Hube, I., Gazzero, E., Birchmeier, C., et al. (2020). Self-organizing 3D
Cell Stem Cell
Previews human trunk neuromuscular organoids. Cell Stem Cell 26, this issue, 172–186. Marton, R.M., and Pașca, S.P. (2019). Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease. Trends Cell Biol., S0962-8924(19) 30200-4.
McCauley, H.A., and Wells, J.M. (2017). Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish. Development 144, 958–962. Tzouanacou, E., Wegener, A., Wymeersch, F.J., Wilson, V., and Nicolas, J.F. (2009). Redefining
the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Dev. Cell 17, 365–376. Verschuuren, J., Strijbos, E., and Vincent, A. (2016). Neuromuscular junction disorders. Handb. Clin. Neurol. 133, 447–466.
Playing Favorites: Integrin avb5 Mediates Preferential Zika Infection of Neural Stem Cells Christine Vazquez1 and Kellie Ann Jurado1,* 1Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2020.01.007
The molecular basis dictating specificity of Zika virus infection in neural stem cells (NSCs) remains elusive. Two recent papers in Cell Stem Cell (Zhu et al., 2020) and Cell Reports (Wang et al., 2020) identify integrin avb5 as an internalization factor that increases susceptibility in NSCs and glioblastoma stem cells.
Zika is a mosquito-transmitted virus that primarily causes acute, febrile illness. In more rare incidences or in particular contexts, such as congenital infection, Zika infection can present with neurological manifestations. Infection studies using human brain organoids (Garcez et al., 2016) or induced pluripotent stem cells (Souza et al., 2016; Tang et al., 2016) found that Zika preferentially targets neural progenitor cells for infection. The increased susceptibility of neural progenitor cells to Zika virus remains unclear. However, in this issue of Cell Stem Cell, Zhu et al. (2020) find that Zika virus tropism for glioblastoma neural stem cells is mediated by the SOX2-integrin avb5 axis. Complementary work by Wang et al. (2020) in Cell Reports further supports a role for integrin avb5 as an internalization factor for Zika infection in neural stem cells. Glioblastoma is an extremely lethal brain cancer that is sustained through self-renewing, highly tumorigenic cancer stem-like cells. Glioblastoma stem cells (GSCs) resist current therapy and contribute to near inevitable tumor recurrence. Since Zika selectively infects and kills neural stem cells, it has been proposed as a therapeutic onco-
lytic agent for glioblastoma therapy. Promisingly, Zika infection specifically depletes patient-derived glioblastoma stem cells as opposed to differentiated tumor or neuronal cells in in vitro systems and leads to improved survival in animal models (Zhu et al., 2017). Due to safety concerns with use of wildtype Zika virus in the clinic, understanding how Zika discriminates between stem cells and differentiated neurons could largely improve oncolytic virus approaches. To define the molecular pathways used by Zika that enable increased susceptibility of GSCs, Zhu et al. (2020) investigated the functional consequences of the transcriptional regulator SOX2. SOX2 expression is important for maintenance of stem-like properties in glioblastoma and was previously found to mark Zika infected neural progenitor cells (Souza et al., 2016). The authors found that silencing of SOX2 levels in GSCs attenuated Zika virus infection and identified two mechanisms by which SOX2 contributes to GSCs selective targeting by Zika virus. First, they observed that in GSCs, SOX2 has an inverse relationship with the innate antiviral immune response suggesting that
GSCs may be less immunogenic than differentiated cells. Second, because SOX2 was found to regulate integrin av (ITGAV) expression, the authors looked at the impact of blocking integrin heterodimer partners. They found that blocking the integrin heterodimer complex avb5 specifically inhibited Zika infection through a reduction of viral internalization. In a separate study aimed at determining mediators of Zika virus entry in GSCs, Wang et al. (2020) independently identified integrin avb5 as an important mediator of Zika virus infection in GSCs through CRISPR-mediated screening. Their analysis extended beyond neoplastic stem cells to assess the functional consequence of avb5 inhibition in neurospheres made from human induced pluripotent stem cells and from primary fetal-derived neural stem cells. Wang et al. (2020) report that avb5 inhibition reduced Zika-mediated cell death in neurospheres. Since avb5 is proposed to mediate virus internalization, the impact of integrin inhibition on viral replication would be a more direct readout, but surprisingly, the authors do not report viral load in neurosphere infections. Further, given that Zhu et al.
Cell Stem Cell 26, February 6, 2020 ª 2020 Elsevier Inc. 133
Previews Organoids Develop Motor Skills: 3D Human Neuromuscular Junctions Justin K. Ichida1,2,3,* and Chien-Ping Ko4 1Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 2Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, CA 90033, USA 3Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 4Section of Neurobiology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2020.01.003
Stem cell technology enables the production of three-dimensional organ-like structures, but engineering multi-tissue anatomy has proven difficult. In this issue of Cell Stem Cell, Martins et al. (2020) show that generating a common progenitor cell for posterior spinal cord and muscle enables the formation of functional neuromuscular junctions in single organoids. Recent advances in pluripotent stem cell technology allow the in vitro generation of three-dimensional organoids that contain the multitude of cell types observed in vivo and self-organize into appropriate substructures (McCauley and Wells, 2017). Within the nervous system, cerebral organoids can recapitulate cortical and subcortical structures, providing new insights into human brain development and disease (Bershteyn et al., 2017). However, reconstructing the neurological circuits that span multiple central nervous system (CNS) regions or connect CNS and non-CNS tissues is a nascent area of study. While fusion of organoids differentiated in separate compartments enables synapse formation between different regions of the nervous system (Marton and Pașca, 2019), it remains unclear whether this strategy is reproducible enough to enable developmental and disease studies. In this issue of Cell Stem Cell, Martins et al. (2020) leverage neuromesodermal progenitor cells, which give rise to both the posterior nervous system and surrounding muscle (Tzouanacou et al., 2009), to generate organoids that contain spinal cord neurons, Schwann cells, and muscle. The resulting organoids self-organize into spatially separated spinal cord and muscle regions, reproducibly form functional neuromuscular junctions (NMJs) decorated with terminal Schwann cells, exhibit central pattern generator-like circuits, and accurately model the NMJ dysfunction observed in myasthenia gravis (MG).
NMJ dysfunction underlies severe neuromuscular diseases including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and MG. Development of patient-specific in vitro NMJ models would provide new insights into disease mechanisms and catalyze the advancement of new therapies for these diseases. Contractile myotubes form in 3D culture (Afshar Bakooshli et al., 2019), suggesting that organoids might enable longer-term culture of muscle. However, differentiating neural and nonneural organoids separately and later fusing them could present reproducibility and scalability challenges. To circumvent this, Martins et al. (2020) devised an elegant strategy based on the fact that in mammals, the posterior spinal cord and at least some of the surrounding muscle originate from a common bipotent progenitor known as the neuromesodermal progenitor cell (Henrique et al., 2015; Tzouanacou et al., 2009). Previous studies showed that WNT and FGF signaling could induce the formation of neuromesodermal progenitors from pluripotent stem cells (Gouti et al., 2014; Henrique et al., 2015), and Martins et al. (2020) used this strategy in 2D human pluripotent stem cell cultures to generate neuromesodermal progenitors (Figure 1). They formed 3D aggregates of the neuromesodermal progenitors and promoted their expansion with FGF, HGF, and IGF. After 5 days, the neuromesodermal progenitors proliferated and differentiated into cells restricted to neural and mesodermal fates which separated
spatially in the neuromuscular organoids (NMOs) (Figure 1). Over the next 50– 100 days, the organoids reached 5–6 mm in diameter and displayed an elongated morphology with neural tissue on one end and muscle cells on the other. More than 80% of the organoids from 4 pluripotent stem cell lines possessed this unique morphology, demonstrating the reproducibility of this technique. Single-cell RNA sequencing at early time points confirmed the posterior identity of the NMOs and the generation of both neural and mesodermal lineages. Single-cell RNA-seq analysis of days 5 and 50 showed that the neural lineages gave rise to neuronal spinal cord motor neurons and interneurons, an early derivative of the neuroectodermal branch resulted in neural crest and Schwann cell production, and the mesodermal progenitors differentiated into skeletal muscle, epithelial cells, and cartilage. RNA sequencing confirmed the reproducibility of forming these different cell types in similar quantities across multiple differentiations. The segregation of neural and muscle compartments was maintained at day 50 when anatomical and functional NMJs became apparent. Fluorescence light microscopy showed clusters of acetylcholine receptors (AChRs) seen at the site of neurites contacting fast-twitch skeletal muscle fibers. In addition, terminal Schwann cells at NMJs, as well as glial cells (GFAP+) and myelination (MBP+), were detected in the NMOs. Furthermore, proliferating muscle progenitors and
Cell Stem Cell 26, February 6, 2020 ª 2020 Elsevier Inc. 131
Cell Stem Cell
Previews Day -3
Day 0 CHIR bFGF
Pluripotent Stem Cells
Day 1
Day 5
bFGF HGF IGF
Neuromesodermal Progenitor Cells
Day 50
Functional NMJs and Muscle Contraction
Neural Muscle
Aggregates
Neural/ Mesodermal Organoids
Neuromuscular Organoids
Myasthenia Gravis Model
Neuronal Circuits
Figure 1. Overview of Neuromuscular Organoid Production and Functional Characterization Stem cell-derived neuromesodermal progenitors differentiated into neural and muscle tissue that developed synchronized neuronal firing and functional neuromuscular junctions capable of modeling myasthenia gravis.
nents. It is also not clear whether the decline of NMJs from day 50 to day 100 seen in NMOs could be due to motor neuron death. Nevertheless, future studies can optimize this NMO model and it seems likely one could integrate other cell types that play key roles in neurodegenerative diseases such as astrocytes and microglia, as well as to include different spinal cord regions and muscle fiber types to address selective vulnerability seen in ALS and SMA. This important study by Martins et al. (2020) will undoubtedly lead to breakthroughs in our understanding of NMJ development and disease mechanisms. ACKNOWLEDGMENTS
satellite-like cells were seen under the basal lamina. Electron microscopy confirmed the tripartite NMJs showing the three key cellular elements: (1) the presynaptic nerve terminals containing synaptic vesicles and active zones, (2) terminal Schwann cells, and (3) the postsynaptic muscle fibers with junctional folds and organized sarcomeres, which are all normal features of mammalian NMJs (Li et al., 2018). Importantly, these NMJs were functional as shown by the fact that signs of muscle contraction, which could be inhibited with curare (AChR blocker), appeared around 50 days in NMOs. Another important finding is the generation of central pattern generator-like circuit in NMOs. Using a multi-electrode array system, Martins et al. (2020) have demonstrated spontaneous calcium oscillations and electrical activities, some of them with synchronous firing seen at day 50. Acute glutamate treatment in day 30 and day 50 organoids significantly increased frequency of spontaneous activity, which was inhibited by glutamate receptor inhibitors (APV and CNQX). Likewise, acetylcholine treatment also increased significantly the electrical activity, consistent with the appearance of AChR clusters at the NMJ at this stage. Furthermore, Martins et al. (2020) showed the presence of interneurons, and treatments with NMDA/5HT, which evoke the rhythmic activity of the locomotor central pattern generators, increased local firings and synchronous network activity from day 30 to day 50 NMOs. 132 Cell Stem Cell 26, February 6, 2020
To test whether the NMOs could model NMJ disorders, Martins et al. (2020) investigated MG, which is caused by autoantibodies primarily against AChRs at the NMJ (Verschuuren et al., 2016). NMOs at day 50 treated with autoantibodies from MG patients showed a significant reduction in the number of AChR clusters, muscle contraction rate, and muscle contraction amplitude as compared with control patients. It would be interesting to use similar approaches to model Lambert-Eaton myasthenic syndrome, which is caused by autoantibodies attacking voltage-gated calcium channels resulting in less ACh release at the NMJ (Verschuuren et al., 2016). When employed using patient-specific iPSCs, the 3D neuromuscular organoids generated by Martins et al. (2020) will be valuable for studying pathogenic mechanisms underlying ALS and SMA. Studies of these diseases have suffered from the lack of human-based models that recapitulate NMJ biology and include critical components such as terminal Schwann cells. The scalable nature of NMOs will provide an ideal platform for drug screening and developing therapies for NMJ diseases. Martins et al. (2020) showed that NMJs form at similar rates in organoids from different batches and lines, which will facilitate translational studies. It is uncertain whether the proportion of NMJs that contain myelinated axons and terminal Schwann cells is consistent between batches and high enough to permit the study of these important NMJ compo-
We are grateful for funding from NIH grant R01NS094721 and the Spinal Muscular Atrophy Foundation to C.-P.K. We also acknowledge funding from NIH grants R01NS097850 and R01DC015530, the New York Stem Cell Foundation, the Tau Consortium, and the Merkin Family Foundation to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator and the Richard N. Merkin Assistant Professor of Stem Cell Biology and Regenerative Medicine at USC. DECLARATION OF INTERESTS C.-P.K. is a Cure SMA Translational Advisory Council member and J.K.I. is a co-founder of AcuraStem, Inc. REFERENCES Afshar Bakooshli, M., Lippmann, E.S., Mulcahy, B., Iyer, N., Nguyen, C.T., Tung, K., Stewart, B.A., van den Dorpel, H., Fuehrmann, T., Shoichet, M., et al. (2019). A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction. eLife 8, 8. Bershteyn, M., Nowakowski, T.J., Pollen, A.A., Di Lullo, E., Nene, A., Wynshaw-Boris, A., and Kriegstein, A.R. (2017). Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia. Cell Stem Cell 20, 435–449.e4. Gouti, M., Tsakiridis, A., Wymeersch, F.J., Huang, Y., Kleinjung, J., Wilson, V., and Briscoe, J. (2014). In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biol. 12, e1001937. Henrique, D., Abranches, E., Verrier, L., and Storey, K.G. (2015). Neuromesodermal progenitors and the making of the spinal cord. Development 142, 2864–2875. Li, L., Xiong, W.C., and Mei, L. (2018). Neuromuscular Junction Formation, Aging, and Disorders. Annu. Rev. Physiol. 80, 159–188. Martins, J.-M.F., Fischer, C., Urzi, A., Vidal, R., Kunz, S., Ruffault, P.-L., Kabuss, L., Hube, I., Gazzero, E., Birchmeier, C., et al. (2020). Self-organizing 3D
Cell Stem Cell
Previews human trunk neuromuscular organoids. Cell Stem Cell 26, this issue, 172–186. Marton, R.M., and Pașca, S.P. (2019). Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease. Trends Cell Biol., S0962-8924(19) 30200-4.
McCauley, H.A., and Wells, J.M. (2017). Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish. Development 144, 958–962. Tzouanacou, E., Wegener, A., Wymeersch, F.J., Wilson, V., and Nicolas, J.F. (2009). Redefining
the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Dev. Cell 17, 365–376. Verschuuren, J., Strijbos, E., and Vincent, A. (2016). Neuromuscular junction disorders. Handb. Clin. Neurol. 133, 447–466.
Playing Favorites: Integrin avb5 Mediates Preferential Zika Infection of Neural Stem Cells Christine Vazquez1 and Kellie Ann Jurado1,* 1Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2020.01.007
The molecular basis dictating specificity of Zika virus infection in neural stem cells (NSCs) remains elusive. Two recent papers in Cell Stem Cell (Zhu et al., 2020) and Cell Reports (Wang et al., 2020) identify integrin avb5 as an internalization factor that increases susceptibility in NSCs and glioblastoma stem cells.
Zika is a mosquito-transmitted virus that primarily causes acute, febrile illness. In more rare incidences or in particular contexts, such as congenital infection, Zika infection can present with neurological manifestations. Infection studies using human brain organoids (Garcez et al., 2016) or induced pluripotent stem cells (Souza et al., 2016; Tang et al., 2016) found that Zika preferentially targets neural progenitor cells for infection. The increased susceptibility of neural progenitor cells to Zika virus remains unclear. However, in this issue of Cell Stem Cell, Zhu et al. (2020) find that Zika virus tropism for glioblastoma neural stem cells is mediated by the SOX2-integrin avb5 axis. Complementary work by Wang et al. (2020) in Cell Reports further supports a role for integrin avb5 as an internalization factor for Zika infection in neural stem cells. Glioblastoma is an extremely lethal brain cancer that is sustained through self-renewing, highly tumorigenic cancer stem-like cells. Glioblastoma stem cells (GSCs) resist current therapy and contribute to near inevitable tumor recurrence. Since Zika selectively infects and kills neural stem cells, it has been proposed as a therapeutic onco-
lytic agent for glioblastoma therapy. Promisingly, Zika infection specifically depletes patient-derived glioblastoma stem cells as opposed to differentiated tumor or neuronal cells in in vitro systems and leads to improved survival in animal models (Zhu et al., 2017). Due to safety concerns with use of wildtype Zika virus in the clinic, understanding how Zika discriminates between stem cells and differentiated neurons could largely improve oncolytic virus approaches. To define the molecular pathways used by Zika that enable increased susceptibility of GSCs, Zhu et al. (2020) investigated the functional consequences of the transcriptional regulator SOX2. SOX2 expression is important for maintenance of stem-like properties in glioblastoma and was previously found to mark Zika infected neural progenitor cells (Souza et al., 2016). The authors found that silencing of SOX2 levels in GSCs attenuated Zika virus infection and identified two mechanisms by which SOX2 contributes to GSCs selective targeting by Zika virus. First, they observed that in GSCs, SOX2 has an inverse relationship with the innate antiviral immune response suggesting that
GSCs may be less immunogenic than differentiated cells. Second, because SOX2 was found to regulate integrin av (ITGAV) expression, the authors looked at the impact of blocking integrin heterodimer partners. They found that blocking the integrin heterodimer complex avb5 specifically inhibited Zika infection through a reduction of viral internalization. In a separate study aimed at determining mediators of Zika virus entry in GSCs, Wang et al. (2020) independently identified integrin avb5 as an important mediator of Zika virus infection in GSCs through CRISPR-mediated screening. Their analysis extended beyond neoplastic stem cells to assess the functional consequence of avb5 inhibition in neurospheres made from human induced pluripotent stem cells and from primary fetal-derived neural stem cells. Wang et al. (2020) report that avb5 inhibition reduced Zika-mediated cell death in neurospheres. Since avb5 is proposed to mediate virus internalization, the impact of integrin inhibition on viral replication would be a more direct readout, but surprisingly, the authors do not report viral load in neurosphere infections. Further, given that Zhu et al.
Cell Stem Cell 26, February 6, 2020 ª 2020 Elsevier Inc. 133