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PreprintCited by 210Neurogenesis and neuroplasticity mechanismsXenotransplantation and immune responseNeuroscience and Neuropharmacology Research

Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits

Open access
Linaro, Daniele, Vermaercke, Ben, Iwata, Ryohei, Ramaswamy, Arjun, Davis, Brittany A., Boubakar, Leila, Libé-Philippot, Baptiste, Bilheu, Angéline +5 more·2019·Neuron
DOI 10.1101/626218PMC PMC6899440

Preprint — published as 10.1016/j.neuron.2019.10.002 · Paper page

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Abstract

Summary: How neural circuits develop in the human brain has remained almost impossible to study at the neuronal level. Here we investigate human cortical neuron development, plasticity and function, using a mouse/human chimera model in which xenotransplanted human cortical pyramidal neurons integrate as single cells into the mouse cortex. Combined neuronal tracing, electrophysiology, and in vivo structural and functional imaging revealed that the human neurons develop morphologically and functionally following a prolonged developmental timeline, revealing the cell-intrinsic retention of juvenile properties of cortical neurons as an important mechanism underlying human brain neoteny. Following maturation, human neurons transplanted in the visual cortex display tuned responses to visual stimuli that are similar to those of mouse neurons, indicating capacity for physiological synaptic integration of human neurons in mouse cortical circuits. These findings provide new insights into human neuronal development, and open novel experimental avenues for the study of human neuronal function and diseases. Highlights: Coordinated morphological and functional maturation of ESC-derived human cortical neurons transplanted in the mouse cortex. Transplanted neurons display prolonged juvenile features indicative of intrinsic species-specific neoteny. Transplanted neurons develop elaborate dendritic arbors, stable spine patterns and long-term synaptic plasticity. In the visual cortex transplanted neurons display tuned visual responses that resemble those of the host cortical neurons.

AI snapshot

Written from the paper’s full text.

TL;DR

Xenotransplanted human ESC-derived cortical pyramidal neurons integrate as single cells into neonatal mouse cortex and mature over months along a prolonged human-like timeline. After maturation they show synaptic plasticity and decorrelated, tuned visual responses resembling host mouse neurons, supporting neuron-intrinsic human neoteny and functional circuit integration.

Key findings

  • Transplanted human cortical neurons integrated as single cells, showed no evidence of fusion (0 of 40 cells; no mouse reads in 10,698 scRNA-seq cells from 2 animals), and were mostly deep-layer identity (>60% FoxP2/CTIP2) with no glial or GABAergic marker expression (0 of 19 and 0 of 27 cells, respectively).
  • Human neurons displayed protracted functional and morphological maturation from 1 to 11 months post-transplantation (MPT): immature APs at 1 MPT, mature-looking APs by 10 MPT, spontaneous firing absent after 6 MPT, and increasing spontaneous synaptic events over months.
  • In vivo two-photon imaging showed increasing spine density up to 10–12 MPT, lower spine turnover at 7–10 MPT than 3–6 MPT, and spine survival increasing from ∼2 weeks at 3–6 MPT to ∼8 weeks at 7–11 MPT.
  • Human neurons showed LTP in 7/10 tested cells at 3–6 MPT; in visual cortex, ∼32% (25/77) were active on gray screen, ∼35% (55/155) during gratings, and 25% (38/155) were visually responsive, with 69% (38/55) of active cells visually responsive; median OSI = 0.36 and DSI = 0.25, with decorrelated activity.

Methods

  • Human ESC H9/H9-GFP cells were differentiated into cortical cells and transplanted intraventricularly with EGTA into neonatal P0/P1 immunodeficient mice (NOD/SCID or Rag2−/−); data came from 65 mice of both sexes.
  • The study combined immunohistology, FACS/scRNA-seq, rabies virus monosynaptic tracing, ex vivo whole-cell patch-clamp at 1–11 MPT, biocytin morphology reconstruction, chronic two-photon structural imaging, LTP recordings, and in vivo two-photon calcium imaging with GCaMP6s/nls-dTomato during visual stimulation.
  • Analyses included Welch one-way ANOVA with Games-Howell post hoc tests, mixed-model ANOVA, Kolmogorov-Smirnov tests, Spearman correlations, and custom MATLAB routines; visual responses were compared with mouse L2/3 pyramidal neurons.

Limitations

  • The paper states that part of the prolonged maturation may reflect a suboptimal xenotransplantation environment, and that transplanted neurons remain less mature than adult human neurons: smaller cell bodies and dendritic arbors, only a fraction showing visual responses, no dendritic spine/synapse pruning, and lower spine density than adult mouse neurons at 10 MPT.
  • The paper notes that the 25% minority of visually responsive cells could reflect connectivity still being too low, excitatory/inhibitory imbalance, or improper connectivity.
  • Evident from the text: the model is a mouse host with immunodeficient neonatal mice, not human cortex; several key analyses rely on small animal numbers (e.g., rabies tracing n = 2 mice, scRNA-seq n = 2 animals, in vivo imaging n = 5 animals, LTP n = 10 human cells and n = 5 mouse cells).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/626218

Cites

98 of 98 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

49 of 83 cited sources checked · 19 supported · 2 flagged · accuracy not yet measured

  • Premisecitation

    Human PSC-derived pyramidal cortical neurons and interneurons transplanted in the mouse brain develop morphologically and synaptically within their host.

    “Human PSC-derived pyramidal cortical neurons and interneurons transplanted in the mouse brain develop morphologically and synaptically within their host”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Anderson and Vanderhaeghen, 2014 source

      The Modeling temporal patterns of corticogenesis section (page not provided) reports that transplanted human PSC-derived pyramidal neurons develop complex dendrites, spines, and functional synaptic activity in mouse cortex, but only after prolonged maturation. For transplanted interneurons, it reports limited terminal differentiation even after six months; the synaptogenesis finding concerns co-culture with mouse neurons, not transplantation. The claim therefore overextends the evidence for interneurons developing synaptically within the host. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Espuny-Camacho et al., 2013 source
    • cites Maroof et al., 2013 source

      The Results section on neuronal differentiation and migration reports that transplanted human interneuron precursors migrated within the mouse cortex, but most retained immature morphologies at 1–2 months and had not become mature interneurons. The Discussion reports limited integration at three months; synaptic activity was demonstrated in vitro, not in the transplanted cells. The paper does not report transplanting human PSC-derived pyramidal neurons. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    Dendritic spines of mouse neocortical neurons display high dynamics and turnover only for a few weeks before adulthood.

    “In contrast, dendritic spines of mouse neocortical neurons display high dynamics and turnover only for a few weeks before adulthood (Hofer et al., 2009, Holtmaat and Svoboda, 2009).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Hofer et al., 2009 source

      The Abstract reports spine gain and loss in adult mouse visual cortex and says that monocular deprivation doubled the rate of spine formation. That contradicts the claim that substantial spine dynamics and turnover occur only for a few weeks before adulthood; the paper also says spines remain sensitive to altered sensory input throughout life. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Holtmaat and Svoboda, 2009 source
  • Interpretationown data

    There is an absence of transplant-to-host cell fusion events.

    “indicating the absence of transplant-to-host cell fusion events”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Ying et al., 2002 source

      The discussion (page not identified in the index) reports spontaneous fusion between embryonic stem cells and brain cells in co-culture and advises checking both donor and host markers. The blastocyst-injection passage describes contributions from hybrids formed before injection; it does not establish whether transplanted cells fused with host cells. A search for transplant-to-host fusion found no passage establishing its absence. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
  • Premisecitation

    Transplanted human neurons develop more slowly than similarly transplanted mouse neurons.

    “transplanted human neurons develop more slowly than similarly transplanted mouse neurons”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Falkner et al., 2016 source
    • cites Gaspard et al., 2008 source

      The “Cortical axonal projections of ESC neurons” section describes neurons grafted into neonatal mice and assessed after one month, but it does not compare transplanted human and mouse neurons. A search for human neurons and species-specific developmental timing found no passage addressing that comparison; page numbers were not available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites Michelsen et al., 2015 source

      The ‘Progressive Axonal Outgrowth’ section (page not available) documents the development of transplanted mouse ESC-derived cortical neurons, including increasing axonal growth over the first month. Our copy does not compare their developmental pace with transplanted human neurons, so it does not establish that human neurons develop more slowly. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    Xenotransplantation of human neurons into the mouse brain can provide a means to study human neuronal maturation under more physiologically realistic conditions.

    “xenotransplantation of human neurons into the mouse brain, which can provide a means to study human neuronal maturation under more physiologically realistic conditions”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Anderson and Vanderhaeghen, 2014 source
    • cites Espuny-Camacho et al., 2013 source
    • cites Maroof et al., 2013 source
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 32
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Alterations of neuronal maturation are associated with neurodevelopmental disorders.

    “alterations of neuronal maturation are associated with neurodevelopmental disorders”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Hutsler and Zhang, 2010 source
    • cites Pfeiffer et al., 2010 source
    • cites Takashima et al., 1981 source

  • Classified as probably not a substantial claim.

    Premisecitation

    The mechanisms underlying the assembly of human neurons into complex neural circuits remain poorly understood.

    “those underlying the assembly of human neurons into complex neural circuits remain poorly understood”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Bae et al., 2015 source

      The Abstract says that understanding of brain development remains incomplete, and “Neurogenetics is Just the Beginning” describes the challenges of studying human-specific circuits. The paper also discusses circuit-assembly molecules in “Noncoding DNA and Human Brain Evolution,” but it does not specifically say that the mechanisms by which human neurons assemble into complex circuits remain poorly understood. No page numbers are available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites Defelipe, 2011 source

      The Introduction discusses distinctive human cortical circuits, and the section on species-specific variations says that further studies may reveal more differences. Neither passage addresses whether the mechanisms by which human neurons assemble into complex circuits are poorly understood; searches for circuit formation and assembly found no passage that does. (LLM, conf=0.70)

      Experimental — accuracy not yet measured
    • cites Geschwind and Rakic, 2013 source

  • May combine two claims.

    Interpretationcitation

    Transplanted human neurons integrating the mouse cortex display protracted physiological maturation over months, until 11 MPT, at which stage their properties resemble those of adult cortical neurons.

    “Thus, transplanted human neurons integrating the mouse cortex display protracted physiological maturation over months, until 11 MPT, at which stage their properties resemble those of adult cortical neurons”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Beaulieu-Laroche et al., 2018 source

      The STAR Methods (Experimental Model and Subject Details; no page given) describe human tissue from adult neurosurgical patients and experiments using rats. The Discussion describes differences between human and rat neurons ex vivo. Searches for transplantation, mouse cortex, and 11 months post-transplantation found no passage addressing the claimed maturation timeline. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites Testa-Silva et al., 2014 source

      The Results and Discussion describe recordings from adult human cortical tissue and separate mouse cortical tissue, not human neurons transplanted into mice. Searches for transplanted neurons, months post-transplantation, and 11 MPT found no passage addressing their maturation or properties at that time point. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    This neoteny may be critical for the acquisition of human-specific cognitive features and primarily implicate cortical neurons.

    “This neoteny, or retention of juvenile traits in a more mature organism, may be critical for the acquisition of human-specific cognitive features”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Gould, 1992 source
    • cites Bufill et al., 2011 source
    • cites Defelipe, 2011 source

      In “Species-specific variations in cortical microstructure” (page unavailable), the paper says specialization of human cortical circuits appears critical to higher mental abilities. Figure 4 in “Brain Size and Intellectual Capabilities” describes maturation of cortical neurons in children. Neither passage links retention of juvenile traits (neoteny) to the acquisition of human-specific cognitive features; searches for “neoteny” and delayed maturation did not find that link. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Human cortical interneurons have been shown to integrate extensively into the host mouse brain thanks to the remarkable migratory properties of this class of neurons.

    “Human cortical interneurons have been shown to integrate extensively into the host mouse brain thanks to the remarkable migratory properties of this class of neurons”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Cunningham et al., 2014 source
    • cites Fandel et al., 2016 source
    • cites Huo et al., 2018 source

      The Results (page unavailable) show that transplanted human GABAergic interneurons survived in mouse brains and migrated along endogenous pathways to the olfactory bulb and hippocampus. They do not establish extensive integration into host circuitry or show that migration caused such integration; the Results also report few grafted cells in the cortex. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    A recent study using in vivo imaging showed limited synaptic integration of human cortical pyramidal neurons in the mouse cortex.

    “A recent study using in vivo imaging showed limited synaptic integration of human cortical pyramidal neurons in the mouse cortex”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Real et al., 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Comparison with the chimpanzee also indicates human neoteny, although less pronounced.

    “comparison with the chimpanzee also indicates human neoteny, although less pronounced”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Bianchi et al., 2013 source
    • cites Liu et al., 2012 source

      The paper’s Discussion reports delayed prefrontal cortical synaptic development in humans relative to chimpanzees, supporting the comparison as evidence of human neoteny. It does not establish that the delay relative to chimpanzees is less pronounced than the delay relative to macaques; it describes synaptic-gene expression in both comparison species as peaking shortly after birth. No page number is available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    A critical feature of juvenile neurons is their structural dynamics, which precedes the establishment of synaptic connectivity.

    “A critical feature of juvenile neurons is their structural dynamics, which precedes the establishment of synaptic connectivity”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Grutzendler et al., 2002 source
    • cites Zuo et al., 2005 source

      The Results show that dendritic filopodia in young mice are highly dynamic and can become spines, supporting a role for structural changes before the formation of individual synapses. But the paper does not say that juvenile structural dynamics precedes the establishment of synaptic connectivity generally; its Introduction says synaptic density already increases rapidly before birth. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    Dendritic spines of transplanted human neurons follow a prolonged timeline that resembles the maturation time of human cortical neurons and that is largely intrinsic to these neurons, as they still display juvenile-like dynamic patterns at 3-6 MPT (e.g., in a mature mouse brain environment).

    “These data support the notion that dendritic spines of transplanted human neurons follow a prolonged timeline that resembles the maturation time of human cortical neurons (Petanjek et al., 2011) and that is largely intrinsic to these neurons, as they still display juvenile-like dynamic patterns at 3–6 MPT (e.g., in a mature mouse brain environment)”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Petanjek et al., 2011 source
    • cites Falkner et al., 2016 source
    • cites Holtmaat et al., 2005 source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The transplanted neurons displayed a prolonged timeline of morphogenesis, corresponding to late fetal neurons, which was strongly correlated with functional maturation parameters.

    “The transplanted neurons displayed a prolonged timeline of morphogenesis, corresponding to late fetal neurons (Huttenlocher, 1979, Huttenlocher et al., 1982, Huttenlocher et al., 1982–1983, Mrzljak et al., 1990, Petanjek et al., 2011), which was strongly correlated with functional maturation parameters”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Huttenlocher, 1979

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Huttenlocher et al., 1982 source
    • cites Petanjek et al., 2011 source

      The opening section (p. 1) reports prolonged remodeling of dendritic spines in human prefrontal neurons into the third decade of life, supporting the general point that neuronal development can be prolonged. The paper studies postnatal human tissue, not transplanted neurons or late-fetal neuronal morphogenesis; its Discussion (p. 4) links structural and functional changes but does not report a strong correlation with functional maturation parameters. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Neurons in mouse visual cortex are characterized by diversely tuned responses to specific properties of visual stimuli, such as stimulus orientation and stimulus direction.

    “Neurons in mouse visual cortex are characterized by diversely tuned responses to specific properties of visual stimuli (e.g., stimulus orientation stimulus direction) (Niell and Stryker, 2008).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Niell and Stryker, 2008 source

  • May combine two claims.

    Findingown data

    The spine turnover ratio decreased at 7-10 MPT, indicating that the neurons had reached a more stable stage, although still more unstable than the adult mouse cortex.

    “It then decreased at 7–10 MPT, indicating that the neurons had reached a more stable stage (Figure 4E), although still more unstable than the adult mouse cortex”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Grutzendler et al., 2002 source
    • cites Zuo et al., 2005 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Recent work has shown faster development of chimpanzee versus human neurons xenotransplanted in the mouse brain.

    “However, it is interesting to note in this context that recent work has shown faster development of chimpanzee versus human neurons xenotransplanted in the mouse brain (Marchetto et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Marchetto et al., 2019 source

      The Results section on cross-species dendritic maturation reports that some aspects of transplanted chimpanzee neurons developed earlier than those of human neurons in the mouse brain. The claim is broader than the finding: the paper says this pattern did not hold for all dendritic measures, and human neurons surpassed chimpanzee neurons on some measures later in development. The indexed copy provides no page numbers. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The transplantation of human PSC-derived cortical pyramidal neurons into the lesioned adult cortex has been shown to be promising.

    “The transplantation of human PSC-derived cortical pyramidal neurons into the lesioned adult cortex has been shown to be promising (Espuny-Camacho et al., 2018, Tornero et al., 2013, Tornero et al., 2017)”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Espuny-Camacho et al., 2018 source
    • cites Tornero et al., 2013 source
    • cites Tornero et al., 2017

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingcitation

    These neurons fire in a decorrelated fashion and display visually tuned responses resembling those of mouse cortical neurons.

    “These neurons fire in a decorrelated fashion and display visually tuned responses resembling those of mouse cortical neurons (Andermann et al., 2011).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Andermann et al., 2011 source

      The Results section (“Identification and Targeting of Functionally Distinct Higher Cortical Areas”) reports visually driven responses and tuning to spatial and temporal frequency, speed, orientation, and motion direction in mouse cortical neurons. The cited paper does not establish the separate claim that these neurons fire in a decorrelated fashion; searches for decorrelated firing and neuronal correlations found no supporting passage. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    Dendritic spine dynamics suggest that transplanted human neurons are still undergoing synaptic growth and have not yet reached the stage of dendritic spine/synapse pruning.

    “Similarly, dendritic spine dynamics suggest that neurons are still undergoing synaptic growth and have not yet reached the stage of dendritic spine/synapse pruning, which is supposed to last for more than a decade in some cortical areas (Petanjek et al., 2011).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Petanjek et al., 2011 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Morphologies were reconstructed using the semi-automated procedure provided by ShuTu, followed by manual curation.

    “The morphologies were reconstructed using the semi-automated procedure provided by ShuTu (Jin et al., 2019), followed by manual curation.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jin et al., 2019 source

  • May combine two claims.

    Premisecitation

    PSC-derived human cortical neurons transplanted as bulk cell populations displayed rapid dendritic spine turnover throughout the experiment and reached very limited levels of synaptic connectivity with the host brain.

    “This contrasts with recent findings in PSC-derived human cortical neurons transplanted as bulk cell populations, which displayed rapid dendritic spine turnover throughout the experiment (spine half-life <4 days versus 2 weeks at 3–4 months), and reached very limited levels of synaptic connectivity with the host brain (Real et al., 2018).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Real et al., 2018 source

      In “Human synaptic development imaged in vivo” (page unavailable), the paper reports substantial spine turnover at 3 months, but lower turnover at 4 months; it could not quantify spine dynamics before 3 months. It does not establish a spine half-life of less than 4 days throughout the experiment. “Functional human cortical networks imaged in vivo” (page unavailable) supports limited host input: tracing found that 7.5% ± 1.5% of inputs came from host neurons. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    At 24 h, transplanted cells were found within the cortical tissue and appeared to migrate along the radial glia processes still present at this stage.

    “At 24 h, transplanted cells were found within the cortical tissue and appeared to migrate along the radial glia processes still present at this stage”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The progressive maturation of human neurons was accompanied by progressive maturation of intrinsic properties, including membrane potential hyperpolarization, decrease in input resistance, increase in maximum sodium currents, and shortening of AP half-width.

    “These changes were accompanied by progressive maturation of intrinsic properties, including membrane potential hyperpolarization, decrease in input resistance, increase in maximum sodium currents, and shortening of AP half-width”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The majority of transplanted cells (>60%) expressed markers of deep cortical layer identity (FoxP2/CTIP2) and were located primarily in layer 5/6 (L5/6), although some were found in L2/3.

    “The majority of cells (>60%) expressed markers of deep cortical layer identity (FoxP2/CTIP2) and were located primarily in layer 5/6 (L5/6), although some were found in L2/3”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A minority of transplanted cells expressed upper-layer marker Cux1, and these were found exclusively in L2/3.

    “A minority of cells expressed upper-layer marker Cux1, and these were found exclusively in L2/3”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    None of the transplanted cells expressed markers of glial cells (0 of 19 cells, n = 2 animals) or GABAergic neurons (0 of 27 cells, n = 2 animals).

    “None of the cells expressed markers of glial cells (0 of 19 cells, n = 2 animals) or GABAergic neurons (0 of 27 cells, n = 2 animals)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    These changes were paralleled by a decrease in spontaneous activity and increased peak firing rates.

    “These changes were paralleled by a decrease in spontaneous activity (Figures 2F and 2G) and increased peak firing rates (Figure 2E)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The distributions of preferred orientations and direction selectivity indices of human and mouse neurons were similar.

    “The distributions of preferred orientations and direction selectivity indices of human and mouse neurons were similar.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Compared to mouse neurons, visually responsive human neurons showed weaker selectivity for orientation but not for direction of motion.

    “Compared to mouse neurons, visually responsive human neurons showed weaker selectivity for orientation but not for direction of motion (Table S1), which may reflect the maturing nature of the underlying circuitry (Rochefort et al., 2011).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Orientation and direction selectivity were pronounced, with 60% and 78% of visually responsive cells showing tuned responses for stimulus orientation and direction (OSI and DSI >0.2), similar to mouse neurons.

    “Orientation and direction selectivity were pronounced, with 60% and 78% of visually responsive cells showing tuned responses for stimulus orientation and direction (OSI and DSI >0.2; Figures 7B and 7F), similar to mouse neurons (Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Xenotransplanted human neurons fired independently, with cells showing calcium transients at distinct time points.

    “Xenotransplanted neurons fired independently, with cells showing calcium transients at distinct time points (Figure 6D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Spine sizes and neck lengths of xenotransplanted neurons were higher than those of host mouse neurons and in the range of those reported in the human cortex.

    “Spine sizes and neck lengths of xenotransplanted neurons were higher than those of host mouse neurons (Figures 3I–3K) and in the range of those reported in the human cortex”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Benavides-Piccione et al., 2002 source

  • May combine two claims.

    Findingown data

    Xenotransplanted human cortical neurons show robust spontaneous and visually driven activity in more than one-fourth of sampled cells, and more than two-thirds of spontaneously active cells respond to visual stimulation.

    “These experiments revealed robust spontaneous and visually driven activity in more than one-fourth of the sampled xenotransplanted cells, with more than two-thirds of spontaneously active cells displaying responses to visual stimulation (Figures 6C–6J; Video S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Over time (1-7 MPT), transplanted neurons showed pronounced changes of dendritic length and complexity, as well as dendritic spine density and morphology.

    “We observed over time (1–7 MPT) pronounced changes of dendritic length and complexity (Figures 3A–3C and S3A–S3C), as well as dendritic spine density and morphology (Figures 3D–3F)”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Visual stimulation increases the frequency and amplitude of calcium transients in human neurons.

    “Note how visual stimulation increases the frequency and amplitude of calcium transients (red lines)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Approximately 25% of xenotransplanted human neurons showed clear visual responses, and 69% of neurons that showed calcium activity were deemed visually responsive.

    “Approximately 25% of xenotransplanted neurons (38 of 155 labeled neurons) showed clear visual responses (median ΔF/F0 response >3 SDs over baseline for >1 s), resulting in 69% (38 of 55) of neurons that showed calcium activity being deemed visually responsive.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Up to 25% of xenotransplanted neurons and 69% of those that show calcium transients display functional responses to visual sensory stimulation.

    “We report that up to 25% of the xenotransplanted neurons and 69% of those that show calcium transients display functional responses to visual sensory stimulation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Under the conditions used, 50%-70% of cells express both GCaMP6s and nls-dTomato under doxycycline treatment.

    “In these conditions, 50%–70% cells express both GCaMP6s and nls-dTomato under doxycycline treatment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Human neurons are initially as highly dynamic as juvenile neurons and then become progressively more stable over a prolonged 6- to 11-month-long period.

    “Moreover, human neurons are initially as highly dynamic as juvenile neurons and then become progressively more stable, albeit only over a prolonged 6- to 11-month-long period.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Transplanted human cortical neurons integrated as single cells in the mouse cortex follow a coordinated program of functional and morphological maturation that is similar to the one observed in the human cortex in vivo, which is distinct from the much faster-developing mouse cortical tissue.

    “These data indicate that transplanted human cortical neurons integrated as single cells in the mouse cortex follow a coordinated program of functional and morphological maturation that is similar to the one observed in the human cortex in vivo, which is distinct from the much faster-developing mouse cortical tissue”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    As transplanted human neurons develop according to a prolonged timeline, they appear to retain juvenile properties in an otherwise mature mouse brain.

    “As transplanted human neurons develop according to a prolonged timeline, they appear to retain juvenile properties in an otherwise mature mouse brain”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Human cortical pyramidal neurons are characterized by high spine density and large spine size compared to mouse neurons.

    “Human cortical pyramidal neurons are characterized by high spine density and large spine size compared to mouse neurons (Benavides-Piccione et al., 2002)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Benavides-Piccione et al., 2002 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    During spine analysis, the 3D structure of the spine was taken into account by scrolling through the volume, which allowed distinguishing actual spines from processes passing above and below the branch of interest.

    “During this process we took the 3D structure of the spine into account by scrolling through the volume. This allowed us to distinguish between actual spines and processes passing above and below the branch of interest.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The intrinsic retention of juvenile properties in human cortical neurons may constitute the cellular basis of the brain neoteny that is thought to have played a major role in human evolution.

    “The intrinsic retention of juvenile properties in human cortical neurons may constitute the cellular basis of the brain neoteny that is thought to have played a major role in human evolution (Gould, 1992).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gould, 1992 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Titer check was performed on HEK293T cell culture for every batch of lentiviral preparation.

    “Titer check was performed on HEK293T cell culture for every batch of lentiviral preparation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Two-photon movies for all experiments collected during one session were motion registered to a common reference image constructed by registering and averaging 1200 frames from the center of the session.

    “Two-photon movies for all experiments collected during 1 session were motion registered to a common reference image. This image was constructed by registering and then averaging 1200 frames from the center of the session.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Regions-of-interest were manually segmented using information from both red and green channels.

    “We manually segmented regions-of-interest (ROIs) using information from both red and green channels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Xenotransplanted human neurons can integrate in the adult mouse brain and gain information-processing functioning.

    “This shows that xenotransplanted human neurons can integrate in the adult mouse brain and gain information-processing functioning.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Results are shown as mean ± standard error (SEM) of at least three biologically independent experiments.

    “Results are shown as mean ± standard error (SEM) of at least three biologically independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mouse anti-human nucleus antibody was used to confirm the human identity of the recorded cells.

    “using as primary antibodies chicken anti-GFP (Abcam, 1:2000) and occasionally mouse anti-human nucleus (Merck, 1:500) to confirm the human identity of the recorded cells.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Differentiated cortical cells were validated for neuronal and cortical markers by immunostaining.

    “Differentiated cortical cells were validated for neuronal and cortical markers by immunostaining”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Juvenile neurons could engage functionally into a mature cortical circuit in relatively old animals and yet display responses highly similar to those of the host neurons.

    “implying that juvenile neurons could engage functionally into a mature cortical circuit in relatively old animals and yet display responses highly similar to those of the host neurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The host brain provides not only a permissive environment but also instructive cues for spine morphogenesis and synaptogenesis.

    “suggesting that the host brain provides not only a permissive environment but also instructive cues for spine morphogenesis and synaptogenesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    While xenotransplanted human neurons have achieved unprecedented maturation, some of their properties still correspond to a less mature stage than adult human neurons.

    “While the xenotransplanted human neurons described here have achieved unprecedented maturation, some of their properties still correspond to a less mature stage than adult human neurons.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Human cortical neurons take up to several years to reach full maturity.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    To reduce effects of shot noise, 50 frames collected per section were averaged and a 3D median filter was applied over the entire stack.

    “To reduce effects of shot noise, we averaged over 50 frames collected per section and applied a 3D median filter (Fiji, kernel size = [1.5, 1.5, 4]) over the entire stack.”
    Quote in the paperNot a substantial claim
52 of 52

  • The paper relies on this without support.

    Transplants of cortical pyramidal neurons tend to form poorly integrated lumps in the host tissue.

    “A major limitation of transplants of cortical pyramidal neurons is that they tend to form poorly integrated lumps in the host tissue”

  • The paper relies on this without support.

    The functional maturation of neurons is coordinated with their morphogenesis.

    “The functional maturation of neurons is coordinated with their morphogenesis”

  • The paper relies on this without support.

    Synaptic integration within the host cortex may allow xenotransplanted neurons to respond to internal and external events and encode information.

    “Synaptic integration within the host cortex may allow xenotransplanted neurons to respond to internal and external events and encode information”

  • The paper relies on this without support.

    Human cortical neurons take up to several years to reach full maturity.

    “in line with the fact that human cortical neurons take up to several years to reach full maturity.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Human xenotransplanted CPNs from 2 to 18 months post-transplantation revealed a much more protracted pattern of dendritic spine development than mouse CPNs, confirming cell-intrinsic neuronal neoteny.

    “Similar analysis of dendritic spine development in human xenotransplanted CPNs (from 2 to 18 MPT) revealed a much more protracted pattern, confirming the cell-intrinsic neuronal neoteny described previously.15,16,17,18,20”

    From Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition (2024) · cites this paper as [20]

    The Results section on dendritic spine dynamics reports prolonged maturation in xenotransplanted human cortical neurons, with increasing spine density up to 10–12 months post-transplantation (MPT); the Discussion contrasts their months-long spine dynamics with the few weeks seen in mouse neurons. The paper describes this pattern as largely intrinsic, but allows that the transplantation environment may contribute. It does not document dendritic spine development through 18 MPT, so the claimed range and unqualified confirmation go beyond this source. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Xenotransplanted human CPNs mature following a protracted timeline, taking months to develop complex dendritic morphology, mature dendritic spines, functional synapses, and cortical circuit integration.

    “Using this experimental approach, xenotransplanted human CPNs mature following a protracted timeline, taking months to develop complex dendritic morphology, mature dendritic spines, functional synapses, and cortical circuit integration.20”

    From Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition (2024) · cites this paper as [20]

  • The xenotransplantation model in the neonatal mouse cerebral cortex recapitulates some features of CPN development.

    “To assess the role of SRGAP2B/C in neuronal differentiation and synaptic development of human CPNs in vivo, we used a model of xenotransplantation in the neonatal mouse cerebral cortex that recapitulates some of the features of CPN development15,20 (Figure 1B).”

    From Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition (2024) · cites this paper as [20]

  • Human cortical pyramidal neurons develop along a prolonged neotenic timescale when xenotransplanted in the fast-developing mouse cortex, pointing to cell-intrinsic mechanisms.

    “Human cortical pyramidal neurons (CPNs) develop along a prolonged neotenic timescale when xenotransplanted in the fast-developing mouse cortex, pointing to cell-intrinsic mechanisms.15,16,17,18,19,20”

    From Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition (2024) · cites this paper as [20]

  • Human CPNs found in the mouse cortex expressed mostly deep-layer 5/6 markers, while few expressed upper layer 2/3 markers, suggesting that the majority correspond to deep-layer fate neurons.

    “Human CPNs found in the mouse cortex were found to express mostly deep-layer 5/6 markers, while few of them expressed upper layer 2/3 markers (Figure S2C), suggesting that in our experimental setting, the majority of the xenotransplanted neurons found in the mouse cortex correspond to deep-layer fate neurons, as expected.20”

    From Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition (2024) · cites this paper as [20]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting counts of visually responsive human neurons

    Major

    Figure 6 describes 68 visually responsive neurons, but the Results state that 38 neurons were visually responsive and that only 55 showed calcium activity. The reported visual-response counts cannot all describe the same dataset.

  • Conflicting maximum duration of spine imaging

    Minor

    The Results say the same cells and dendrites were imaged for up to 12 weeks, whereas the Figure 4 legend says imaging lasted up to 15 weeks.

2 of 2

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Authors
Linaro, D., Vermaercke, B., Iwata, R., Ramaswamy, A., Davis, B. A., Boubakar, L., Libé-Philippot, B., Bilheu, A., Bruyne, L. D., Gall, D., Conzelmann, K., Bonin, V., & Vanderhaeghen, P.
Affiliations
  • Université Libre de Bruxelles (BE)
  • Neuroelectronics Research Flanders (BE)
  • IMEC (BE)
  • VIB-KU Leuven Center for Brain & Disease Research (BE)
Corresponding author
  • Bonin, Vincent
  • Vanderhaeghen, Pierre
ORCID
Venue
Neuron
Publisher
openRxiv
Published
2019-05-02
Volume
104
Issue
5
Pages
972-986.e6
ISSN
0896-6273 (ISSN-L)
DOI
10.1101/626218
PMC
PMC6899440
OpenAlex
W2991249110
Semantic Scholar
694cd976e07f383d45e3db9ade75bae4870cda22
Cited by
210
References
98
Citations by year
2026: 25 · 2025: 31 · 2024: 42 · 2023: 32 · 2022: 34 · 2021: 20 · 2020: 21 · 2019: 4 · 2018: 1
FWCI
6.39
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Neuron (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Lirias (submittedVersion, other-oa)
  • Virtual Community of Pathological Anatomy (University of Castilla La Mancha) (submittedVersion, other-oa)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • Dépôt institutionnel de l'Université libre de Bruxelles (Université Libre de Bruxelles) (submittedVersion, other-oa)
  • UCL Discovery (University College London) (submittedVersion)
Funders
  • Waalse Gewest
  • AXA Research Fund
  • Fonds De La Recherche Scientifique - FNRS
  • Fonds Wetenschappelijk Onderzoek (G092915, FWO G.0929.15, 1237220N)
  • KU Leuven (C14/19, C14/16/048)
  • Vlaams Instituut voor Biotechnologie
  • Fondation Roger de Spoelberch
  • Vlaamse regering (C14/19/111, AKUL/15/37_GOH1816N)
  • Walloon excellence in life sciences and biotechnology
  • European Research Council
Topics
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Health Sciences › Medicine › Surgery › Xenotransplantation and immune response
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
xenotransplantation, neoteny, visual cortex, synaptic integration, neuroplasticity
MeSH terms
Animals, Cell Differentiation, Humans, Visual Cortex, Pyramidal Cells, Mice, Neurogenesis, Heterografts

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-17.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-17.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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