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Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex
Journal Pre-proof Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex Rosa D'Alessio, Fani Koukouli, Stéphane Blanchard, Julie Catteau, Célia Raïs, Thomas Lemonnier, Olivier Féraud, Annelise Bennaceur-Griscelli, Matthias Groszer, Uwe Maskos PII:
S0012-1606(19)30362-8
DOI:
https://doi.org/10.1016/j.ydbio.2020.01.009
Reference:
YDBIO 8200
To appear in:
Developmental Biology
Received Date: 6 July 2019 Revised Date:
26 December 2019
Accepted Date: 17 January 2020
Please cite this article as: D'Alessio, R., Koukouli, F., Blanchard, Sté., Catteau, J., Raïs, Cé., Lemonnier, T., Féraud, O., Bennaceur-Griscelli, A., Groszer, M., Maskos, U., Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex, Developmental Biology (2020), doi: https://doi.org/10.1016/j.ydbio.2020.01.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex
Rosa D’Alessio1, Fani Koukouli1,2, Stéphane Blanchard1, Julie Catteau1, Célia Raïs1,2, Thomas Lemonnier3, Olivier Féraud4,5, Annelise Bennaceur-Griscelli4,5, Matthias Groszer3, Uwe Maskos1* 1
Institut Pasteur, Neurobiologie intégrative des systèmes cholinergiques, Département de Neuroscience, CNRS UMR 3571, 25 rue du Dr Roux, 75724 Paris cedex 15, France
2
Sorbonne Université, Collège doctoral, 75005 Paris, France
3
Institut du Fer à Moulin, Sorbonne Université, INSERM UMR-S 1270, 75005 Paris, France
4
INSERM UMR 935, ESTeam Paris Sud, SFR André Lwoff, Université Paris Sud, Villejuif, France
5
Infrastructure Nationale INGESTEM, Université Paris Sud, INSERM, Paris, France.
*Correspondence: [email protected]
Abstract One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using twophoton in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a “protracted” human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in “real time”.
Introduction The development of the human brain is a challenging topic in neuroscience. The genetic heterogeneity of humans makes it even more complex to relate a given genome to developmental programs, and identify environmental factors interacting with the genetic profile of individuals. Molecular and cellular studies of human brain development have significantly advanced with the introduction of model systems derived from human pluripotent stem cells (hPSCs). As a first step towards addressing these issues, several studies have demonstrated that human embryonic stem cells (hESCs) are able to mature and differentiate into functional neurons and glia, by using mechanisms similar to in vivo development (Hu et al., 2009; Perrier et al., 2004). However, the issue of the “protracted” developmental period for humans is very difficult to model in vitro. A recent review by the Sestan laboratory highlights the complexity of the in vivo situation that experimental approaches will still be facing (Silbereis et al., 2016). It will be especially important to find a match between the developmental time frame under study, and a clinically relevant developmental period for example for autism spectrum disorder (ASD) or schizophrenia (SCZ), that are considered to occur separately in the human brain (Silbereis et al., 2016). The transplantation of hESC has already proven to be a valuable tool to study mechanisms of human cortical development (Gaillard et al., 1998; Gaspard et al., 2008) and is summarized by Thompson and Björklund (2015). Espuny-Camacho and colleagues transplanted hESC-derived cortical neurons into the newborn mouse brain and showed that they integrated robustly and established defined axonal projections and dendritic patterns corresponding to native cortical neurons. Over several months following transplantation in vivo, the human neurons showed a progressive maturation of their differentiation and connectivity (Espuny-Camacho et al., 2013). However, the genetic and cellular heterogeneity and long developmental time of hPSC-derived neurons are major limiting factors for their wide use as model system. Longitudinal observation of their development in vivo may provide a more efficient, experimentally robust means for discovery.
In addition, ethical issues have limited the use of hESCs (Aboody et al., 2011; Ramos-Zúñiga et al., 2012). The drawbacks of using hESCs could be overcome by instead using human induced pluripotent stem cells (hiPSCs), being derived from human somatic cells (Park et al., 2008; Takahashi and Yamanaka, 2006). The finding that neurons can be generated from hiPSCs has led to the expectation that neurological conditions such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), stroke or spinal cord injury can be better modeled, and may be treatable using cellbased transplantation strategies (Dimos et al., 2008; Muotri and Gage, 2006; Pluchino and Peruzzotti-Jametti, 2013; Suzuki and Svendsen, 2008). Furthermore, the advent of hiPSCs makes it possible to study various psychiatric and neurological syndromes, including ASD and SCZ, which are complex genetic disorders of human neurodevelopment (Brennand and Gage, 2011). Recently, an example of modeling early events in transplanted hiPSC from Down syndrome patients has confirmed the power of this approach (Real et al., 2018). We are pursuing here an approach using grafted human iPSC-derived neurons and nonneuronal cells, to explore their potential for in vivo integration into the host architecture, and how this human neurodevelopmental programme can be altered. Induced PSCs allow the study of individual genomes, from identified patients, and are also able to be modified further by genome editing techniques, like the CRISPR/Cas9 technology and related approaches. It is therefore important to establish a “baseline” for further comparative studies. Here, the morphological development of the hiPSC-derived graft was studied by postmortem immunohistochemistry, and also tracked over several months post-injection (mpi), by in vivo two-photon microscopy, thus offering the potential for in vivo modeling and study of human cortical development, in real time.
Materials and Methods
Generation of an hiPSC line expressing GFP constitutively The iPS cell line used in this study was generated by reprogramming human foreskin fibroblasts (Millipore) by daily transfection of messenger RNA encoding the reprogramming factors according to the manufacturer's recommendations (mRNA Reprogramming Kit, Stemgent) (Warren et al., 2010). The reporter iPS line expressing GFP was generated by co-transfection with Fugene-HD (Promega) of the plasmids expressing respectively human Cas9 (hCas9 was a gift from George Church, Addgene plasmid # 41815) (Mali et al., 2013), the RNA guide targeting the locus AAVS1 (gRNA_AAVS1-T2 was a gift from George Church, Addgene plasmid # 41818) (Hockemeyer et al., 2009) and the plasmid allowing the insertion of GFP under the control of the promoter CAG in the AAVS1 locus (AAV-CAGGS-EGFP) was a gift from Rudolf Jaenisch, Addgene plasmid # 22212) (Mali et al., 2013). The iPS colonies were further expanded in KOSR medium (KnockOut™ Serum Replacement, cat. no. 10828028, Gibco) onto inactivated mouse embryonic fibroblasts (MEF). All the cultures were performed at 37°C in a 5% CO2 atmosphere. The iPSC lines were characterized by FACS analysis for the expression of pluripotent stem cell surface markers. Briefly, 105 cells were stained with a combination of BD Horizon™ V450-conjugated mouse anti-human SSEA4 (Clone MC81370; BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human HESCA1 (clone 051007-4A5; Millipore), phosphatidylethanolamine (PE) - conjugated rat anti-mouse SSEA3 (clone MC631; BD Biosciences) and Alexa Fluor® 647-conjugated mouse anti-human TRA-1-60 (clone TRA-1-60; BD Biosciences) according to the manufacturer’s recommendations. Cells were analyzed on a MACSQuant flow cytometer (Miltenyi Biotec) using the MACSQuantify software. The genomic integrity was assessed by karyotyping according to standard procedures. Pluripotency of the iPSC line was assessed by teratoma formation assays. Six week-old NSG mice ("NOD Scid gamma", NOD/ShiLtJ; Prkdcscid; Il2rgtm1Wjl ) were subjected to intra-muscular injection of 2x106 to 3x106 hiPSCs. After 8 weeks, teratomas were dissected, fixed in 4% paraformaldehyde (PFA) and samples embedded in paraffin and stained with hematoxylin-eosin.
In vitro generation of hiPSC-derived neuronal precursors Human Neuronal Precursor Cells (NPCs) destined for cortical fate were obtained as previously described (Paşca et al., 2011), with some modifications: On day in vitro 0 (DIV0), hiPSCs were partially detached with collagenase and placed in a 25 cm2 tissue culture flask to generate embryoid bodies (EBs). The first night, EBs were cultured in E8 medium with Y27632 10µM (Miltenyi Biotec). On DIV1, medium was changed for a mix of EB medium 25% and E8 medium 75%, on DIV2 the medium was changed for EB medium 50% and E8 medium 50%, on DIV3 the medium was changed for EB medium 75% and E8 medium 25%. From DIV4 to DIV7, medium was changed with EB medium. The EB medium contained: DMEM/F12 (ThermoFischer, cat. no. 31330038), KnockOut Serum Replacement (20%), Penicillin-Streptomycin 1%, β-mercapto-ethanol 0.1%, supplemented with SB431542 10µM (Sigma) and Dorsomorphin 5 µM (Sigma). On DIV7, EBs were collected and plated on polyornithine/laminin-coated plates in Neural Precursor Medium (NPM) with FGF2 (20 ng/µl) and EGF (20 ng/µl). Medium was changed every two days during seven days for neural rosette formation. NPM contained: Neurobasal, B-27® Supplement Minus Vitamin A (50x), GlutaMAX (ThermoFischer, 100×), Penicillin-Streptomycin 1%. On DIV14, "Neural Rosettes" were selectively isolated using the STEMdiffTM Neural Rosette Selection reagent (Stem Cell Technologies) according to the manufacturer’s instructions, or manual picking. Neural Rosettes were dissociated with Accutase (ThermoFischer, cat. no. A1110501) and plated on polyornithine/laminin. NPCs formed a monolayer and were considered ready for the first passage when cultures were approximately 80 to 90% confluent. NPCs were split with Accutase and seeded at 3-4×106 in 60 mm Petri dishes. NPCs were ready for injections between passage 6 to 10.
Intracerebral Grafting Dissociated hiPSC-derived NPCs were transplanted either into the motor cortex of neonatal NOD/SCID mice, on postnatal day 0 (P0) to P2, or into the lateral ventricle. Neonatal mice were anesthetized with isofluorane. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under 05119.02. The mice were then immobilized on wet gauze with the head in the horizontal position. A Hamilton syringe filled with hiPSC-derived neuronal precursors was used to inject ~200,000 cells/µl into the right cortex, or lateral ventricle. Bregma was used as landmark to localize the injection site. We used as coordinates dorso-lateral (DL) = +1 mm, antero-posterior (AP) = -1 mm, depth = -1 mm, or depth = -2mm to target the ventricle. After injection, the pups were kept under a warming lamp. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under APAFIS#8245-2016121616055720 v2. Analysis at each time point after transplantation was performed in order to confirm the correct injection point. A total number of 29 animals were analyzed to establish the in vivo model, n = 6 for 15 days after injection, n = 8 for 1 month post-injection (1 mpi), n = 4 for 2 mpi, n = 4 for 4 mpi and n = 5 for 6 mpi.
Chronic cranial windows and in vivo two-photon imaging A chronic cranial window was prepared four weeks before the start of the two-photon imaging sessions, when the mice were three months of age. In detail, mice were anesthetized with ketamine (Imalgen® 1000, 10% in PBS; Rhône-Mérieux) and xylazine (Rompun®, 2% in PBS; Bayer AG), 10 ml/kg i.p. and placed in a stereotaxic frame. Their eyes were protected with artificial tear ointment. Betadine and 70% ethanol were used to clean the scalp, the scalp was retracted and 1% xylocaine was applied to the periosteum. The skull was thinned using a dental drill and a 5mm bone
area was removed centred over the motor cortex area. The dura was left intact. The cranial window was carefully covered with a circular coverglass (5mm diameter) and the perimeter was sealed with dental cement (Coffret SUPERBOND complet, Phymep, Paris). During the surgery, the body temperature was maintained at 37°C. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0.8 NA water immersion objective (Nikon) with a femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View, CA, USA) tuned to 920 nm for the imaging of GFP expressing cells. Z-stack Video 1 was acquired at a xy resolution of 0.32 um/pixel with a z-step of 0.2 um. Z-stack Video 2 was acquired at a xy resolution of 1.28 um/pixel with a z-step of 0.4 um. During the imaging, the mice were anesthetized with isoflurane (1.3%/O2) and kept on a thermal blanket with the temperature stable at 37°C. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under APAFlS#6349-20 16052416089693 v2.
Immunofluorescence For conventional microscopy analysis and quantification, the mice were deeply anesthetized with ketamine/xylazine and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA, Sigma-Aldrich, Saint Louis, MO, USA). After dissection, the brains were post-fixed overnight in 4% PFA and sectioned at 50µm thickness using a vibratome (Leica Biosystems, VT1000S). After permeabilization and blocking with 0.2% Triton X-100 and 5% horse serum in PBS, the freefloating sections were incubated at 4°C overnight in primary antibodies. For control experiments, stainings were carried out without primary antibodies. To visualize the dendrites and axons of GFP-positive neurons, we used a rabbit anti-GFP antibody (1:750, Life Technologies) or chicken anti-GFP antibody (1:2000, Abcam). For the staining of astrocytes, microglia cells, somatostatin (SOM) and parvalbumin (PV) expressing interneurons, we
used the rabbit anti-GFAP (1:1000, Chemicon, AB5804, Temecula, CA), rabbit anti-Iba1 (1:1000, Wako laboratory chemicals), goat anti-SOM (1:500, sc-7819, Santa-Cruz Biotechnology) and rabbit anti-PV (1:5000, Swant PV27) antibodies, respectively. For the staining of human nuclei, STEM101 was used (1:100, Takara Bio Y40400), and for the identification of upper layer pyramidal neurons, CUX1/CUTL1 (1:500, Abcam ab54583). For the staining of endothelial cells, we used rabbit anti CD31 (1:800, Abcam), and all buffers were supplemented with 1% bovine serum albumin (BSA) to decrease non-specific binding. Appropriate cyanin-3 (Cy3) (1:200, Sigma-Aldrich) or Alexa Fluor® 599 (1:200, Life Technologies) secondary antibodies were used at room temperature for 3h. After DAPI (SigmaAldrich, Saint Louis, MO, USA) incubation, the slices were mounted on slides in ProLong Gold Antifade Reagent mounting medium (Life Technologies, Molecular Probes, Carlsbad, CA, USA).
Quantification and statistics of histological and morphological analysis For the histological and morphological analysis of neurons, the transplanted mice (males and females) were sacrificed at three different time points (2, 4 and 6 mpi). After slicing, images were taken with a confocal spinning disk system (Cell Voyager CV1000, Yokogawa) using a dry 20x objective. Because of the bright labeling of the graft, in order to avoid the bleaching of the signal stemming from the neuronal projections, we used for all the images the same set of microscopy parameters (Exposition=120%; Gain=50%). After the acquisition, each image was adapted for analysis by using FIJI Plugins Stitching. Morphological development of neurons. Images were analyzed with automated detection of processes, using the neurite detection module from Perkin-Elmer Acapella™ (Vallotton et al., 2007), generating morphological and neuronal characteristics per cell. The analysis was performed with a total number of n = 166 cells for 2 mpi, n = 769 cells for 4 mpi, and n = 468 for 6 mpi.
For each mouse, we analyzed the same brain slice section, figure 16 from the Mouse Brain Atlas (Paxinos and Franklin, 2003). For each image, the software calculated by segmenting the cell its body area, maximum neurite length, number of Nodes1 and Nodes2, and number of segments.
Statistical analysis Because the data were not normally distributed and we compared three unmatched groups the statistical analysis was performed using Kruskal-Wallis in Prism6 (GraphPad) with significance set at p<0.05. The exact values for the groups with p<0.0001were determined using Kruskal-Wallis test in MATLAB (Mathworks 2018a). Data are presented as means ± SEM. Graphs were generated in Prism6 and figures were generated with FIJI (ImageJ), Photoshop 2017 (Adobe) and Illustrator 2017 (Adobe).
Results Efficient transplantation of hiPSC-derived neuronal precursors into newborn mice To evaluate the integration and differentiation over time of hiPSC-derived neuronal precursor cells (NPCs) in the mouse brain, hiPSCs were derived from human fibroblasts. In order to detect the graft and follow the neuronal maturation over time in the host brain, we generated a hiPSC line ubiquitously expressing green fluorescent protein (GFP). Fluorescent imaging revealed that all cells were expressing GFP in a constitutive manner (Fig. 1). For this study we used NOD/SCID newborn mice since an immune-deficient host was necessary to achieve integration beyond several weeks post-grafting, similar to Mattis et al. (2014) and Espuny-Camacho et al. (2013). We established two injection paradigms (Fig. 1C-E) that allowed us to obtain major contributions of human neurons to the brain parenchyma. Direct transplantation into different cortical areas generated highly chimeric brains (Fig. 2). When analyzing injected mice at different time-points, we observed the localization of large supra-cortical grafts extending from rostral to
caudal cortical regions (Fig. 2A, B). A graft typically spanned across cortical areas, from prefrontal via motor to somatosensory cortex. (Fig. 2). The supra-cortical part of the human graft is sending axonal projections into the mouse cortex, as early as 2 months post-injection (mpi, Fig. 2C), and a number of human iPSC-derived neurons have descended into the upper layers of mouse cortex (Fig. 2D). In contrast, targeting the injection to the lateral ventricle led to extensive human grafts in the different parts of the ventricular system, including lateral ventricle (Fig. 3A), fourth ventricle and aquaeduct. From there, extensive colonization of the brain parenchyma was also observed (Fig. 3B, C). By injecting the NPCs at more anterior levels, the neuronal precursors appear to have entered the subventricular zone (SVZ), situated along the lateral wall of the lateral ventricle (Doetsch and Alvarez-Buylla, 1996), and migrated trough the rostral migratory stream to the olfactory bulb (Fig. 3D, E), where they integrated into the granule and glomerular cell layers, respectively. Fig. 3F, G show examples of the olfactory bulb neuron morphologies adopted by the human neurons.
Human iPSC-derived NPCs integrate efficiently, differentiate into pyramidal neurons, and mature over time We first performed double immunostainings for GFP, GFAP (Glial Fibrillary Acid Protein, an astrocytic marker) and Iba1 (microglia marker) in order to evaluate the presence of human nonneuronal cells in the grafts (Fig. 4A, B). As expected, we found co-localization in only a small number of cells, supporting the efficiency of our directed differentiation protocol, in that the majority of GFP labeled cells are neurons. We further aimed to characterize and identify the GFPexpressing neurons. For this purpose, we performed immunostainings for defined interneuronal types, such as somatostatin (SOM+) and parvalbumin (PV+) expressing interneurons. As shown in Fig. 4C, we were able to detect a few individual SOM neurons that colocalized with GFP. However, no colocalization of PV+ neurons with GFP-expressing cells was observed. Fig. 4D highlights this
absence of PV+ neurons in the human-only part of the graft, on top of mouse cortex, that itself shows the typical high density of this interneuron class. There were no signs of rejection of transplanted cells, as had been shown by another group for hESC-derived neurons (EspunyCamacho et al., 2017, 2013). We next examined the evolution of the graft. Importantly, a considerable number of cells were found outside the graft, many of which had settled in the host cortex (Fig. 5). These express markers corresponding to the culture method used, favoring the development of upper-layer pyramidal neurons, and the location of the integrated neurons, cortical laminae 2 to 4 in mouse: using the CUTL1/CUX1 marker resulted in extensive colocalization in GFP-expressing human neurons (Fig. 5A). More than 99% of these neurons displayed a pyramidal morphology, with one larger apical dendrite oriented radially within the cortex (Fig. 5B, C). The transplanted human neurons all express the STEM101 marker, specific for human neuronal nuclei (Cummings et al., 2005). Fig. 5D demonstrates this specific co-labelling, and Fig. 5E indicates the net difference between the human graft and the mouse brain. We decided to follow the neuronal maturation and integration into the host brain of isolated human iPSC-derived pyramidal cells in mouse cortex, up to 6 mpi. GFP immunofluorescence detection and morphological analysis revealed evident maturation over time, represented by the increasing number of ramifications and lengthening of the projections, extending into the cortex (Fig. 6A-D). When examined at 1 and 2 mpi, the grafted cells displayed an immature neurite structure, however, this pattern became more complex over time with more dendritic branches at 4 and 6 mpi (Fig. 6A3, A4). Quantitative analysis at 2, 4, and 6 mpi of the grafted cells’ structure was carried out with the Acapella™ software, as detailed in Methods. The neurons integrated into the mouse parenchyma showed an increase in the cell body area (2 mpi, 412.7 ± 14.3 with n = 166 cells; 4 mpi, 400.7 ± 5.1 with n= 769 cells; 6 mpi, 456 ± 10.9 with n= 468 cells). No significant difference was found between 2 and 4 mpi (p = 0.4), whereas the cell body area was significantly increased
from 2 to 6 mpi (p = 0.001) and 4 to 6 mpi with p = 1.16 x 10-7 (Kruskal-Wallis test, Fig. 6E). Importantly, we observed an increase in neurite length by 6 mpi (2 mpi, 340.6 ± 28.5; 4 mpi, 321.3 ± 13.5; 6 mpi, 492.1 ± 22.9; 2 mpi vs. 4 mpi, p = 0.59; 2 mpi vs. 6 mpi, p= 2.57 x 10-6; 4 mpi vs. 6 mpi, p= 2.92 x 10-14 , Kruskal-Wallis test, Fig. 6F). Using the Acapella™ software further, we were able to quantify the number of segments and nodes, and observed a gradual increase over time. The number of segments increased from 65.7 ± 8.7 at 2 mpi to 96.3 ± 7.0 at 4 mpi and 178 ± 14.7 at 6 mpi, with a significant difference between the groups (2 mpi vs. 4 mpi, p = 0.00056; 2 mpi vs. 6 mpi, p = 6.59 x 10-13; 4 mpi vs. 6 mpi, p = 5.64 x 10-4, Kruskal-Wallis test, Fig. 6G). The number of Nodes1 increased from 34.0 ± 4.8 at 2 mpi to 61.8 ± 5.4 at 4 mpi and 115.2 ± 12.2 at 6 mpi with a significant difference between the groups (2 mp vs. 4 mpi, p = 1.36 x 10-3; 2 mpi vs. 6 mpi, p = 9 x 10-13; 4 mpi vs. 6 mpi, p = 1.36 x 10-3, Kruskal-Wallis test, Fig. 6H). Finally, we quantified the number of Nodes2 and found an increase from 30.9 ± 4.3 at 2 mpi to 57.4 ± 5.3 at 4 mpi and 106.7 ± 11.9, with a significant difference between the different timepoints (2 mpi vs. 4 mpi, p = 2.31 x 10-3; 2 mpi vs. 6 mpi, p = 1.98 x 10-12; 4 mpi vs. 6 mpi, p = 2.31 x 10-3, KruskalWallis test, Fig. 6I).
In vivo maturation of supra-cortical, exclusively hiPSC-derived neurons followed by twophoton imaging We then decided to also follow the development and maturation of human neurons in the supracortical part of the graft. This allowed us to observe, in vivo, how human pyramidal neurons “selforganize” to create “human” parenchyma. In order to track these processes in the host brain over time, in real time, in the same animal, we performed in vivo two-photon microscopy. In vivo imaging was first performed at 4 mpi in the human-specific graft overlying the mouse motor cortex area (Fig. 7A, B), since the transplants before 3 mpi did not display a mature morphology. At 4 mpi the cell bodies were well labeled in the
cortex, but at this stage we could not detect any projections or interconnections between cells. But, by repeating the in vivo imaging at 7 and 8 mpi, we observed more mature dendritic structures that we could track down to 200 µm from the surface, thus also demonstrating that the human neurons continue to mature throughout the supra-cortical graft. Video 1 shows an example of the humanonly part of the brain with easily identifiable pyramidal-like dendrites. The human neurons form a densely packed structure that is fully vascularized (Fig. 7B, C). In Video 2, a z-scan through this area shows blood vessels of different diameters, and the passage of the unlabeled blood cells can be observed over the fluorescent background.
Discussion During the last decade, stem cell therapy has developed as a new medical frontier to prevent or rescue brain damage. Several studies have attempted to demonstrate the possibilities of hESCs and hiPSCs in neurobiology as reliable source for cell replacement therapies (Aboody et al., 2011). In particular, previous work showed how hESCs, derived from established embryonic cell lines, can be efficiently transplanted into the mouse cortex to generate cortical pyramidal neurons (EspunyCamacho et al., 2013; Gaspard et al., 2008). This has allowed the field to appreciate the major differences that exist between in vitro stem cell-derived model systems, and the power of having human stem cells participate in neurodevelopment in vivo. Thus, a second major use of hPSCs will be to further our understanding of human neural development in vivo at unprecedented resolution. As it is precisely at these embryonic and early post-natal stages that some major pathologies occur, like ASD and SCZ (Silbereis et al., 2016). Here, we present a grafting paradigm that allowed the targeted engraftment of human induced PSC-derived neuronal precursors into different cortical areas of neonatal mice. In addition, by injecting into the ventricles, we were able to visualize human NPC-derived cells in areas close to the ventricles, like the hippocampus, striatum and thalamus, and the olfactory bulbs. There, very
efficient integration was observed, and the development and function of human olfactory bulb neurons merits further study. Our data show that hiPSC-derived NPCs efficiently transplanted into the motor cortex also robustly integrated into the host brain by differentiating and maturing over time. Although the majority of the cells were confined within the bulk of the graft, we were able to perform morphological analysis on a considerable group of neurons outside the graft, fully integrated into the host architecture. In many brains, these represent on the order of hundreds of neurons per section. Most of these cells displayed long axonal and dendritic projections. Our findings suggest that after transplantation, the grafted neurons follow a progressive and continued integration into the host brain. Up to 6 mpi, they exhibited increasing neuronal maturation and axonal outgrowth. Further evidence of their differentiation was the detection of well-defined dendritic spines, thus suggesting that active synaptogenesis had taken place as seen before for embryonic PSCs (EspunyCamacho et al., 2013). In this study, we tracked in vivo the morphological development of human transplanted neurons into the motor cortex of mouse brain. Our grafting coordinates and the strong constitutive expression of GFP allowed us to image by two-photon microscopy the cell bodies and dendritic structure of the grafted neurons. The combination of the novel technique targeting the different areas of cortex and the promising use of hiPSC-derived neuronal precursor transplantation, together with the possibility to perform two-photon imaging in vivo of neuronal morphology and structure, may thus pave the way for an enhanced use of hiPSCs in studies of pathological mechanisms and also therapeutic screening.
Conclusion hiPSC-derived neurogenesis models have significantly advanced the study of human neurodevelopment at the molecular and cellular level, in vitro. However, the exceptionally long duration of human brain development is often not fully recapitulated in iPSC-derived in vitro
systems and presents a substantial challenge. Novel model systems are needed to access and experimentally manipulate the embryonic to postnatal period. Here, we present an efficient method to follow protracted human cortical neuron maturation over time using in vivo two-photon imaging and slice histology, after neo-natal transplantation of human iPSC-derived NPCs. This system now allows the detailed study of both genetic, environmental and pharmacological intervention at this early stage.
Acknowledgements We would like to thank Stéphanie Pons for continued support throughout this study, and Pascal Roux and Anne Danckaert from the Photonic BioImaging plateform for image acquisition support and quantitative data analysis performed with Acapella software, respectively. This work was funded by Institut Pasteur Programme PasteurInnov (IS/fc/13/160, and NICOBLOC 2018), the Centre National de la Recherche Scientifique (UMR 3571), the French National Cancer Institute INCa (2016-1-Tabac-01-IP-1), the Fondation de la Recherche Médicale (FRM, Grants SPF20140129365, DPA20140629803, FDT201805005198 and EQU201903007822), the Institut Pasteur priority programme GPF "Microbes&Brain", the ERANET programme iPS&BRAIN, the Fondation Alzheimer-INSERM, and the Fondation Vaincre Alzheimer, all to U.M. This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 7202070 (HBP SGA1). M.G. acknowledges support from Fondation pour la Recherche Médicale FRM (ING20111223378), l'Association "Schizo oui", Institut de France – Fondation NRJ (NRJ2010), Association ‘Vaincre les Maladies Lysosomales-VML’ (VML2016-1004), Fondation Lejeune (FJL1389), Fondation de France (Eng° 00060522) and Fondation Motrice (N°2016/4). U.M. and M.G. are members of the Laboratory of Excellence, LABEX BIO-PSY. The laboratories of U.M. and M.G. are part of the École des Neurosciences de Paris Ile-de-France Thematic Network for Advanced Research (RTRA).
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Figure legends Figure 1 Human iPSC-derived neuronal precursor line expressing green fluorescent protein (GFP) used for the grafting, and intracranial injection of hiPSC-derived neuronal precursors into the cortex of neonatal mice. (A, B) GFP is expressed ubiquitously and constitutively. Scale bars = 200 µm in (A) for 5x magnification, 50 µm in (B) for 20x magnification. (C) Intracranial injection of hiPSC derived neuronal precursors into the cortex of neonatal mice. The two landmarks used to target the motor cortex for injection are easily visible through the translucent skin of NOD/SCID neonatal pups. (D) Schematic view of the mouse head shows the target site for the injection (red arrow). The injection site is located approximately 1 mm posterior to bregma and 1 mm lateral to the sagittal suture, with 1 mm of depth. (E) Whole mouse brain and indication of the graft (white delineated area).
Figure 2 Efficient human iPSC derived neuronal precursor transplantation into the cortex of newborn mice. (A) Representative engraftment in the cortex of adult mice after neonatal transplantation. Anti-GFP immunofluorescence (in green) to detect the graft localization in the cortex. It extends from the most rostral to caudal part of the brain. The graft localization includes the prelimbic cortex, somatosensory cortex and motor cortex. (B) Higher magnification shows that most of the injected cells remain confined in the bulk of the graft. Scale bars = 100 µm in (B) for a 10x magnification. (C, D) Human iPSC-derived neuronal precursors and axons emanating from the solid graft over time. GFP-positive cells detected by anti-GFP immunofluorescence (in white) in the motor cortex and analyzed at 2 months post injection (mpi). For each time point most of the
neurons remain confined within the bulk of the graft. Most cells at 2 mpi differentiated by extending long axonal projections. Scale bar = 200 µm for 20x magnification.
Figure 3 Intra-ventricular injection of human NPCs allows to target several brain parenchyma, and the olfactory bulb. (A) Brain slice with transplant in the lateral ventricle, analyzed at 5 mpi. (B, C) Grafts in the lateral ventricle, between the posterior part of the CA1 area of the hippocampus and entorhinal cortex. (D-G) Presence of hiPSC derived neurons in the olfactory bulb revealed by GFP immunofluorescence (in red). Most of the human neurons reside in the granule cell layer, but also in the glomerular layer. By injecting the NPCs into the ventricles, the cells have migrated from the graft trough the rostral migratory stream towards the olfactory bulb. (F, G) Images showing the typical morphology of olfactory bulb cells. Scale bars = 1 mm in (A), 200 µm in (B-E) and 20 µm in (F, G).
Figure 4 Immunofluorescence analysis for the detection of astrocytes, microglia, and somatostatinexpressing human interneurons. (A) High-magnification confocal analysis of human astrocytes 4 mpi of the hiPSC-derived neuronal precursors. Panel, from left to right: Astrocytes in red, labelled with anti-GFAP antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (B) High-magnification confocal analysis of microglia 4 mpi of the hiPSC-derived neuronal precursors. Panel, from left to right: Microglial cells in red, labelled with anti-Iba1
antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (C) High-magnification confocal analysis of somatostatin-expressing human interneurons, analyzed at 6 mpi. Panel, from left to right: SOM+ interneurons in red, labelled with antisomatostatin antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (D) Low-magnification confocal analysis of parvalbumin-expressing human interneurons, analyzed at 6 mpi. This is an example of a human graft on top of mouse cortex. PV+ labeling is only detectable in the mouse, not the human cells. Panel, from left to right: PV+ interneurons in red, labelled with anti-parvalbumin antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 50 µm.
Figure 5 Immunofluorescence analysis for upper-layer pyramidal neurons, cortical laminae 2 to 4 in mouse 4 mpi of the hiPSC-derived neuronal precursors. (A) High-magnification confocal analysis of human pyramidal neurons expressing the CUTL1/CUX1 marker. The CUTL1/CUX1 marker resulted in extensive colocalization in GFP-expressing human neurons. Scale bar = 20 µm. (B, C) GFP-expressing human neurons displaying a pyramidal morphology. Scale bar = 20 µm. (D) GFP-expressing human pyramidal neurons exhibit extensive co-localization with the human neuronal nuclei-specific marker, STEM101. Arrow heads indicate examples of colocalization. STEM101 nuclear labeling in red, DAPI staining of nuclei in blue. Scale bar = 20 µm. (E) Absence of human neuronal nuclei-specific marker, STEM101, in the mouse brain. Arrow heads indicate examples of co-localization. STEM101 nuclear labeling in red, DAPI
staining of nuclei in blue, in the human graft. Asterisks demarcate the boundary of the mouse cortex. Scale bar = 20 µm.
Figure 6 Human iPSC-derived neuronal precursor maturation and differentiation over time. (A) Representative images of hiPSC-derived neuronal precursors (GFP positive) displaying immature dendritic patterns at 1 and 2 mpi. At 4 mpi the neurons start displaying a more mature dendritic pattern until achieving a complex and mature differentiation at 6 mpi. Scale bar = 20 µm. (B, C) Representative confocal images of GFP immunostained neurons used for morphological analysis with the Acapella software. (D) Schematic neuron representation showing the parameters used for the branching analysis. (E-I) Quantification of the morphological development over time performed at 2, 4 and 6 mpi. n=166 for 2 mpi, n=769 for 4 mpi, and n=468 for 6 mpi. **p<0.01, ****p<0.0001.
Figure 7 In vivo integration and maturation of hiPSC-derived NPCs by two-photon imaging. (A) In vivo two-photon images of hiPSC-derived NPCs in the "human-only" part of the transplant overlying mouse cortex at 8 mpi, as detailed in Video 1. The dendritic maturation of the pyramidal neurons is indicated (arrows). Scale bar = 20 µm. (B) In vivo two-photon image of hiPSC-derived neuronal precursors performed at 4mpi, as detailed in Video 2. Arrows indicate the blood vessels. Scale bar = 20 µm. (C) High magnification image of a blood vessel in a human graft. Arrows point to endothelial cells identified with the CD31 marker (red). Cell bodies are DAPI stained (blue), human neurons express GFP (green). Scale bar = 20 µm.
Video 1 Higher magnification video of developed dendrites at 8mpi. Z-scan through the supra-cortical graft of human-only neurons at high resolution. Note the developed dendritic architecture. Scale bar = 20 µm.
Video 2 Z-scan through the supra-cortical graft of human-only neurons. The blood vessels are clearly visible, in black. With the fluorescent background, the movement of the unlabeled blood cells can be made out. Scale bar = 20 µm.
Highlights
Development of an efficient in vivo system to study human neurons Human induced pluripotent stem-cell derived cells integrate into the mouse brain Human neurons exhibit a protracted in vivo developmental program Transplants of human neuronal precursors can be followed by live two-photon imaging
S0012-1606(19)30362-8
DOI:
https://doi.org/10.1016/j.ydbio.2020.01.009
Reference:
YDBIO 8200
To appear in:
Developmental Biology
Received Date: 6 July 2019 Revised Date:
26 December 2019
Accepted Date: 17 January 2020
Please cite this article as: D'Alessio, R., Koukouli, F., Blanchard, Sté., Catteau, J., Raïs, Cé., Lemonnier, T., Féraud, O., Bennaceur-Griscelli, A., Groszer, M., Maskos, U., Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex, Developmental Biology (2020), doi: https://doi.org/10.1016/j.ydbio.2020.01.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex
Rosa D’Alessio1, Fani Koukouli1,2, Stéphane Blanchard1, Julie Catteau1, Célia Raïs1,2, Thomas Lemonnier3, Olivier Féraud4,5, Annelise Bennaceur-Griscelli4,5, Matthias Groszer3, Uwe Maskos1* 1
Institut Pasteur, Neurobiologie intégrative des systèmes cholinergiques, Département de Neuroscience, CNRS UMR 3571, 25 rue du Dr Roux, 75724 Paris cedex 15, France
2
Sorbonne Université, Collège doctoral, 75005 Paris, France
3
Institut du Fer à Moulin, Sorbonne Université, INSERM UMR-S 1270, 75005 Paris, France
4
INSERM UMR 935, ESTeam Paris Sud, SFR André Lwoff, Université Paris Sud, Villejuif, France
5
Infrastructure Nationale INGESTEM, Université Paris Sud, INSERM, Paris, France.
*Correspondence: [email protected]
Abstract One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using twophoton in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a “protracted” human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in “real time”.
Introduction The development of the human brain is a challenging topic in neuroscience. The genetic heterogeneity of humans makes it even more complex to relate a given genome to developmental programs, and identify environmental factors interacting with the genetic profile of individuals. Molecular and cellular studies of human brain development have significantly advanced with the introduction of model systems derived from human pluripotent stem cells (hPSCs). As a first step towards addressing these issues, several studies have demonstrated that human embryonic stem cells (hESCs) are able to mature and differentiate into functional neurons and glia, by using mechanisms similar to in vivo development (Hu et al., 2009; Perrier et al., 2004). However, the issue of the “protracted” developmental period for humans is very difficult to model in vitro. A recent review by the Sestan laboratory highlights the complexity of the in vivo situation that experimental approaches will still be facing (Silbereis et al., 2016). It will be especially important to find a match between the developmental time frame under study, and a clinically relevant developmental period for example for autism spectrum disorder (ASD) or schizophrenia (SCZ), that are considered to occur separately in the human brain (Silbereis et al., 2016). The transplantation of hESC has already proven to be a valuable tool to study mechanisms of human cortical development (Gaillard et al., 1998; Gaspard et al., 2008) and is summarized by Thompson and Björklund (2015). Espuny-Camacho and colleagues transplanted hESC-derived cortical neurons into the newborn mouse brain and showed that they integrated robustly and established defined axonal projections and dendritic patterns corresponding to native cortical neurons. Over several months following transplantation in vivo, the human neurons showed a progressive maturation of their differentiation and connectivity (Espuny-Camacho et al., 2013). However, the genetic and cellular heterogeneity and long developmental time of hPSC-derived neurons are major limiting factors for their wide use as model system. Longitudinal observation of their development in vivo may provide a more efficient, experimentally robust means for discovery.
In addition, ethical issues have limited the use of hESCs (Aboody et al., 2011; Ramos-Zúñiga et al., 2012). The drawbacks of using hESCs could be overcome by instead using human induced pluripotent stem cells (hiPSCs), being derived from human somatic cells (Park et al., 2008; Takahashi and Yamanaka, 2006). The finding that neurons can be generated from hiPSCs has led to the expectation that neurological conditions such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), stroke or spinal cord injury can be better modeled, and may be treatable using cellbased transplantation strategies (Dimos et al., 2008; Muotri and Gage, 2006; Pluchino and Peruzzotti-Jametti, 2013; Suzuki and Svendsen, 2008). Furthermore, the advent of hiPSCs makes it possible to study various psychiatric and neurological syndromes, including ASD and SCZ, which are complex genetic disorders of human neurodevelopment (Brennand and Gage, 2011). Recently, an example of modeling early events in transplanted hiPSC from Down syndrome patients has confirmed the power of this approach (Real et al., 2018). We are pursuing here an approach using grafted human iPSC-derived neurons and nonneuronal cells, to explore their potential for in vivo integration into the host architecture, and how this human neurodevelopmental programme can be altered. Induced PSCs allow the study of individual genomes, from identified patients, and are also able to be modified further by genome editing techniques, like the CRISPR/Cas9 technology and related approaches. It is therefore important to establish a “baseline” for further comparative studies. Here, the morphological development of the hiPSC-derived graft was studied by postmortem immunohistochemistry, and also tracked over several months post-injection (mpi), by in vivo two-photon microscopy, thus offering the potential for in vivo modeling and study of human cortical development, in real time.
Materials and Methods
Generation of an hiPSC line expressing GFP constitutively The iPS cell line used in this study was generated by reprogramming human foreskin fibroblasts (Millipore) by daily transfection of messenger RNA encoding the reprogramming factors according to the manufacturer's recommendations (mRNA Reprogramming Kit, Stemgent) (Warren et al., 2010). The reporter iPS line expressing GFP was generated by co-transfection with Fugene-HD (Promega) of the plasmids expressing respectively human Cas9 (hCas9 was a gift from George Church, Addgene plasmid # 41815) (Mali et al., 2013), the RNA guide targeting the locus AAVS1 (gRNA_AAVS1-T2 was a gift from George Church, Addgene plasmid # 41818) (Hockemeyer et al., 2009) and the plasmid allowing the insertion of GFP under the control of the promoter CAG in the AAVS1 locus (AAV-CAGGS-EGFP) was a gift from Rudolf Jaenisch, Addgene plasmid # 22212) (Mali et al., 2013). The iPS colonies were further expanded in KOSR medium (KnockOut™ Serum Replacement, cat. no. 10828028, Gibco) onto inactivated mouse embryonic fibroblasts (MEF). All the cultures were performed at 37°C in a 5% CO2 atmosphere. The iPSC lines were characterized by FACS analysis for the expression of pluripotent stem cell surface markers. Briefly, 105 cells were stained with a combination of BD Horizon™ V450-conjugated mouse anti-human SSEA4 (Clone MC81370; BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human HESCA1 (clone 051007-4A5; Millipore), phosphatidylethanolamine (PE) - conjugated rat anti-mouse SSEA3 (clone MC631; BD Biosciences) and Alexa Fluor® 647-conjugated mouse anti-human TRA-1-60 (clone TRA-1-60; BD Biosciences) according to the manufacturer’s recommendations. Cells were analyzed on a MACSQuant flow cytometer (Miltenyi Biotec) using the MACSQuantify software. The genomic integrity was assessed by karyotyping according to standard procedures. Pluripotency of the iPSC line was assessed by teratoma formation assays. Six week-old NSG mice ("NOD Scid gamma", NOD/ShiLtJ; Prkdcscid; Il2rgtm1Wjl ) were subjected to intra-muscular injection of 2x106 to 3x106 hiPSCs. After 8 weeks, teratomas were dissected, fixed in 4% paraformaldehyde (PFA) and samples embedded in paraffin and stained with hematoxylin-eosin.
In vitro generation of hiPSC-derived neuronal precursors Human Neuronal Precursor Cells (NPCs) destined for cortical fate were obtained as previously described (Paşca et al., 2011), with some modifications: On day in vitro 0 (DIV0), hiPSCs were partially detached with collagenase and placed in a 25 cm2 tissue culture flask to generate embryoid bodies (EBs). The first night, EBs were cultured in E8 medium with Y27632 10µM (Miltenyi Biotec). On DIV1, medium was changed for a mix of EB medium 25% and E8 medium 75%, on DIV2 the medium was changed for EB medium 50% and E8 medium 50%, on DIV3 the medium was changed for EB medium 75% and E8 medium 25%. From DIV4 to DIV7, medium was changed with EB medium. The EB medium contained: DMEM/F12 (ThermoFischer, cat. no. 31330038), KnockOut Serum Replacement (20%), Penicillin-Streptomycin 1%, β-mercapto-ethanol 0.1%, supplemented with SB431542 10µM (Sigma) and Dorsomorphin 5 µM (Sigma). On DIV7, EBs were collected and plated on polyornithine/laminin-coated plates in Neural Precursor Medium (NPM) with FGF2 (20 ng/µl) and EGF (20 ng/µl). Medium was changed every two days during seven days for neural rosette formation. NPM contained: Neurobasal, B-27® Supplement Minus Vitamin A (50x), GlutaMAX (ThermoFischer, 100×), Penicillin-Streptomycin 1%. On DIV14, "Neural Rosettes" were selectively isolated using the STEMdiffTM Neural Rosette Selection reagent (Stem Cell Technologies) according to the manufacturer’s instructions, or manual picking. Neural Rosettes were dissociated with Accutase (ThermoFischer, cat. no. A1110501) and plated on polyornithine/laminin. NPCs formed a monolayer and were considered ready for the first passage when cultures were approximately 80 to 90% confluent. NPCs were split with Accutase and seeded at 3-4×106 in 60 mm Petri dishes. NPCs were ready for injections between passage 6 to 10.
Intracerebral Grafting Dissociated hiPSC-derived NPCs were transplanted either into the motor cortex of neonatal NOD/SCID mice, on postnatal day 0 (P0) to P2, or into the lateral ventricle. Neonatal mice were anesthetized with isofluorane. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under 05119.02. The mice were then immobilized on wet gauze with the head in the horizontal position. A Hamilton syringe filled with hiPSC-derived neuronal precursors was used to inject ~200,000 cells/µl into the right cortex, or lateral ventricle. Bregma was used as landmark to localize the injection site. We used as coordinates dorso-lateral (DL) = +1 mm, antero-posterior (AP) = -1 mm, depth = -1 mm, or depth = -2mm to target the ventricle. After injection, the pups were kept under a warming lamp. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under APAFIS#8245-2016121616055720 v2. Analysis at each time point after transplantation was performed in order to confirm the correct injection point. A total number of 29 animals were analyzed to establish the in vivo model, n = 6 for 15 days after injection, n = 8 for 1 month post-injection (1 mpi), n = 4 for 2 mpi, n = 4 for 4 mpi and n = 5 for 6 mpi.
Chronic cranial windows and in vivo two-photon imaging A chronic cranial window was prepared four weeks before the start of the two-photon imaging sessions, when the mice were three months of age. In detail, mice were anesthetized with ketamine (Imalgen® 1000, 10% in PBS; Rhône-Mérieux) and xylazine (Rompun®, 2% in PBS; Bayer AG), 10 ml/kg i.p. and placed in a stereotaxic frame. Their eyes were protected with artificial tear ointment. Betadine and 70% ethanol were used to clean the scalp, the scalp was retracted and 1% xylocaine was applied to the periosteum. The skull was thinned using a dental drill and a 5mm bone
area was removed centred over the motor cortex area. The dura was left intact. The cranial window was carefully covered with a circular coverglass (5mm diameter) and the perimeter was sealed with dental cement (Coffret SUPERBOND complet, Phymep, Paris). During the surgery, the body temperature was maintained at 37°C. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0.8 NA water immersion objective (Nikon) with a femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View, CA, USA) tuned to 920 nm for the imaging of GFP expressing cells. Z-stack Video 1 was acquired at a xy resolution of 0.32 um/pixel with a z-step of 0.2 um. Z-stack Video 2 was acquired at a xy resolution of 1.28 um/pixel with a z-step of 0.4 um. During the imaging, the mice were anesthetized with isoflurane (1.3%/O2) and kept on a thermal blanket with the temperature stable at 37°C. This protocol was approved by the Institut Pasteur CETEA animal ethics committee, and the French Ministère de l'Education nationale, de le Recherche, et de l'Innovation, under APAFlS#6349-20 16052416089693 v2.
Immunofluorescence For conventional microscopy analysis and quantification, the mice were deeply anesthetized with ketamine/xylazine and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA, Sigma-Aldrich, Saint Louis, MO, USA). After dissection, the brains were post-fixed overnight in 4% PFA and sectioned at 50µm thickness using a vibratome (Leica Biosystems, VT1000S). After permeabilization and blocking with 0.2% Triton X-100 and 5% horse serum in PBS, the freefloating sections were incubated at 4°C overnight in primary antibodies. For control experiments, stainings were carried out without primary antibodies. To visualize the dendrites and axons of GFP-positive neurons, we used a rabbit anti-GFP antibody (1:750, Life Technologies) or chicken anti-GFP antibody (1:2000, Abcam). For the staining of astrocytes, microglia cells, somatostatin (SOM) and parvalbumin (PV) expressing interneurons, we
used the rabbit anti-GFAP (1:1000, Chemicon, AB5804, Temecula, CA), rabbit anti-Iba1 (1:1000, Wako laboratory chemicals), goat anti-SOM (1:500, sc-7819, Santa-Cruz Biotechnology) and rabbit anti-PV (1:5000, Swant PV27) antibodies, respectively. For the staining of human nuclei, STEM101 was used (1:100, Takara Bio Y40400), and for the identification of upper layer pyramidal neurons, CUX1/CUTL1 (1:500, Abcam ab54583). For the staining of endothelial cells, we used rabbit anti CD31 (1:800, Abcam), and all buffers were supplemented with 1% bovine serum albumin (BSA) to decrease non-specific binding. Appropriate cyanin-3 (Cy3) (1:200, Sigma-Aldrich) or Alexa Fluor® 599 (1:200, Life Technologies) secondary antibodies were used at room temperature for 3h. After DAPI (SigmaAldrich, Saint Louis, MO, USA) incubation, the slices were mounted on slides in ProLong Gold Antifade Reagent mounting medium (Life Technologies, Molecular Probes, Carlsbad, CA, USA).
Quantification and statistics of histological and morphological analysis For the histological and morphological analysis of neurons, the transplanted mice (males and females) were sacrificed at three different time points (2, 4 and 6 mpi). After slicing, images were taken with a confocal spinning disk system (Cell Voyager CV1000, Yokogawa) using a dry 20x objective. Because of the bright labeling of the graft, in order to avoid the bleaching of the signal stemming from the neuronal projections, we used for all the images the same set of microscopy parameters (Exposition=120%; Gain=50%). After the acquisition, each image was adapted for analysis by using FIJI Plugins Stitching. Morphological development of neurons. Images were analyzed with automated detection of processes, using the neurite detection module from Perkin-Elmer Acapella™ (Vallotton et al., 2007), generating morphological and neuronal characteristics per cell. The analysis was performed with a total number of n = 166 cells for 2 mpi, n = 769 cells for 4 mpi, and n = 468 for 6 mpi.
For each mouse, we analyzed the same brain slice section, figure 16 from the Mouse Brain Atlas (Paxinos and Franklin, 2003). For each image, the software calculated by segmenting the cell its body area, maximum neurite length, number of Nodes1 and Nodes2, and number of segments.
Statistical analysis Because the data were not normally distributed and we compared three unmatched groups the statistical analysis was performed using Kruskal-Wallis in Prism6 (GraphPad) with significance set at p<0.05. The exact values for the groups with p<0.0001were determined using Kruskal-Wallis test in MATLAB (Mathworks 2018a). Data are presented as means ± SEM. Graphs were generated in Prism6 and figures were generated with FIJI (ImageJ), Photoshop 2017 (Adobe) and Illustrator 2017 (Adobe).
Results Efficient transplantation of hiPSC-derived neuronal precursors into newborn mice To evaluate the integration and differentiation over time of hiPSC-derived neuronal precursor cells (NPCs) in the mouse brain, hiPSCs were derived from human fibroblasts. In order to detect the graft and follow the neuronal maturation over time in the host brain, we generated a hiPSC line ubiquitously expressing green fluorescent protein (GFP). Fluorescent imaging revealed that all cells were expressing GFP in a constitutive manner (Fig. 1). For this study we used NOD/SCID newborn mice since an immune-deficient host was necessary to achieve integration beyond several weeks post-grafting, similar to Mattis et al. (2014) and Espuny-Camacho et al. (2013). We established two injection paradigms (Fig. 1C-E) that allowed us to obtain major contributions of human neurons to the brain parenchyma. Direct transplantation into different cortical areas generated highly chimeric brains (Fig. 2). When analyzing injected mice at different time-points, we observed the localization of large supra-cortical grafts extending from rostral to
caudal cortical regions (Fig. 2A, B). A graft typically spanned across cortical areas, from prefrontal via motor to somatosensory cortex. (Fig. 2). The supra-cortical part of the human graft is sending axonal projections into the mouse cortex, as early as 2 months post-injection (mpi, Fig. 2C), and a number of human iPSC-derived neurons have descended into the upper layers of mouse cortex (Fig. 2D). In contrast, targeting the injection to the lateral ventricle led to extensive human grafts in the different parts of the ventricular system, including lateral ventricle (Fig. 3A), fourth ventricle and aquaeduct. From there, extensive colonization of the brain parenchyma was also observed (Fig. 3B, C). By injecting the NPCs at more anterior levels, the neuronal precursors appear to have entered the subventricular zone (SVZ), situated along the lateral wall of the lateral ventricle (Doetsch and Alvarez-Buylla, 1996), and migrated trough the rostral migratory stream to the olfactory bulb (Fig. 3D, E), where they integrated into the granule and glomerular cell layers, respectively. Fig. 3F, G show examples of the olfactory bulb neuron morphologies adopted by the human neurons.
Human iPSC-derived NPCs integrate efficiently, differentiate into pyramidal neurons, and mature over time We first performed double immunostainings for GFP, GFAP (Glial Fibrillary Acid Protein, an astrocytic marker) and Iba1 (microglia marker) in order to evaluate the presence of human nonneuronal cells in the grafts (Fig. 4A, B). As expected, we found co-localization in only a small number of cells, supporting the efficiency of our directed differentiation protocol, in that the majority of GFP labeled cells are neurons. We further aimed to characterize and identify the GFPexpressing neurons. For this purpose, we performed immunostainings for defined interneuronal types, such as somatostatin (SOM+) and parvalbumin (PV+) expressing interneurons. As shown in Fig. 4C, we were able to detect a few individual SOM neurons that colocalized with GFP. However, no colocalization of PV+ neurons with GFP-expressing cells was observed. Fig. 4D highlights this
absence of PV+ neurons in the human-only part of the graft, on top of mouse cortex, that itself shows the typical high density of this interneuron class. There were no signs of rejection of transplanted cells, as had been shown by another group for hESC-derived neurons (EspunyCamacho et al., 2017, 2013). We next examined the evolution of the graft. Importantly, a considerable number of cells were found outside the graft, many of which had settled in the host cortex (Fig. 5). These express markers corresponding to the culture method used, favoring the development of upper-layer pyramidal neurons, and the location of the integrated neurons, cortical laminae 2 to 4 in mouse: using the CUTL1/CUX1 marker resulted in extensive colocalization in GFP-expressing human neurons (Fig. 5A). More than 99% of these neurons displayed a pyramidal morphology, with one larger apical dendrite oriented radially within the cortex (Fig. 5B, C). The transplanted human neurons all express the STEM101 marker, specific for human neuronal nuclei (Cummings et al., 2005). Fig. 5D demonstrates this specific co-labelling, and Fig. 5E indicates the net difference between the human graft and the mouse brain. We decided to follow the neuronal maturation and integration into the host brain of isolated human iPSC-derived pyramidal cells in mouse cortex, up to 6 mpi. GFP immunofluorescence detection and morphological analysis revealed evident maturation over time, represented by the increasing number of ramifications and lengthening of the projections, extending into the cortex (Fig. 6A-D). When examined at 1 and 2 mpi, the grafted cells displayed an immature neurite structure, however, this pattern became more complex over time with more dendritic branches at 4 and 6 mpi (Fig. 6A3, A4). Quantitative analysis at 2, 4, and 6 mpi of the grafted cells’ structure was carried out with the Acapella™ software, as detailed in Methods. The neurons integrated into the mouse parenchyma showed an increase in the cell body area (2 mpi, 412.7 ± 14.3 with n = 166 cells; 4 mpi, 400.7 ± 5.1 with n= 769 cells; 6 mpi, 456 ± 10.9 with n= 468 cells). No significant difference was found between 2 and 4 mpi (p = 0.4), whereas the cell body area was significantly increased
from 2 to 6 mpi (p = 0.001) and 4 to 6 mpi with p = 1.16 x 10-7 (Kruskal-Wallis test, Fig. 6E). Importantly, we observed an increase in neurite length by 6 mpi (2 mpi, 340.6 ± 28.5; 4 mpi, 321.3 ± 13.5; 6 mpi, 492.1 ± 22.9; 2 mpi vs. 4 mpi, p = 0.59; 2 mpi vs. 6 mpi, p= 2.57 x 10-6; 4 mpi vs. 6 mpi, p= 2.92 x 10-14 , Kruskal-Wallis test, Fig. 6F). Using the Acapella™ software further, we were able to quantify the number of segments and nodes, and observed a gradual increase over time. The number of segments increased from 65.7 ± 8.7 at 2 mpi to 96.3 ± 7.0 at 4 mpi and 178 ± 14.7 at 6 mpi, with a significant difference between the groups (2 mpi vs. 4 mpi, p = 0.00056; 2 mpi vs. 6 mpi, p = 6.59 x 10-13; 4 mpi vs. 6 mpi, p = 5.64 x 10-4, Kruskal-Wallis test, Fig. 6G). The number of Nodes1 increased from 34.0 ± 4.8 at 2 mpi to 61.8 ± 5.4 at 4 mpi and 115.2 ± 12.2 at 6 mpi with a significant difference between the groups (2 mp vs. 4 mpi, p = 1.36 x 10-3; 2 mpi vs. 6 mpi, p = 9 x 10-13; 4 mpi vs. 6 mpi, p = 1.36 x 10-3, Kruskal-Wallis test, Fig. 6H). Finally, we quantified the number of Nodes2 and found an increase from 30.9 ± 4.3 at 2 mpi to 57.4 ± 5.3 at 4 mpi and 106.7 ± 11.9, with a significant difference between the different timepoints (2 mpi vs. 4 mpi, p = 2.31 x 10-3; 2 mpi vs. 6 mpi, p = 1.98 x 10-12; 4 mpi vs. 6 mpi, p = 2.31 x 10-3, KruskalWallis test, Fig. 6I).
In vivo maturation of supra-cortical, exclusively hiPSC-derived neurons followed by twophoton imaging We then decided to also follow the development and maturation of human neurons in the supracortical part of the graft. This allowed us to observe, in vivo, how human pyramidal neurons “selforganize” to create “human” parenchyma. In order to track these processes in the host brain over time, in real time, in the same animal, we performed in vivo two-photon microscopy. In vivo imaging was first performed at 4 mpi in the human-specific graft overlying the mouse motor cortex area (Fig. 7A, B), since the transplants before 3 mpi did not display a mature morphology. At 4 mpi the cell bodies were well labeled in the
cortex, but at this stage we could not detect any projections or interconnections between cells. But, by repeating the in vivo imaging at 7 and 8 mpi, we observed more mature dendritic structures that we could track down to 200 µm from the surface, thus also demonstrating that the human neurons continue to mature throughout the supra-cortical graft. Video 1 shows an example of the humanonly part of the brain with easily identifiable pyramidal-like dendrites. The human neurons form a densely packed structure that is fully vascularized (Fig. 7B, C). In Video 2, a z-scan through this area shows blood vessels of different diameters, and the passage of the unlabeled blood cells can be observed over the fluorescent background.
Discussion During the last decade, stem cell therapy has developed as a new medical frontier to prevent or rescue brain damage. Several studies have attempted to demonstrate the possibilities of hESCs and hiPSCs in neurobiology as reliable source for cell replacement therapies (Aboody et al., 2011). In particular, previous work showed how hESCs, derived from established embryonic cell lines, can be efficiently transplanted into the mouse cortex to generate cortical pyramidal neurons (EspunyCamacho et al., 2013; Gaspard et al., 2008). This has allowed the field to appreciate the major differences that exist between in vitro stem cell-derived model systems, and the power of having human stem cells participate in neurodevelopment in vivo. Thus, a second major use of hPSCs will be to further our understanding of human neural development in vivo at unprecedented resolution. As it is precisely at these embryonic and early post-natal stages that some major pathologies occur, like ASD and SCZ (Silbereis et al., 2016). Here, we present a grafting paradigm that allowed the targeted engraftment of human induced PSC-derived neuronal precursors into different cortical areas of neonatal mice. In addition, by injecting into the ventricles, we were able to visualize human NPC-derived cells in areas close to the ventricles, like the hippocampus, striatum and thalamus, and the olfactory bulbs. There, very
efficient integration was observed, and the development and function of human olfactory bulb neurons merits further study. Our data show that hiPSC-derived NPCs efficiently transplanted into the motor cortex also robustly integrated into the host brain by differentiating and maturing over time. Although the majority of the cells were confined within the bulk of the graft, we were able to perform morphological analysis on a considerable group of neurons outside the graft, fully integrated into the host architecture. In many brains, these represent on the order of hundreds of neurons per section. Most of these cells displayed long axonal and dendritic projections. Our findings suggest that after transplantation, the grafted neurons follow a progressive and continued integration into the host brain. Up to 6 mpi, they exhibited increasing neuronal maturation and axonal outgrowth. Further evidence of their differentiation was the detection of well-defined dendritic spines, thus suggesting that active synaptogenesis had taken place as seen before for embryonic PSCs (EspunyCamacho et al., 2013). In this study, we tracked in vivo the morphological development of human transplanted neurons into the motor cortex of mouse brain. Our grafting coordinates and the strong constitutive expression of GFP allowed us to image by two-photon microscopy the cell bodies and dendritic structure of the grafted neurons. The combination of the novel technique targeting the different areas of cortex and the promising use of hiPSC-derived neuronal precursor transplantation, together with the possibility to perform two-photon imaging in vivo of neuronal morphology and structure, may thus pave the way for an enhanced use of hiPSCs in studies of pathological mechanisms and also therapeutic screening.
Conclusion hiPSC-derived neurogenesis models have significantly advanced the study of human neurodevelopment at the molecular and cellular level, in vitro. However, the exceptionally long duration of human brain development is often not fully recapitulated in iPSC-derived in vitro
systems and presents a substantial challenge. Novel model systems are needed to access and experimentally manipulate the embryonic to postnatal period. Here, we present an efficient method to follow protracted human cortical neuron maturation over time using in vivo two-photon imaging and slice histology, after neo-natal transplantation of human iPSC-derived NPCs. This system now allows the detailed study of both genetic, environmental and pharmacological intervention at this early stage.
Acknowledgements We would like to thank Stéphanie Pons for continued support throughout this study, and Pascal Roux and Anne Danckaert from the Photonic BioImaging plateform for image acquisition support and quantitative data analysis performed with Acapella software, respectively. This work was funded by Institut Pasteur Programme PasteurInnov (IS/fc/13/160, and NICOBLOC 2018), the Centre National de la Recherche Scientifique (UMR 3571), the French National Cancer Institute INCa (2016-1-Tabac-01-IP-1), the Fondation de la Recherche Médicale (FRM, Grants SPF20140129365, DPA20140629803, FDT201805005198 and EQU201903007822), the Institut Pasteur priority programme GPF "Microbes&Brain", the ERANET programme iPS&BRAIN, the Fondation Alzheimer-INSERM, and the Fondation Vaincre Alzheimer, all to U.M. This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 7202070 (HBP SGA1). M.G. acknowledges support from Fondation pour la Recherche Médicale FRM (ING20111223378), l'Association "Schizo oui", Institut de France – Fondation NRJ (NRJ2010), Association ‘Vaincre les Maladies Lysosomales-VML’ (VML2016-1004), Fondation Lejeune (FJL1389), Fondation de France (Eng° 00060522) and Fondation Motrice (N°2016/4). U.M. and M.G. are members of the Laboratory of Excellence, LABEX BIO-PSY. The laboratories of U.M. and M.G. are part of the École des Neurosciences de Paris Ile-de-France Thematic Network for Advanced Research (RTRA).
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Figure legends Figure 1 Human iPSC-derived neuronal precursor line expressing green fluorescent protein (GFP) used for the grafting, and intracranial injection of hiPSC-derived neuronal precursors into the cortex of neonatal mice. (A, B) GFP is expressed ubiquitously and constitutively. Scale bars = 200 µm in (A) for 5x magnification, 50 µm in (B) for 20x magnification. (C) Intracranial injection of hiPSC derived neuronal precursors into the cortex of neonatal mice. The two landmarks used to target the motor cortex for injection are easily visible through the translucent skin of NOD/SCID neonatal pups. (D) Schematic view of the mouse head shows the target site for the injection (red arrow). The injection site is located approximately 1 mm posterior to bregma and 1 mm lateral to the sagittal suture, with 1 mm of depth. (E) Whole mouse brain and indication of the graft (white delineated area).
Figure 2 Efficient human iPSC derived neuronal precursor transplantation into the cortex of newborn mice. (A) Representative engraftment in the cortex of adult mice after neonatal transplantation. Anti-GFP immunofluorescence (in green) to detect the graft localization in the cortex. It extends from the most rostral to caudal part of the brain. The graft localization includes the prelimbic cortex, somatosensory cortex and motor cortex. (B) Higher magnification shows that most of the injected cells remain confined in the bulk of the graft. Scale bars = 100 µm in (B) for a 10x magnification. (C, D) Human iPSC-derived neuronal precursors and axons emanating from the solid graft over time. GFP-positive cells detected by anti-GFP immunofluorescence (in white) in the motor cortex and analyzed at 2 months post injection (mpi). For each time point most of the
neurons remain confined within the bulk of the graft. Most cells at 2 mpi differentiated by extending long axonal projections. Scale bar = 200 µm for 20x magnification.
Figure 3 Intra-ventricular injection of human NPCs allows to target several brain parenchyma, and the olfactory bulb. (A) Brain slice with transplant in the lateral ventricle, analyzed at 5 mpi. (B, C) Grafts in the lateral ventricle, between the posterior part of the CA1 area of the hippocampus and entorhinal cortex. (D-G) Presence of hiPSC derived neurons in the olfactory bulb revealed by GFP immunofluorescence (in red). Most of the human neurons reside in the granule cell layer, but also in the glomerular layer. By injecting the NPCs into the ventricles, the cells have migrated from the graft trough the rostral migratory stream towards the olfactory bulb. (F, G) Images showing the typical morphology of olfactory bulb cells. Scale bars = 1 mm in (A), 200 µm in (B-E) and 20 µm in (F, G).
Figure 4 Immunofluorescence analysis for the detection of astrocytes, microglia, and somatostatinexpressing human interneurons. (A) High-magnification confocal analysis of human astrocytes 4 mpi of the hiPSC-derived neuronal precursors. Panel, from left to right: Astrocytes in red, labelled with anti-GFAP antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (B) High-magnification confocal analysis of microglia 4 mpi of the hiPSC-derived neuronal precursors. Panel, from left to right: Microglial cells in red, labelled with anti-Iba1
antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (C) High-magnification confocal analysis of somatostatin-expressing human interneurons, analyzed at 6 mpi. Panel, from left to right: SOM+ interneurons in red, labelled with antisomatostatin antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 20 µm. (D) Low-magnification confocal analysis of parvalbumin-expressing human interneurons, analyzed at 6 mpi. This is an example of a human graft on top of mouse cortex. PV+ labeling is only detectable in the mouse, not the human cells. Panel, from left to right: PV+ interneurons in red, labelled with anti-parvalbumin antibody; GFP-expressing human cells; DAPI staining of cell nuclei in blue; overlay. Scale bar = 50 µm.
Figure 5 Immunofluorescence analysis for upper-layer pyramidal neurons, cortical laminae 2 to 4 in mouse 4 mpi of the hiPSC-derived neuronal precursors. (A) High-magnification confocal analysis of human pyramidal neurons expressing the CUTL1/CUX1 marker. The CUTL1/CUX1 marker resulted in extensive colocalization in GFP-expressing human neurons. Scale bar = 20 µm. (B, C) GFP-expressing human neurons displaying a pyramidal morphology. Scale bar = 20 µm. (D) GFP-expressing human pyramidal neurons exhibit extensive co-localization with the human neuronal nuclei-specific marker, STEM101. Arrow heads indicate examples of colocalization. STEM101 nuclear labeling in red, DAPI staining of nuclei in blue. Scale bar = 20 µm. (E) Absence of human neuronal nuclei-specific marker, STEM101, in the mouse brain. Arrow heads indicate examples of co-localization. STEM101 nuclear labeling in red, DAPI
staining of nuclei in blue, in the human graft. Asterisks demarcate the boundary of the mouse cortex. Scale bar = 20 µm.
Figure 6 Human iPSC-derived neuronal precursor maturation and differentiation over time. (A) Representative images of hiPSC-derived neuronal precursors (GFP positive) displaying immature dendritic patterns at 1 and 2 mpi. At 4 mpi the neurons start displaying a more mature dendritic pattern until achieving a complex and mature differentiation at 6 mpi. Scale bar = 20 µm. (B, C) Representative confocal images of GFP immunostained neurons used for morphological analysis with the Acapella software. (D) Schematic neuron representation showing the parameters used for the branching analysis. (E-I) Quantification of the morphological development over time performed at 2, 4 and 6 mpi. n=166 for 2 mpi, n=769 for 4 mpi, and n=468 for 6 mpi. **p<0.01, ****p<0.0001.
Figure 7 In vivo integration and maturation of hiPSC-derived NPCs by two-photon imaging. (A) In vivo two-photon images of hiPSC-derived NPCs in the "human-only" part of the transplant overlying mouse cortex at 8 mpi, as detailed in Video 1. The dendritic maturation of the pyramidal neurons is indicated (arrows). Scale bar = 20 µm. (B) In vivo two-photon image of hiPSC-derived neuronal precursors performed at 4mpi, as detailed in Video 2. Arrows indicate the blood vessels. Scale bar = 20 µm. (C) High magnification image of a blood vessel in a human graft. Arrows point to endothelial cells identified with the CD31 marker (red). Cell bodies are DAPI stained (blue), human neurons express GFP (green). Scale bar = 20 µm.
Video 1 Higher magnification video of developed dendrites at 8mpi. Z-scan through the supra-cortical graft of human-only neurons at high resolution. Note the developed dendritic architecture. Scale bar = 20 µm.
Video 2 Z-scan through the supra-cortical graft of human-only neurons. The blood vessels are clearly visible, in black. With the fluorescent background, the movement of the unlabeled blood cells can be made out. Scale bar = 20 µm.
Highlights
Development of an efficient in vivo system to study human neurons Human induced pluripotent stem-cell derived cells integrate into the mouse brain Human neurons exhibit a protracted in vivo developmental program Transplants of human neuronal precursors can be followed by live two-photon imaging