HUMAN iPSC-DERIVED BRAIN ORGANOIDS AND USES THEREOF
20210300998 · 2021-09-30
Assignee
Inventors
Cpc classification
C12N5/0062
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C12N2506/45
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C07K16/085
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Disclosed are methods of producing human iPSC-derived brain organoids and uses thereof to detect and develop treatment for HCMV-induced brain deformation in developing fetus.
Claims
1. A method of treating or preventing a neurodevelopmental deficit or HCMV-induced brain malformation in a developing fetus, comprising administering to the mother carrying the fetus a therapeutically effective amount of: one or more neutralizing antibodies that target HCMV pentamer complex; one or more neutralizing antibodies that target HCMV gH; a vaccine comprising the HCMV pentamer complex and capable of eliciting neutralizing antibodies; a vaccine comprising gH and capable of eliciting neutralizing antibodies; a platelet-derived growth factor receptor alpha (PDGFRα) inhibitor; an epidermal growth factor receptor (EGFR) inhibitor; or a combination thereof, wherein the mother is infected by HCMV or at an elevated risk of HCMV infection, and wherein the HCMV pentamer complex comprises gH, gL, UL128, UL130, and UL131A.
2. The method of claim 1, wherein the developing fetus is at the transition between the first and second trimester of gestation.
3. The method of claim 1, wherein the neutralizing antibodies include 1B2 and 62-11.
4. The method of claim 1, wherein the neutralizing antibody is a monoclonal antibody.
5. The method of claim 1, wherein the neutralizing antibody is a human antibody or a humanized antibody.
6. (canceled)
7. The method of claim 1, wherein the PDGFRα inhibitor is an siRNA specific for the PDFGRα.
8. The method of claim 1, wherein the EGFR inhibitor is an siRNA specific for the EGFR.
9. The method of claim 1, wherein the administration is by oral administration or intravenous injection.
10. A combinational therapy for treating or preventing a neurodevelopmental deficit or HCMV-induced brain malformation in a developing fetus, comprising administering to the mother carrying the fetus two or more of (i) one or more neutralizing antibodies that target HCMV pentamer complex, (ii) one or more neutralizing antibodies that target HCMV gH, (iii) a vaccine comprising HCMV gH and capable of eliciting neutralizing antibodies, (iv) a vaccine comprising HCMV pentamer complex and capable of eliciting neutralizing antibodies, (v) one or more PDGFRα inhibitors, and (vi) one or more EGFR inhibitors, wherein the mother is infected by HCMV or at an elevated risk of HCMV infection, and wherein the HCMV pentamer complex comprises gH, gL, UL128, UL130, and UL131A.
11. The combinational therapy of claim 10, wherein the developing fetus is at the transition between the first and second trimester of gestation.
12. The combinational therapy of claim 10, wherein the neutralizing antibodies include 1B2 and 62-11.
13. The combinational therapy of claim 10, wherein the neutralizing antibody is a monoclonal antibody.
14. The combinational therapy of claim 10, wherein the neutralizing antibody is a human antibody or a humanized antibody.
15. (canceled)
16. The combinational therapy of claim 10, wherein the PDGFRα inhibitor is an siRNA specific for the PDFGRα.
17. The combinational therapy of claim 10, wherein the EGFR inhibitor is an siRNA specific for the EGFR.
18. The combinational therapy of claim 10, wherein the administration is by oral administration or intravenous injection.
19. An hiPSC-derived brain organoid comprising a layered structure which comprises a core region positive for the neural progenitor markers SOX2 and TLX, an outer layer positive for BRN2, a late-born superficial layer neuronal marker, and MAP2.
20. The brain organoid of claim 19, further comprising PAX6-positive apical progenitors, and TBR2-positive basal progenitors.
21. The brain organoid of claim 19, comprising SOX2-positive progenitors spanning across the ventricular zone and the subventricular zone.
22. The brain organoid of claim 19, wherein the brain organoid is SOX2+ in the ventricle zone, TBR2+ in the subventricular zone, and CTIP2+ in the cortical plate.
23.-24. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.
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DETAILED DESCRIPTION
[0028] Disclosed herein are methods of treating or preventing a neurodevelopmental deficit in a developing fetus, wherein the mother carrying the fetus is infected by HCMV or at an elevated risk of HCMV infection. The methods entail administering to the mother a therapeutically effective amount of one or more of the following: (i) one or more neutralizing antibodies that target HCMV pentamer complex, (ii) one or more neutralizing antibodies that target HCMV gH, (iii) a vaccine comprising HCMV gH and capable of eliciting neutralizing antibodies, (iv) a vaccine comprising HCMV pentamer complex and capable of eliciting neutralizing antibodies, (v) one or more PDGFRα inhibitors, and (vi) one or more EGFR inhibitors.
[0029] The term “effective amount” as used herein refers to an amount of a composition that produces a desired effect. For example, the brain organoids may be infected with an effective amount of a therapeutic agent to study its effect in vitro (e.g., cell culture) or to produce a desired therapeutic effect ex vivo or in vitro. An effective amount of a composition, an antibody or a vaccine may be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In such a case, the effective amount of a composition, an antibody or a vaccine is a “therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.” The precise effective amount or therapeutically effective amount is an amount of the composition, antibody or vaccine that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further, an effective or therapeutically effective amount may vary depending on whether the composition, antibody or vaccine is administered alone or in combination with another composition, drug, therapy or other therapeutic method or modality. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring the brain organoid or subject's response to administration of a composition, antibody or vaccine and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005, which is hereby incorporated by reference as if fully set forth herein.
[0030] “Treating” or “treatment” of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.
[0031] In some embodiments, the antibody, vaccine or other therapeutic agents described herein may be administered by injection, nasal or respiratory inhalation, or by intravascular (i.v.) (e.g., intra-arterial, intravenous, and portal venous), subcutaneous (s.c.), intracutaneously (i.c.), intradermal (i.d.), or intraperitoneal (i.p.) administration. The antibody, vaccine or other therapeutic agents may be administered more than once.
[0032] One of the main limitations in modeling HCMV-induced neurodevelopmental disorders is the lack of an in vitro model that could faithfully recapitulate HCMV infection of the developing human brain. Disclosed herein are hiPSC-derived brain organoids to model effects of HCMV infection on early human brain development in a 3D human brain organoid system. As demonstrated in the working examples, the “clinical-like” HCMV strain TB40/E can efficiently infect hiPSC-derived brain organoids and propagate in these organoids, thereby impairing brain organoid growth and cortical structure, a phenotype reminiscent of HCMV-induced microcephaly. Moreover, HCMV infection of human brain organoids can result in drastically abnormal calcium signaling and neural network activity. The neurodevelopmental defects induced by TB40/E in infected brain organoids can be largely prevented by NAbs that target the HCMV PC, a primary target of protective antibody responses and pivotal component of current subunit vaccine designs to prevent intrauterine HCMV transmission.sup.47. Other publications also describe effects of HCMV infection on early human brain development using human brain organoid models.sup.48,49.
[0033] Human induced pluripotent stem cell (hiPSC)-derived brain organoids have emerged as a powerful in vitro model to study human brain development and neurodevelopmental diseases in a three-dimensional (3D) cellular biosystem.sup.11-14. These so-called “mini brains” can organize into discrete and interdependent brain regions that are reminiscent of the human cortex and form multilayered structures containing inner neural progenitor zones and outer cortical layers.sup.15. Brain organoids have been used to model neurological defects caused by Zika virus.sup.14, which has been linked to microcephalyl.sup.6,17.
[0034] To model the clinical effects of HCMV infection on early human brain development, hiPSC-derived brain organoids at day 45 of differentiation.sup.15,18 were used. This stage of hiPSC-derived brain organoids is reminiscent of early human fetal brain development at the transition between the first and second trimester.sup.19, when congenital HCMV infection is known to be associated with severe neurological consequences.sup.7,50. The observation that HCMV can severely impair the growth, structure, and neural network activity of hiPSC-derived brain organoids provides a possible explanation for HCMV-induced neurological defects during early gestation.sup.2. The data disclosed herein are consistent with the clinical manifestation of HCMV-induced microcephaly, including abnormal cortical development.sup.8,51. Clinical data for neural network activity or brain electrophysiology in patients congenitally infected with HCMV is limited.sup.2. The findings in TB40/E-infected brain organoids disclosed herein may therefore provide novel insights into the dramatic consequences that congenital HCMV infection could have on neural network formation during early human brain development.
[0035] As disclosed in the working examples, brain organoids were generated from human iPSCs to model brain defects caused by HCMV infection in vitro in a 3D human cellular biosystem. The hiPSC-derived brain organoids were infected with the “clinical-like” HCMV strain TB40/E to determine how HCMV infection impacts early human brain development, potential cellular receptors that mediate brain cell infection by HCMV were determined, and the capacity of neutralizing antibodies (NAbs) to prevent HCMV-induced brain malformation were tested.
[0036] In humans, HCMV-immune labeled cells can be found ubiquitously distributed in the ventricular zone, subventricular zone, and cortical plate in the brains of congenitally-infected fetuses.sup.8. Although HCMV can target different cell types in the brains of congenitally infected fetuses, it shows higher tropism to neural progenitor cells.sup.8. It is shown that HCMV primarily infects the TBR2+ cells in the SVZ of the brain organoids and that HCMV infection of the brain organoids disrupts the organization of the SVZ and the formation of proper cortical layers, suggesting that HCMV targets predominantly neural progenitors in the SVZ, thereby impairing cortical development. HCMV infection at early stage of human brain development could dramatically affect the expansion of the neocortex and consequently lead to reduced brain size and impaired brain function. While the brain organoid model may provide a direct link between HCMV infection of the human brain and the development of microcephaly, severe neurological conditions due to congenital HCMV infection may also result from HCMV-induced placental insufficiency, which could lead to nutrition or oxygen deprivation in the fetal compartment.sup.2.
[0037] To support the relevance of the brain organoid model to study HCMV infection of the developing human brain, potential cellular receptors that are involved in brain organoid infection by TB40/E were evaluated. Consistent with recent findings for HCMV entry.sup.39,40,52,53, TB40/E infection of human brain organoid involves PDGFRα and EGFR as cellular receptors either directly or indirectly, whereas the infection does not appear to involve cellular integrins. While PDGFRα has been shown to function as a cellular receptor during HCMV entry into fibroblasts via interaction with a trimeric complex composed of gH/gL/gO.sup.52,54,55, EGFR is thought to be involved in PC-mediated entry into epithelial cells.sup.52,56. As demonstrated in the working examples, both PDGFRα and EGFR are important for TB40/E infection of human brain organoid, as siRNA-mediated knockdown of either PDGFRα or EGFR completely abrogated the susceptibility of brain organoids to TB40/E. Unlike previous findings with fibroblasts and epithelial cells that implicate either PDGFRα or EGFR as important receptor for HCMV entry into host cells, the brain organoid study suggests that both PDGFRα and EGFR are important for efficient HCMV infection of cell types in the developing human brain. This observation suggests that infection of human brain cells by HCMV could be orchestrated by multiple HCMV envelope glycoproteins complexes.
[0038] As demonstrated in the working examples using two previously isolated NAbs, 1B2 and 62-11.sup.36,37 which were raised against the HCMV PC by vaccination, targeting epitopes of the PC can potently prevent the infection of brain organoids by HCMV, thereby allowing normal human brain organoid development. HCMV NAbs targeting the PC or other envelope glycoprotein complexes have been characterized using various cell types, including fibroblasts, epithelial/endothelial cells, monocytes/macrophages, and cells of the human placenta.sup.36,57-59. Yet, the capacity of NAbs to prevent HCMV infection of human brain cells has not been reported. This study demonstrates that both 1B2 and 62-11 NAbs can potently prevent HCMV infection of human brain organoids, suggesting that NAbs targeting the PC may have the capacity to effectively prevent HCMV infection of the developing human brain. Notably, the maximum NAb concentration (4 μg/ml) used to model the prevention of HCMV infection of the brain organoids was more than 50 times lower than monoclonal antibody concentrations found in human serum post intravenous infusion of PC-specific NAbs in healthy adults or kidney transplant recipients.sup.60,61. Considering that fetal IgG concentrations at weeks 17-22 are about 5-10% of the maternal IgG levels.sup.62, this may suggest that maternal monoclonal antibody concentrations of 20-40 μg/ml could potentially reach the fetal compartment at a time point of gestation when HCMV-induced fetal brain damage is most severe′. As the embryo blood-brain-barrier is considered to be immature or “leaky”, antibodies that enter the fetal circulation from the mother during early gestation are thought to have direct access to the fetal brain.sup.63,64, suggesting that vaccine-induced or passively-administered NAbs may have the potential to prevent HCMV-related brain diseases during early fetal development. Given that the antibodies used in this study were isolated from PC-immunized mice, clinical application of these antibodies to reduce or prevent neurodevelopmental defects caused by HCMV would require their humanization to allow for Fc binding and to prevent development of a potentially harmful human anti-mouse antibody response.sup.65.
[0039] There has been controversy about the protective capacity of antibodies in prevention of congenital HCMV infection.sup.32,66, especially in light of imperfect protection by naturally acquired HCMV immunity.sup.3. While a recent study using samples from a high seroprevalence population did not find a correlation between PC-specific or gH/gL/gO-specific antibody titers and the prevention of congenital transmission.sup.67, other studies reported a correlation between PC-specific or gB-specific antibodies and reduced intrauterine HCMV transmission following primary maternal infection.sup.33-35. In addition, a recent study using the highly relevant rhesus macaque model of congenital CMV infection has shown that passively transferred, pre-existing antibodies with high neutralizing activity can protect against CMV vertical transmission and fetal demise.sup.68. While the precise reasons for the difference in the protective capacity of CMV-specific antibodies observed in these studies are unclear, they could be related to differences in epitope specificity, affinity, potency, or function of the antibody response. This study indicates that pre-existing PC-specific or gH-specific NAbs transferred from the mother to the fetus following passive or active immunization may have the capacity to prevent severe fetal brain malformation by HUN that has crossed the fetal-maternal interface.
[0040] Thus, based on hiPSC-derived brain organoids modeling the effects of HCMV infection on early human brain development with different HCMV stains, this disclosure demonstrates that HCMV infection of hiPSC-derived brain organoids can result in severely impaired brain organoid growth and structure as well as altered calcium signaling and neural network activity, phenotypes that mimic HCMV-induced neurological conditions, such as microcephaly. In addition, this disclosure demonstrates that severely impaired brain organoid development caused by HCMV can be effectively prevented by NAbs that target the HCMV PC, suggesting that PC-specific NAbs may have the capacity to prevent HCMV-induced neurodevelopmental deficits. This 3D organoid model provides a powerful system to study HCMV-induced brain malformation and to identify potential antiviral agents that could prevent abnormal brain development due to congenital HCMV infection.
[0041] The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
EXAMPLES
Materials and Methods
[0042] Human iPSC Derivation. AG14048 and IMR90 (190) human fibroblasts were purchased from Coriell. AG14048 iPSCs were generated from AG14048 fibroblasts through episomal reprogramming using episomal plasmids expressing OCT4, SOX2, L-MYC, KLF4, shp53, and EBNA1 (Addgene plasmids pCXLE-hSK, pCXLE-h UL, pCXLE-hOCT3/4-shp53-F, and pCXWB-EBNA1).sup.69,70. Derivation of 190 iPSCs were described previously.sup.71. Specifically, human fibroblast cells were electroporated with the reprogramming factors using 4D Nucleofector (Lonza) and seeded into 6-well plates coated with 1:100 diluted Matrigel (Corning) and maintained in E8 medium (Invitrogen). iPSCs were maintained at 37° C. in Matrigel-coated 6-well plates with daily medium change and passaged every 3-4 days using 0.5 mM EDTA (GIBCO) treatment and manual dissociation. Both AG14048 and 190 iPSC lines generated in this study were authenticated using short tandem repeat (STR) assay.
[0043] Cell Lines. ARPE-19 (male) and MRC-5 (male) cells were purchased from ATCC and maintained in Dulbecco's minimal essential medium (DMEM) or minimal essential medium (MEM), respectively, supplemented with 10% fetal bovine serum (FBS). All cell lines were cultured at 37° C. and 5% CO.sub.2.
[0044] Viruses. GFP-labeled TB40/E and TR viruses that express a GFP reporter under the SV40 promoter were derived from BAC DNA, kindly provided by T. E. Shenk and E. A. Murphy (Princeton University, NJ).sup.25,38. GFP-tagged Towne virus (RC2940) that expresses an IE2/GFP fusion protein, was obtained from E. S. Mocarski (Emory University School of Medicine, GA).sup.26. TB40/E-Gluc virus expressing a luciferase gene under the IE promoter was reconstituted from BAC DNA, kindly provided by S. Sinzger (Ulm University, Germany).sup.31. TB40/E viral stocks were generated following viral propagation in ARPE-19 cells, whereas Towne virus stocks were generated following viral propagation in MRC-5. Viral stocks were generated by standard ultracentrifugation procedures as described previously.sup.37,72 and titrated on MRC-5 by immunostaining using the IE1-specific monoclonal antibody p63-27.sup.73 (kindly provided by William Britt, University of Alabama at Birmingham) and the Vectastain ABC kit and 3,3′-diaminobenzidine (DAB) substrate according to the manufacturer's instructions. Images were taken using a DMi8 inverted microscope equipped with a linear motorized stage.
[0045] Generation of brain organoids from hiPSCs. hiPSC-derived brain organoids were generated based on the protocol described by Lancaster et al..sup.15 with modifications. Briefly, hiPSCs were generated from IMR-90 and AG14048 human fibroblasts, confirmed to be karyotypically normal and negative for mycoplasma contamination. On day 0 of organoid culture, hiPSCs were dissociated with EDTA, and seeded in suspension in a 6-well plate to form embryoid bodies in E8 medium with 5 μM ROCK inhibitor Y-27632. From day 1 to day 4, cells were cultured in E8 medium without ROCK inhibitor with daily medium change. On day 5, E8 medium was replaced by neural induction medium (NIM) containing DMEM-F12, 1×N2 supplement, 1×minimum essential medium NEAA (MEM-NEAA), and 2 μg/ml Heparin. On day 8, the spheres were embedded in 20-25% Matrigel in NIM in a 6-well suspension plate and incubated at 37° C. for 4 hours, followed by gentle addition of 2 ml of the NIM. On day 10-12, brain organoids were lifted and transferred to a new 6-well plate. NIM was changed daily from day 5 to day 15. On day 15, brain organoids were transferred to a T25 suspension culture flask and cultured in differentiation medium containing DMEM-F12, 1×N2 supplement, 2.5 μg/ml Insulin, 1× Glutamax, 0.5×MEM-NEAA, 3.5 μl/L (V/V) 2-Mercaptoethanol, and 1× B27 supplement on an Orbi-Shaker (Benchmark Scientific) at 50 rpm rotating speed. Medium was changed every 2-3 days. Organoids that exhibited similar size and passed the quality control criteria described by Lancaster et al..sup.15,18 were used for the study. The criteria include clear embryoid body border, formation of organized neuroepithelium before embedding, formation of ventricle-like structure, and development of defined bud in Matrigel without premature differentiation.
[0046] The media ingredients and the protocol for producing the hiPSC-derived brain organoids are summarized in Table 1 and Table 2, respectively.
TABLE-US-00001 TABLE 1 Media Ingredients EB medium E8 Neural induction medium DMEM-F12 with N2, MEM-NEAA, and heparin Differential medium DMEM-F12 with N2, B27, insulin, GlutaMAX, MEM-NEAA, 2-mercaptoethanol
TABLE-US-00002 TABLE 2 Brain Organoids Protocol EB medium 5 days Neural induction medium (suspension) 3 days Neural induction medium with 20-25% Matrigel, Matrigel (stationary) 2-4 days Neural induction medium (stationary) 3-5 days Differential medium Shaking for maintaining, shaker Screening Day 20-40
[0047] HCMV infection of brain organoids. Brain organoids at day 45 of differentiation were seeded in 24-well plates and exposed to 5×10.sup.5 pfu/ml of HCMV virus for 24 hours. Following infection, 1 ml medium was replaced, and each organoid was placed in a single well for the duration of the experiment. At different time points post infection, each organoid was evaluated for GFP fluorescence intensity and organoid size in diameter using the same microscopy setting during each experiment.
[0048] Brain organoid treatment with NAbs. 1B2 and 62-11 NAbs at final concentrations ranging from 3.2 ng/ml to 4000 ng/ml were incubated with 5×10.sup.5 pfu/ml of HCMV for 1 hour at 37° C. and then transferred to brain organoid-containing wells. Normal mouse IgG was used as a control at the concentration of 2000 ng/ml. For proliferation assay, brain organoids were incubated with 10 μM BrdU for 2 hours and fixed in 4% paraformaldehyde (PFA) for 1 hour, followed by immunostaining.
[0049] Immunostaining. Cells on coverslip were fixed with 4% PFA in PBS for 15 minutes at room temperature. Brain organoids were fixed with 4% PFA for 1 hour and submerged in 30% sucrose overnight. The samples were embedded in OCT and sectioned at a thickness of 14 or 20 μm using Leica CM3050S. Cells or brain organoid sections were permeabilized and blocked with blocking solution (1×PBS containing 0.1% Triton X-100 and 5% normal donkey serum) for 1 hour. Primary antibodies in blocking solution were then added and incubated at 4° C. for overnight, followed by washing and incubation with secondary antibodies. Cells were counterstained with DAPI before mounting.
[0050] The following antibodies were used: TUJ1 (Covance PRB-435P, rabbit or mouse, 1:20,000), MAP2 (Abcam ab5392, chicken, 1:5000), SOX2 (Santa Cruz sc-17320, goat, 1:1000), CTIP2 (Abcam, ab18465, rat, 1:300), SABT2 (Abcam ab92446, mouse, 1:300), TBR2 (Abcam ab31940, rabbit, 1:300); BRN2 (Santa Cruz sc-28594, rabbit, 1:200), TLX (Shi lab20, rabbit, 1:1000), BrdU (Accurate Chemical & Scientific Corp OBT0030, Rat, 1:6000), Cleaved Caspase3 (Cell Signaling 9661, rabbit, 1:200).
[0051] Images were obtained with a Carl Zeiss LSM700 confocal microscope or Nikon Eclipse TE-2000-S microscope. Cortical layer thicknesses, brain organoid size and GFP fluorescence intensity of brain organoids were measured by Image J. For measuring the layer thickness, small rectangles were drawn to ensure two opposite sides to align with both layer marker dashed lines.
[0052] Calcium imaging. Brain organoids at day 45 of differentiation were partially dissociated and seeded on Matrigel-coated Ibidi p-slide 8-well-chamber slides and allowed to grow for 3 days until calcium imaging was performed. Brain organoids were rinsed in artificial-cerebrospinal fluid (ACSF) (124 mM NaCl, 2.5 mM KCl, 26 mM NaHCO.sub.3, 1 mM MgCl2, 2 m M CaCl.sub.2), 1.25 mM NaH.sub.2PO.sub.4 and 10 mM D-glucose solution) at 37° C. for 10 minutes and then incubated in fresh 95% 02 oxygenated ACSF containing 2 μM Fluo-4 AM for 20 minutes. Subsequently, brain organoids were visualized using a Zeiss Axio Observer Z1 microscope for serial time lapse imaging. Time lapse imaging was acquired at 10× magnification and at 16 frames per second speed for 5 minutes using a Hamamatsu EMCCD model C9100-13. Glutamate (3 μM) stimulation during imaging progress was performed 5 seconds after start. Ca.sup.2+ imaging videos were captured and processed using ZEN software and quantification was performed using Image-Pro Premier 9.1. Fluorescence intensity change over time is defined as: ΔF/F=(F−Fo)/Fo, where F is the fluorescence intensity at any time point, and Fo is the baseline fluorescence intensity averaged across the whole movie for each cell. For calcium imaging of HCMV (TB40/E-Gluc without GFP)-infected brain organoids at day 45 of differentiation, organoids were partially dissociated and allowed to attach overnight onto Matrigel-coated Ibidi p-Slide 8 Well chamber slide. Brain organoids were either exposed to HCMV at 5×10.sup.5 pfu/ml per brain organoid or to a mixture of HCMV and NAb 1B2. For NAb treatment, the same amount of virus was incubated with 4,000 ng/ml 1B2 antibody at 37° C. for 1 hour before addition to the brain organoid culture. As a control, brain organoids were infected with 5×10.sup.5 pfu/ml UV-inactivated HCMV. Calcium imaging was performed 3 days post infection. In order to identify cells that were infected by HCMV, calcium imaging data of each area were recorded with the associated coordinates using most bottom-right corner as a reference (0, 0). Right after calcium imaging, the brain organoids were immediately fixed with 4% PFA and immunostained for HCMV IE. For imaging, the fields were chosen based on the calcium imaging video coordinates and the positions were adjusted manually to match the original calcium imaging video pictures. By using this procedure, the HCMV positive cells were located by 1E1 positive staining.
[0053] Microelectrode Arrays (MEA). Brain organoids at day 45 of differentiation were partially dissociated and seeded onto 12-well transparent MEA plates at three brain organoids per well. Brain organoids were cultured in BrainPhys medium, including 1)(1327, 1× N2, 20 ng/ml GDNF, 20 ng/ml BDNF, 500 μg/ml Dibutyryl-cAMP, 1× Glutamax, and 1×NEAA. MEA recordings were performed at 37° C. in a Maestro MEA system with AxIS software using a bandwidth with a filter for 10 Hz to 2.5 kHz cutoff frequencies. Spike detection was performed using an adaptive threshold set to 5.5 times of the standard deviation of the estimated noise on each electrode. For recordings, following a 5-minute resting time in the Maestro instrument, each plate was recorded for 10 minutes to calculate the spike rate per well. When a recording of 5 spikes over the length of 1 minute (5 spikes per minute) was obtained, the electrode was considered active. Individual electrode bursts were identified using an adaptive Poisson surprise algorithm, while network bursts were identified for each well using a non-adaptive algorithm requiring a minimum of 10 spikes with a maximum inter-spike interval of 100 ms. Multielectrode data analysis was performed using the Axion Biosystems NeuralMetrics Tool. Synchrony index was calculated by NeuralMetric Tool with synchrony window set as 20 ms. For the pharmacological experiment, CNQX (10 μM) or bicuculline (10 μM) were applied to plate immediately before recording. For MEA recording, brain organoids treated with UV-irradiated TB40/E were included as the control organoids. Brain organoids were exposed to TB40/E or UV-irradiated TB40/E at 5×10.sup.5 pfu/ml per brain organoid in the presence of 4000 ng/ml IgG. For NAb treatment, 5×10.sup.5 pfu/ml of TB40/E was incubated with 4000 ng/ml NAb 1B2 for 1 hour and then added to brain organoids cultures. Wave forms of spike were generated from exported recording data on single electrode and the graph was created in Excel. The phase contrast and GFP fluorescent images of organoids seeded in the MEA plates were taken after MEA recording.
[0054] Generation of NPCs from human iPSCs. hiPSC-derived NPCs were generated according to previously described procedures.sup.74. Briefly, IMR90 hiPSCs were dissociated with Accutase into single cells and seeded onto a Matrigel-coated 6-well plate at 1×10.sup.5 cells per well in E8 medium containing 1 μM Y-27632. On the next day, the E8 medium was substituted with NPC induction medium, including E6 medium, 100 nM ATRA, 10 μM SB431542, 250 nM LDN-193189, and the NPC induction medium was changed every day for 8 days. Cells were then transferred to a T25 or T75 flask and maintained in NPC maintenance medium containing 1× B27, 1× N2 supplement, 1×NEAA, 1× Glutamax, 100 nM ATRA, 3 μM CHIR99021, 2 μM SB431542, 10 ng/ml EGF and 10 ng/ml FGF, with daily medium change.
[0055] Neuronal differentiation from NPCs. Three million hiPSC-derived NPCs were dissociated with Accutase and seeded on Matrigel-coated 10 cm plates in neural induction medium containing DMEM/F12, 1× N2, 1)(1327, 1×NEAR, 1×Glutamax. Cells were cultured in this medium for 3 weeks and then transferred to BrainPhys medium and maintained in the medium with medium change every 4-5 days.
[0056] RNA-sequencing. hiPSC-derived brain organoids at day 45 differentiation were infected with 5×10.sup.5 pfu/ml TB40/E or UV-irradiated TB40/E as a control. Total RNA was isolated from TB40/E-infected organoids or control organoids 15 days post infection using Trizol. RNA quality control was performed by the Integrative Genomics Core at City of Hope. RNA-sequencing reads were aligned against the human genome (hg19) using TopHat275. Read counts were quantified using htseq-count with UCSC known gene annotations. Aligned reads were counted using GenomicRanges. Genes were filtered to only include transcripts with RPKM values greater than 0.1 (after a rounded log 2-transformation) in at least 50% samples. Genes smaller than 150 bp were removed prior to differential expression analysis. Log 2 (RPKM+0.1) expression values were used for visualization and fold-change calculations. Normalization of heatmap values was performed as the following: RNA seq of control sample was converted by Log 10 of the RPKM value, the HCMV infected sample value was determined by sum=log 10(RPKM value)+DEseq2−log 2 (fold change value). Heatmap was generated by ClustVis and image was prepared using Photoshop.
[0057] RT-qPCR analysis. For siRNA knockdown of receptors in iPSC-derived NPCs, total RNA was extracted using Trizol Reagent. cDNAs were reverse transcribed using Tetro cDNA synthesis kit. RT-qPCR was performed using DyNAmo Flash SYBR Green qPCR mix on a StepOnePlus system and normalized to β-actin. Primers used in the qPCR are listed in Table 3.
TABLE-US-00003 TABLE 3 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-CMV IE-1 clone p63- Andreoni et al, N/A 27 1989 Mouse monoclonal anti-CMV PC clone 1B2 Chiuppesi et al., N/A 2015 Mouse monoclonal anti-CMV gH clone 62-11 Chiuppesi et al., N/A 2015 Normal mouse IgG Santa Cruz Cat# sc-2025; Biotechnologies RRID: AB_737182 Rabbit anti-TUJ1 Covance Cat# PRB-435P; RRID: AB_10616742 Chicken anti-MAP2 Abcam Cat# Abcam ab5392; RRID: AB_2138153 Goat anti-SOX2 Santa Cruz Cat# sc-17320; Biotechnologies RRID: AB_2286684 Rat anti-CTIP2 Abcam Cat# ab18465; RRID: AB_2064130 Mouse anti-SABT2 Abcam Cat# ab92446; RRID: AB_10563678 Rabbit anti-TRB2 Abcam Cat# ab31940; RRID: AB_2200219 Rabbit anti-BRN2 Santa Cruz Cat# sc-28594 Biotechnologies RRID: AB_2167382 Rabbit anti-TLX Li et al., 2008 N/A MAb Rat anti-BrdU Accurate Chemical Cat# OBT0030 & Scientific Corp RID: AB_2341179 Rabbit anti-cleaved Caspase-3 Cell Signaling Cat# 9661 RRID: AB_2341188 Bacterial and Virus Strains TB40/E-GFP O'Connor and N/A Murphy, 2012 TR-GFP Murphy et al., N/A 2003 TOWNE-GFP (clone RC2940) Cherrington and N/A Mocarski, 1988 TB40/E-Gluc Falk et al., 2016 N/A Chemicals, Peptides, and Recombinant Proteins ROCK inhibitor Y-27632 dihydrochloride Stemgent / Cat # 04-0012-10 Reprocell Bicuculline Tocris Cat # 0130 Retinoic acid Sigma Cat # R2625 CHIR99021 Cellagen Cat # C2447-2s Technology LDN-193189 Cellagen Cat # C5361-2s Technology SB431542 Cellagen Cat # C7243-5 Technology Matrigel Corning Cat # 354230 CNQX disodium salt Tocris Cat # 1045 N2 supplement Life Technologies Cat # 17502048 B27 supplement Thermo Fisher Cat # 17504044 Scientific Essential 8 Medium Life Technologies Cat # A1517001 Human Recombinant Insulin solution Sigma Cat # I9278 MEM NEAA Thermo Fisher Cat # 11140076 Scientific DMEM-F12 Gibco Cat # 11330-032 BrainPhys medium STEMCELL Tech Cat # 05790 GDNF PeproTech Cat # 450-10 BDNF PeproTech Cat # 450-02 Dibutyryl-cAMP Sigma Aldrich Cat # D0627 ATRA Sigma Aldrich Cat # R2625 EGF PeproTech Cat # 100-15 FGF PeproTech Cat # 100-18B Trizol Invitrogen Cat # 15596018 Gibco GlutaMAX Supplement Invitrogen Cat # 35050079 siLentFect Lipid Reagent for RNAi Bio-Rad Cat # 1703361 DyNAmo Flash SYBR Green qPCR mix Thermo Fisher Cat # F416 Scientific PMSF Roche Cat # 837091 Fluo-4, AM Thermo Fisher Cat # F14201 Scientific Critical Commercial Assays VECTASTAIN Elite ABC HRP Kit (Peroxidase, Vector Cat # PK-6102 Mouse IgG) Laboratories DAB Peroxidase (HRP) Substrate Kit, 3,3′- Vector Cat # SK-4100 diaminobenzidine Laboratories Tetro cDNA synthesis kit Bioline Cat # Bio-65043 Amaxa P3 primary 4D-Nucleofector X kit L Lonza Cat # V4XP-3024 Deposited Data RNAseq data of mock and hCMV infected This study GSE145415 brain organoids Experimental Models: Cell Lines Human: IMR-90 fibroblasts Coriell Institute Cat # I90-10 Human: AG14048 fibroblasts Coriell Institute Cat # AG14048 Human: ARPE-19 ATCC Cat # CRL-2302 Human: MRC-5 ATCC Cat # CCL-171 Experimental Models: Organisms/Strains Oligonucleotides EGFR-siRNA: 5′-GAC GGC GUC CGC AAG This study N/A UGU A -3′ (sense, SEQ ID NO: 1) and 5′-UAC ACU UGC GGA CGC CGU CUU-3′ (antisense, SEQ ID NO: 2) PDGFRα-specific siRNA: 5′-CCA CCU UCA This study N/A UCA AGA GAG A-3′(sense, SEQ ID NO: 3) and 5′-UCU CUC UUG AUG AAG GUG GAA-3′ (antisense, SEQ ID NO: 4) ITGA3-specific siRNA: 5′-GCU ACA UGA UUC This study N/A AGC GCA A-3′ (sense, SEQ ID NO: 5) and 5′- UUG CGC UGA AUC AUG UAG CUG-3′ (antisense, SEQ ID NO: 6) ITGA5-specific siRNA: 5′-GGG AAC CUC ACU Dharmacon Cat# L-004124-00- UAC GGC U-3′ (sense, SEQ ID NO: 7) and 5′- 0005 AGC CGU AAG UGA GGU UCC CUU-3′ (antisense, SEQ ID NO: 8) Integrin β3 siRNA: 5′-CCA GAU GCC UGC This study N/A ACC UUU A-3′ (SEQ ID NO: 9), 5′-GCC AAC CCA CUG UAU A-3′ (SEQ ID NO: 10), 5′-GAA GAA CGC GCC AGA GCA A-3′ (SEQ ID NO: 11), and 5′-GCA GUG AAU UGU ACC UAU A-3′ (SEQ ID NO: 12) Recombinant DNA pHIV-T2A-DsRed plasmid Dr. Xiuli Wang N/A from City of Hope pBABE-EGFR WT plasmid Greulich et al., Addgene Cat# 2005 11011; RRID: Addgene_110 11 pDONR223-PDGFRA plasmid Johannessen et Addgene Cat# al., 2010 23892; RRID: Addgene_238 92 pCXLE-hSK Addgene Cat# 27078 pCXLE-hUL Addgene Cat# 27080 pCXLE-hOCT3/4-shp53-F Addgene Cat# 27077 pCXWB-EBNA1 Addgene Cat# 37624 pcDNA-PDGFRα This study N/A pHIV-EGFR-T2A-DsRed This study N/A Software and Algorithms Prism version 7 Graphpad Prism https://www.graphpad. com/scientific- software/prism/ ZEN software Carl Zeiss https://www.zeiss.com/ microscopy/us/ products/microscope- software/zen.html Image-Pro Premier 9.1 Media Cybernetics http://www.mediacy. com/support/ imagepropremier AxIS software Axion Biosystems https://www.axionbio systems.com/products/ axis-software AxIS Metrics Tool Axion Biosystems https://www.axionbio systems.com/products/ axis-software NeuralMetric Tool Axion Biosystems https://www.axionbio systems.com/products/ axis-software ClustVis N/A https://biit.cs.ut.ee/ Other clustvis/ CytoView MEA 12 plate Axion BioSystems Cat # M768-GL1- 30Pt200-5 Maestro MEA system Axion BioSystems Equipment Hamamatsu EMCCD Hamamatsu Model C9100-13 Orbi-Shaker Benchmark NC0483060 Scientific
[0058] RNA interference. Oligonucleotides for siRNA-mediated RNA interference were synthesized by Integrated Device Technology. Oligonucleotides used for RNA interference were listed in Table 1. For RNA interference in brain organoids, four brain organoids with similar size were seeded in ultralow 24-well plates (one organoid per well) and transfected with siRNA using siLentFect. After 96 hours incubation, each organoid was infected with TB40/E at 5×10.sup.5 pfu/ml. Medium was changed the following day. Brain organoids were cultured for 20 days on shaker with continuous medium change every 3-4 days.
[0059] Western blot analysis. Cells were lysed in 0.1 M Na.sub.2CO.sub.3 containing 2 mM PMSF, and cell lysates were sonicated using Sonic Dismembrator to disintegrate genomic DNA. Protein concentrations were measured by Bradford Assay. Forty μg total proteins of NPCs and neurons were loaded for Western blot analysis. EGFR and PDGFRα antibodies were used at 1:1000 dilution.
[0060] Plasmids. EGFR WT was a gift from Matthew Meyerson (Addgene plasmid #11011; n2t.net/addgene:11011; RRID:Addgene_11011). The pHIV7-EGFR-T2A-DsRed vector was generated by cloning the human EGFR coding region via SwaI and NotI sites into the plasmid pHIV-T2A-DsRed (kindly provided by Dr. Xiuli Wang from City of Hope), resulting in the EGFR expression vector. pDONR223-PDGFRA was a gift from William Hahn & David Root (Addgene plasmid #23892; n2t.net/addgene:23892; RR ID: Addgene_23892). The pcDNA-PDGFRα vector was created by cloning the human PDGFRα cDNA into pcDNA3.1(+) neo vector using NheI and SmaI sites. The sequences of the cDNA were verified by Sanger sequencing.
[0061] hiPSC electroporation and HCMV infection. Control DsRed plasmid DNA or the mix of EGFR-T2A-DsRed and PDGFRα plasmid DNAs were introduced into hiPSCs by electroporation using 4D-Nucleofector following manufacturer's instructions. Briefly, 3 million hiPSCs in single cell suspension were mixed with P3 solution and subjected to electroporation using program CA137. The electroporated iPSCs were immediately transferred onto Matrigel-coated 24-well plate in E8 medium containing 1 μM Y27632. The hiPSCs were subcultured twice in order to remove dead cells and subsequently allowed to attach on a Matrigel-coated 24-well plate at 2×10.sup.4 cells/well. hiPSCs were infected with TB40/E at a MO I of 5 and harvested at 2 days after infection. The percent of CMV IE1-positive and DsRed-positive (CMV+DsRed+) cells out of total DsRed+ cells was quantified and plotted.
[0062] Statistical analysis. All statistical analyses were performed using GraphPad Prism7.0. Statistical details of experiments can be found in the figure legends. All data are shown as mean±SD or SE. The statistical significance of experiment outcome when comparing two or more groups was calculated using two-way ANOVA followed by Tukey's, Dunnet's or Sidak's multiple comparison tests or Student's t-test. The difference between experimental groups was considered significant when p<0.05.
[0063] Data and code availability. The RNA-seq dataset generated during this study is available at NCBI. The GEO Accession Super Series ID is GEO: GSE145415.
Example 1: Generation and Characterization of hiPSC-Derived Brain Organoids
[0064] To study the effects of HCMV infection on early human brain development, hiPSC-derived brain organoids were generated using a protocol similar to that described by Lancaster et al..sup.15,18 the brain organoids were characterized at day 45 to day 75 of differentiation (
Example 2: HCMV TB40/E Impairs the Growth and Cortical Structure of hiPSC-Derived Brain Organoids
[0065] Using recombinant viruses of HCMV strains TB40/E and Towne that express a GFP reporter.sup.25,26, the consequences of HCMV infection on the development of hiPSC-derived brain organoids were evaluated. In contrast to the laboratory HCMV strain Towne, the “clinical-like” HCMV strain TB40/E expresses the envelope pentamer complex (PC) and was able to efficiently infect both fibroblasts and epithelial cells, whereas Towne was only able to efficiently infect fibroblast, but not epithelial cells (
[0066] To determine the effect of HCMV infection on brain organoid growth, hiPSC-derived brain organoids at day 45 of differentiation were exposed to GFP-labeled TB40/E or Towne virus. The GFP fluorescence intensity and the brain organoid size were evaluated over a period of 20 days after viral exposure. Mock-infected brain organoids were used as a negative control. It was found that the “clinical-like” HCMV strain TB40/E could efficiently infect and propagate in the brain organoids and severely compromise the brain organoid growth, as revealed by an increase in GFP fluorescence intensity in the infected organoids and substantially reduced size of the infected brain organoids, when compared to the mock-infected brain organoid controls (
[0067] Further characterization of the mock-infected and TB40/E-infected brain organoids 15 days after infection revealed that TB40/E compromised the formation of the outer layer of the SOX2-positive progenitor core and the TUJ1-positive neuronal layer in human brain organoids (
Example 3: NAbs Effectively Prevent HCMV TB40/E-Induced Abnormal Brain Organoid Growth and Structure
[0068] Although NAbs are considered important for preventing congenital HCMV infection.sup.32-35, their capacity to interfere with HCMV infection of the developing human brain remains unknown. To gain insight into the potential of NAbs to interfere with HCMV infection of human brain cells, the prevention of TB40/E infection of hiPSC-derived brain organoids was evaluated by two previously isolated NAbs, 1B2 and 62-11, that were raised in mice against the envelope PC by vaccination.sup.36,37. NAb 1B2 is an antibody that targets the UL128/130/131A subunits of the PC. 1B2 is unable to prevent HCMV infection of fibroblasts, but confers exceptionally high potency to inhibit HCMV infection of epithelial cells.sup.36. In contrast, NAb 62-11 is a gH-specific antibody that can potently interfere with both fibroblast and epithelial cell infection.sup.36,37.
[0069] Using the GFP-labeled TB40/E virus, human brain organoids were infected at day 45 of differentiation in the presence of different concentrations of the 1B2 or 62-11 antibody (3.2-2000 ng/ml) and measured GFP fluorescence intensity and brain organoid size for up to 30 days post infection. Mock-infected and TB40/E-infected brain organoids treated with IgG control were used as control organoids. Compared to brain organoids infected with TB40/E in the presence of IgG control, organoids infected with TB40/E in the presence of either the 1B2 or 62-11 NAb exhibited substantially reduced GFP fluorescence intensity and markedly increased growth kinetics in a dose-dependent manner. More dramatic effects were observed with higher concentrations of the 1B2 or 62-11 NAb (
[0070] In addition to preventing brain organoid growth restriction by TB40/E, the NAb treatment also inhibited abnormal cortical structure formation induced by TB40/E. Cortical layers in brain organoids were defined by layer-specific marker staining (
Example 4: PDGFRα and EGFR are Involved in HCMV TB40/E Infection of Human Brain Organoids
[0071] To support the relevance of the brain organoid model to study HCMV infection of the developing human brain, potential cellular receptors that are involved in brain organoid infection by TB40/E were investigated. Several receptors, such as platelet-derived growth factor receptor alpha (PDGFRα), epidermal growth factor receptor (EGFR), and integrins (α3, α5, and β3), have been previously described to be either directly or indirectly involved in HCMV host cell entry.sup.39-41.
[0072] To determine the importance of these cellular receptors for brain organoid infection by TB40/E, the expression of individual receptors was knocked down using receptor-specific siRNAs. The knockdown efficiency of each receptor was verified in hiPSC-derived NPCs by qRT-PCR (
Example 5: HCMV TB40/E Disrupts Calcium Signaling and Neural Network Activity in Brain Organoids
[0073] To uncover mechanisms of HCMV-induced neurodevelopmental defects, RNA sequencing analysis was performed in control and HCMV TB40/E-infected brain organoids. Interestingly, it was found that at least three out of the top ten down-regulated genes in HCMV TB40/E-infected organoids were related to calcium signaling (
[0074] The downregulation of genes involved in calcium signaling in TB40/E-treated brain organoids led to the test whether calcium response is impaired in HCMV-infected organoids. Using calcium imaging, Ca.sup.2+ oscillations in control brain organoids and TB40/E-treated brain organoids were measured, as well as brain organoids infected with TB40/E in the presence of 1B2 NAb. Because the calcium signal was monitored using a green fluorescent calcium dye, TB40/E virus without a GFP reporter was used to infect human brain organoids, and the HCMV-infected cells were detected by immunostaining for the HCMV 1E1 protein. While control brain organoids exhibited frequent bursts of calcium surges, no calcium activity was detected in HCMV-positive regions (identified by 1E1+ staining) in HCMV-infected brain organoids (
[0075] In addition to measuring neural activity using calcium imaging, neural network activity in TB40/E-infected brain organoids was evaluated by MEA (
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