עבודה זו נתמכה על ידי פריץ תיסן קרן (Az.10.14.2.152). אנו מודים למתקן הטבעת רקמות ואת מתקן גרעין מיקרוסקופ של CMMC. אנו מודים על הדיונים ותמיכה טכנית מסופק על ידי חברי המעבדה לביולוגיה centrosome ו נוגדנים. אנו מודים לי מינג Gooi להגהה את כתב היד.

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The authors declare that they have no competing financial interests.

MCPH היא הפרעה נוירו-התפתחותיות מורכבות אנושית שלא ניתן לסכם במודלים של בעלי חיים in vivo או תרבית תאים אנושיים פשוטים גישות במבחנה. הביטוי הקליני של MCPH מתחיל להופיע במהלך הטרימסטר הראשון, כאשר בתחילת נוירוגנסיס מתחיל. לפיכך, organoids מוח 3D מייצג מערכת ניסיונית אמי?...

פרעות התפתחותיות אדם, כגון ממיקרוצפלוס-, ניתן ללמוד בצורה גרועה רק במודלים של בעלי חיים בשל העובדה כי מוח האנושי יש משטח קליפת מוח מורחב, תכונה ייחודית ושונת חיות לא אנושיות. היבט זה עושה התפתחות המוח האנושי תהליך מורכב כי לא ניתן ללמוד מספיק בתוך 2D, במערכת תרבית תאים במבחנה. מתעוררים טכניקות התרבות 3D לאפשר לדור של organoids דמויי רקמה מתאי גזע מושרים (iPSCs). במבחנת הבידול של תאי גזע פלוריפוטנטיים בתרבות השעית 3D מאפשר ההיווצרות של סוגי תאים שונים במועד ובאזור ספציפי, והוליד רקמות מאורגנות, מרובדת 1, 2, 3. הודות מעבד כי חלוץ בטכנולוגיות תרבות 3D ו demystified המורכבת של היווצרות איברים, החלה מתאי גזע,פתחנו שיטה חזקה של יצירת organoids המוח להתוות אירועים מוקדמים של התפתחות מוח אנושית מודל ממיקרוצפלוס- במבחנה 1, 2, 3. ראוי לציין כי התאמנו בשיטה המקורית שפיתח לנקסטר ואח. 1 כדי ליצור organoids מוחין. שיטה זו שונתה על פי דרישות הניסוי שלנו. מטרת מחקר מ ואח &#39;גבריאל. היה לנתח את המנגנונים התאיים ומולקולריים של תחזוקת תאי גזע עצבית במהלך התפתחות המוח. על מנת לעשות זאת, מחקר המכניסטי בוצע באמצעות ניתוח ובתאים עצביים (NPCs) ב organoids מוח 3D נגזר חולה ממיקרוצפלוס- 4. החולה הזה נשא מוטציה CPAP, חלבון centrosomal נשמר הנדרשים biogenesis centrosome 5. היפו מקובלהתזה היא כי ממיקרוצפלוס- הוא התוצאה של דלדול של ברכת NPC, זה עלול לנבוע גם למוות של תאים או בידול מוקדם 1, 6, 7, 8, 9. על ידי ניתוח אזורי חדרית (VZs) של organoids המוח ממיקרוצפלוס-, הודגם כי מספר משמעותי של NPCs לעבור חלוקת התא סימטרית, בניגוד organoids המוח נגזר מתורם בריא 4. ניתוחים מיקרוסקופים וביוכימיים נרחבים של organoids מוח microcephalic חשפו תפקיד לא צפוי עבור CPAP מבולגן ריסים בזמן 4. באופן ספציפי, CPAP מוטציה מזוהה עם פירוק cilium מפגר מחדש כניסת מחזור התא מתעכבת, מה שמוביל את הבידול המוקדם של NPCs 4. תוצאות אלו מצביעות על תפקיד עבור ריסים ב ממיקרוצפלוס- ו המעורבות EIR במהלך מלא הנוירוגנזה גודל המוח 10. חלקו הראשון של פרוטוקול זה הוא תיאור של שיטת שלושת השלבים ליצירת organoids המוח הומוגנית. כפי שהוזכר קודם לכן, הפרוטוקול לנקסטר המקורי הותאם ו שונה שמתאים למטרות שלנו 1. ראשית, iPSCs אדם מתורבת מצב מזין ללא מוגדר על Engelbreth-הולם-הנחיל (EHS) מטריקס. צעד זה ימנע את הווריאציות של תרביות תאי גזע פלוריפוטנטיים מזין תלוי. בפרוטוקול זה, בדומה להשפעת בידול העצבי להקים אפיתל עצבית מתחילה ישירות iPSCs. אם תדלג גוף embryoid (EB) צעד ההיווצרות, את ההתמיינות העצבית בצורה מבוקרת יותר וביים. גישה זו מגבילה את ההיווצרות הספונטנית undirected של שכבות תאי ניבט אחרות, כגון המזודרם ו endoderm. על ידי יישום פרוטוקול זה, neurospheres המכיל רוזטות עצבית ניתן לקצור ביום 5 עבור EHS הטבעת מטריקס ותרבות השעיה נייחת. מדיום organoid המשמש את הצעד השלישי של הפרוטוקול שלנו הוא השלים עם dorsomorphin ו SB431542. Dorsomorphin הוא מעכב מולקולות קטנות של חלבון העצם morphogenic (BMP), וכן SB431542 מעכב את TGFβ / Activin / מסלול איתות קטרים. השילוב של גורמים אלה יכול לקדם בידול עצבי בצורה יעילה יותר מאשר חומצה רטינואית לבד 11, 12, 13, 14. בסך הכל, שינויים אלה מאפשרים לדור לשחזור של organoids המוח, עם וריאציות מינימאליות ברחבי organoids. חשוב לציין, שיטה זו יושמה כדי ליצור organoids המוח microcephalic וחסונה מן iPSCs החולה, הנושאות מוטציות בגנים המשפיעים centrosomes והדינמיקה מחזור התא. החלק השני של פרוטוקול זה נותן הוראות להכנת brAin organoids לניתוח ופרשנות של ליקויים הסלולר ממיקרוצפלוס-. זה כולל קיבוע, cryosectioning, מכתים immunofluorescent, ואת ניתוח מיקרוסקופי confocal. פרוטוקול זה יספק את הקורא עם תיאור מפורט של תוצאות צפויות ובליווי לפרשנות.

<table><tbody><tr><td>Anti-mouse 488</td><td>Invitrogen</td><td>A-11001</td><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td></tr><tr><td>Anti-rabbit 647</td><td>Invitrogen</td><td>A-21245</td><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647</td></tr><tr><td>Arl13b</td><td>proteintech</td><td>17711-1-AP</td><td>ARL13B rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>CELLSPIN system</td><td>IBS Integra Bioscience</td><td>183001</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, US</td><td>32670</td><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers</td></tr><tr><td>DMEM/F-12</td><td>Gibco, US</td><td>31331093</td><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12&amp;nbsp;</td></tr><tr><td>Dorsomorphin</td><td>Sigma-Aldrich, US</td><td>P5499</td><td>Compound C; multiple suppliers</td></tr><tr><td>Embedding medium</td><td>AppliChem</td><td>A9011, 0100</td><td>Mowiol; embedding medium; multiple suppliers</td></tr><tr><td>Engelbreth-Holm-Swarm (EHS) matrix</td><td>Corning</td><td>354277</td><td>Matrigel hESC-qualified matrix; important: hESC qualified</td></tr><tr><td>Fish gelatin&amp;nbsp;</td><td>Sigma-Aldrich, US</td><td>G7765-250ML</td><td>Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4 &amp;deg;C</td></tr><tr><td>Glycine</td><td>AppliChem</td><td>A1067,1000</td><td>Glycine for molecular biology; multiple suppliers&amp;nbsp;</td></tr><tr><td>Inoculation loop with needle, disposable (1 &amp;micro;L)</td><td>Sigma Aldrich, US</td><td>BR452201-1000EA</td><td>multiple suppliers&amp;nbsp;</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich, US</td><td>I3536-100MG</td><td>multiple suppliers</td></tr><tr><td>L-glutamine</td><td>Gibco, US</td><td>25030081</td><td>L-glutamine (200 mM)</td></tr><tr><td>Medium A</td><td>Stem cell technologies</td><td>#05850</td><td>mTeSR1 (hiPSC medium)</td></tr><tr><td>Medium B</td><td>Stem cell technologies</td><td>#05835</td><td>Neural induction medium (NIM); neural differentiation medium</td></tr><tr><td>Medium C</td><td>Gibco, US</td><td>21103049</td><td>Neural Basal Medium</td></tr><tr><td>MEM</td><td>Gibco, US</td><td>11140035</td><td>MEM non-essential amino acids solution (100x)</td></tr><tr><td>MycoAlert Mycoplasma Detection Kit</td><td>Lonza, Switzerland</td><td>#LT07-218</td><td>Mycoplasma detection kit; multiple suppliers</td></tr><tr><td>Nestin</td><td>Novus biologicals</td><td>NBP1-92717</td><td>Nestin mouse monoclonal antibody (4D11)</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>AppliChem</td><td>A3813, 0500</td><td>4% in PBS, store solution at -20 &amp;deg;C; caution: wear skin and eye protection and work under hood&amp;nbsp;</td></tr><tr><td>PBS tablets</td><td>Gibco, US</td><td>18912014</td><td>See manufacturer&amp;acute;s instructions; multiple suppliers</td></tr><tr><td>Penicillin-Streptomycin (10.000 U/mL)</td><td>Gibco, US</td><td>15140122</td><td>Multiple suppliers</td></tr><tr><td>Poly-L-lysine solution (PLL)</td><td>Sigma-Aldrich, US</td><td>P8920-100ML</td><td>Multiple suppliers</td></tr><tr><td>pVim</td><td>MBL</td><td>D076-3S</td><td>Phospho-Vimentin (Ser55) mAb</td></tr><tr><td>Reagent A&amp;nbsp;</td><td>Stem cell technologies</td><td># 05872</td><td>Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&amp;acute;s protocol; alternative products from muliple suppliers available</td></tr><tr><td>Reagent B&amp;nbsp;</td><td>Sigma-Aldrich, US</td><td>A6964-100ML</td><td>Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &quot;enzymatic cell dissociation solution&amp;rdquo;&amp;nbsp;</td></tr><tr><td>Research Cryostat Leica CM3050 S</td><td>Leica biosystems</td><td>CM3050 S</td><td>Multiple suppliers</td></tr><tr><td>SB431542</td><td>Selleckchem.com</td><td>S1067</td><td>Multiple suppliers</td></tr><tr><td>Spinner flask 250 mL</td><td>IBS Integra Bioscience</td><td>182026</td><td></td></tr><tr><td>Sucrose</td><td>AppliChem</td><td>A4734, 1000</td><td>Multiple suppliers</td></tr><tr><td>Superfrost ultra plus microscope slides</td><td>Thermo scientific, US</td><td>J3800AMNZ</td><td>Slides should be labeled with a &quot;+&quot; and positively charged</td></tr><tr><td>Supplement 1</td><td>Gibco, US</td><td>17502048</td><td>N-2 supplement (100x)</td></tr><tr><td>Supplement 2 w/o Vitamin A</td><td>Gibco, US</td><td>12587010</td><td>B-27 supplement (50x), minus vitamin A; multiple suppliers</td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, NL</td><td>4565</td><td>Multiple suppliers</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura, NL</td><td>4583</td><td>Multiple suppliers</td></tr><tr><td>Triton X-100</td><td>AppliChem</td><td>A1388,0500</td><td>Multiple suppliers</td></tr><tr><td>TUJ1</td><td>Sigma-Aldrich, US</td><td>T2200</td><td>&amp;beta;-Tubulin III (rabbit polyclonal)</td></tr><tr><td>TUNEL assay</td><td>Promega, US</td><td>G3250</td><td>DeadEnd Fluorometric TUNEL system; multiple suppliers</td></tr><tr><td>Tween 20 for molecular biology</td><td>AppliChem</td><td>A4974,0500</td><td>Multiple suppliers</td></tr><tr><td>waterproof sheet</td><td>BEMIS company, inc.</td><td>PM996</td><td>Parafilm &amp;ldquo;M&amp;rdquo;; multiple suppliers</td></tr><tr><td>Y-27632&amp;nbsp;</td><td>Selleckchem.com</td><td>S1049</td><td>ROCK-inhibitor (Y-27632 2HCL); multiple suppliers</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco, US</td><td>31350010</td><td>2-mercaptoethanol (50 mM); multiple suppliers</td></tr></tbody></table>

generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

הדור של organoids המוח דורש לפחות שלושה שבועות של culturing רציפה (איור 1A). כדי להשיג תוצאות לשחזור, אנו ממליצים כי החוקר מתעד כל צעד, חשוב, ימנע שינויים כלשהם לגבי רכיבים בינוניים, בנקודות זמן, וטיפול תא. הנה, אנחנו נותנים תמצית כיצד להעריך אם אבני דרך...

Watch this Scientific Journal Video about Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders at JoVE.com

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

הדור של Organoids Human Brain iPSC הנגזרות למודל הפרעה נוירו-התפתחותית מוקדמת

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

generation-ipsc-derived-human-brain-organoids-to-model-early

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

19.9K Views.  University of Cologne. Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described. 

Video: Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

זיהוי מהיר של פנוטיפים התפתחותיות בתאי האדם קודמן עצבית (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

דור של Organoids מוחי סטנדרטית ו לשחזור הקדמי-סוג של האדם המושרה בתאי גזע Pluripotent

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

שיטת בימוי עצמי סטטי ליצירת אורגנואידים המוח מתאי גזע האדם העובריים

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

מודלים מבוססי תאי גזע פלוריפוטנטיים דו-ממדיים ותלת-ממדיים אנושיים לניתוח מעורבות סיליום ראשוני במהלך התפתחות ניאוקורטיקלית

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

דור אורגנויד במוח מתאי גזע פלוריפוטנטים מושרים במיני ביו-רהקטורים תוצרת בית

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

<meta charset="utf8"></head><body>יצירת אורגנואידים במוח האנושי עם מיקרוגליה מקומית באמצעות HPCs שמקורם ב-iPSC

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

<meta charset="utf8"></head><body>גזירה של אורגנואיד מוח אנושי עם התפתחות מיקרוגליה

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

גזירה של אורגנואיד מוח אנושי עם התפתחות מיקרוגליה

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

יצירת אורגנואידים תלת מימדיים של המוח התיכון מתאי גזע פלוריפוטנטיים המושרים על ידי בני אדם

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

דור אורגנוידים במוח האדם למידול מחלות מיטוכונדריאליות

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

הדור של Organoids Human Brain iPSC הנגזרות למודל הפרעה נוירו-התפתחותית מוקדמת

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זיהוי מהיר של פנוטיפים התפתחותיות בתאי האדם קודמן עצבית (NPCs)

דור של Organoids מוחי סטנדרטית ו לשחזור הקדמי-סוג של האדם המושרה בתאי גזע Pluripotent

שיטת בימוי עצמי סטטי ליצירת אורגנואידים המוח מתאי גזע האדם העובריים

מודלים מבוססי תאי גזע פלוריפוטנטיים דו-ממדיים ותלת-ממדיים אנושיים לניתוח מעורבות סיליום ראשוני במהלך התפתחות ניאוקורטיקלית

דור אורגנויד במוח מתאי גזע פלוריפוטנטים מושרים במיני ביו-רהקטורים תוצרת בית

גזירה של אורגנואיד מוח אנושי עם התפתחות מיקרוגליה

גזירה של אורגנואיד מוח אנושי עם התפתחות מיקרוגליה

גזירה של אורגנואיד מוח אנושי עם התפתחות מיקרוגליה

יצירת אורגנואידים תלת מימדיים של המוח התיכון מתאי גזע פלוריפוטנטיים המושרים על ידי בני אדם

דור אורגנוידים במוח האדם למידול מחלות מיטוכונדריאליות