Name: Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells (IPSCs)
Uploaded: 2023-03-24T12:10:00
Description: Watch this Scientific Journal Video about Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells IPSCs at JoVE.com

The video outlines a detailed protocol for culturing human induced pluripotent stem cells (iPSCs) into cerebral organoids. It includes steps for cell detachment, creating embryoid bodies, embedding them in a basement membrane matrix, and culturing organoids in a specific environment for optimal development and maintenance.

generation of cerebral organoids from primate-induced pluripotent stem cells (ipscs)

प्राइमेट सेरेब्रल ऑर्गेनोइड का आनुवंशिक संशोधन

Investigating the (patho)physiological development and evolution of the cerebral cortex is a formidable task that is hampered by the lack of suitable model systems. Previously, such studies were confined to two-dimensional cell culture models (such as primary neural progenitor or neuronal cell cultures) and evolutionarily distant animal models (such as rodents)1,2. While these models are useful for addressing certain questions, they are limited in modeling the complexity, cell type composition, cellular architecture, and gene expression patterns of the developing human neocortex in healthy and diseased states. These limitations lead, for example, to the poor translatability of mouse models of human diseases to the human situation, as described for certain cases of microcephaly (e.g., Zhang et al.3). Recently, transgenic non-human primates, which are an evolutionarily, functionally, and morphologically closer model of human neocortex development, have come into focus4,5,6,7,8 as they overcome many limitations of cell culture- and rodent-based models. However, the use of non-human primates in research is not only highly expensive and time-consuming but also raises ethical concerns. More recently, the development of brain organoid technology9,10 has emerged as a promising alternative that solves many of the limitations of previous models11,12,13,14,15,16.
Brain organoids are three-dimensional (3D), multicellular structures that emulate the main features of the cytoarchitecture and cell-type composition of one or multiple brain regions for a defined developmental time window11,12,13,14,17. These 3D structures are generated either from induced pluripotent stem cells (iPSCs) or, if available for the species of interest, from embryonic stem cells (ESCs). In general, two types of brain organoids can be distinguished based on the methodology used: unguided and regionalized (guided) brain organoids18. In generating the latter type of organoids, small molecules or factors are provided that guide the differentiation of the pluripotent stem cells to organoids of a particular brain region (e.g., forebrain organoids)18. By contrast, in unguided organoids, the differentiation is not guided by the addition of small molecules but rather relies exclusively on the spontaneous differentiation of the iPSCs/ESCs. The resulting brain organoids consist of cell types representing different brain regions (e.g., cerebral organoids)18. Brain organoids combine many key features of brain development with relatively cost- and time-efficient generation from any species of interest for which iPSCs or ESCs are available11,12,13,14. This makes brain organoids an excellent model for many kinds of neurobiological studies, ranging from evolutionary and developmental questions to disease modeling and drug testing15,16. However, addressing such questions using brain organoids strongly depends on the availability of different methods for genetic modification.
One key aspect of studying neocortex (patho)physiological development and its evolution is the functional analysis of genes and gene variants. This is usually achieved by (ectopic) expression and/or by knock-down (KD) or knock-out (KO) of those genes. Such genetic modifications can be classified into stable and transient genetic modification, as well as into the modifications being temporally and spatially restricted or not restricted. Stable genetic modification is defined by the introduction of a genetic alteration into the host genome that is passed on to all subsequent cell generations. Depending on the time point of genetic modification, it can affect all the cells of an organoid or can be restricted to certain cell populations. Most frequently, stable genetic modification is achieved in brain organoids at the iPSC/ESC level by applying lentiviruses, transposon-like systems, and the CRISPR/Cas9 technology (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20). This has the advantage that all cells of the brain organoid carry the genetic modification and that it is not temporally or spatially restricted. However, the generation and characterization of these stable iPSC/ESC lines are very time-consuming, often taking several months until the first modified brain organoids can be analyzed (reviewed by e.g., Fischer et al.17, Kyrousi et al.19, or Teriyapirom et al.20).
In contrast, transient genetic modification is defined by the delivery of genetic cargo (e.g., a gene expression plasmid) that does not integrate into the host genome. While this modification can, in principle, be passed on to subsequent cell generations, the delivered genetic cargo will be progressively diluted with each cell division. Therefore, this type of genetic modification is usually temporally and spatially restricted. Transient genetic modification can be carried out in brain organoids by adeno-associated viruses or by electroporation (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20), with the latter being described in detail in this article. In contrast to stable genetic modification, this approach is very fast and cost-efficient. Indeed, electroporation can be performed within minutes, and, depending on the target cell population(s), electroporated organoids are ready for analysis within days (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19). However, gross morphological changes of the brain organoid, such as differences in size, cannot be detected using this method, as this type of genetic modification is temporally and spatially restricted. This restriction can also be an advantage, for example, in the case of studying individual cell populations within the organoid or the effects on brain organoids at specific developmental time points (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19).
A classical approach to study gene function during brain development and evolution is in utero electroporation. In utero electroporation is a well-known and useful technique for the delivery of gene expression constructs into rodent21,22,23 and ferret24,25 brains. First, a solution containing the expression construct(s) of interest is microinjected through the uterine wall into a certain ventricle of the embryonic brain, depending on the region to be targeted. In the second step, electric pulses are applied to transfect the cells directly lining the targeted ventricle. This approach is not only limited to ectopic expression or the overexpression of genes, as it can also be applied in KD or KO studies by microinjecting short hairpin (shRNA) or CRISPR/Cas9 (in the form of expression plasmids or ribonucleoproteins [RNPs]), respectively26,27. However, the in utero electroporation of mouse, rat, and ferret embryos has the same limitations as described above for these animal models.
Ideally, one would like to perform in utero electroporation directly in primates. While this is, in principle, technically possible, in utero electroporation is not conducted in primates due to ethical concerns, high animal maintenance costs, and small litter sizes. For certain primates, such as great apes (including humans), this is not possible at all. However, these primates have the greatest potential for the study of human (patho)physiological neocortex development and its evolution. One solution to this dilemma is to apply the electroporation technique to primate brain organoids28.
This paper presents a protocol for the electroporation of a subtype of primate brain organoids, primate cerebral organoids. This approach allows the fast and cost-efficient genetic modification of cell populations within the ventricle-like structures of the organoids. Specifically, we describe a unified protocol for the generation of primate cerebral organoids from human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and common marmoset (Callithrix jacchus) iPSCs. Moreover, we describe the microinjection and electroporation technique in detail and provide &#34;go&#34; and &#34;no-go&#34; criteria for performing primate cerebral organoid electroporation. This approach is an effective tool for studying (patho)physiological neocortex development and its evolution in a model especially close to the human situation.

लेखक स्पॉटलाइट: जेनेटिक संशोधन के लिए प्राइमेट सेरेब्रल ऑर्गेनोइड के लक्षित माइक्रोइंजेक्शन और इलेक्ट्रोपोरेशन  

आनुवंशिक संशोधन के लिए प्राइमेट सेरेब्रल ऑर्गेनोइड्स का लक्षित माइक्रोइंजेक्शन और इलेक्ट्रोपोरेशन

Our field of research is primate brain development and evolution. We are trying to understand the factors underlying neocortical expansion in primates. To address this question in a practical and ethically justifiable way, we use brain organoids as our research model.Differences in brain development between modern humans, us, and neanderthals have recently emerged. These differences are due to a small number of amino acid changes in proteins with key roles in brain development. Cerebral organoids have been a crucial model system in these studies.The study of primate development from an evolutionary point of view conducting gain and loss of function studies is important. However, apes including humans naturally cannot be used for such experiments, so genetic modification of organoids plays a vital role in this field. While working as a postdoc in my lab, Michael Heide demonstrated that the human specific gene ARHGAP11B was a key player in increasing brain size during human evolution.As a group leader at the German Primate Center now, Michael contributed crucial insight by comparing human versus chimpanzee cerebral organoids. Our protocol offers a targeted approach for genetic modifications by specific micro injecting ventricle-like structures instead of commercial electroporation cuvettes. This approach uses a cost efficient setup including square wave electroporator and Petri dish electrode chamber instead of expensive nuclear effector solutions.While studying function and brain development in primates by genetic modification is technically possible, it is methodologically demanding, expensive, and not allowed for great apes. The electroporation of primate cerebral organoids provides a fast and cost efficient approach to introduce genetic modifications in a model close to primate brain development. In the future, we would like to focus on the characterization of differentially expressed genes in the cortex of different primate species.We will study these genes by the electroporation of primate cerebral organoids and analyze their potential effects on cortical progenitor cells.

Watch this Scientific Journal Video about Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells IPSCs at JoVE.com

Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells (IPSCs)  

प्राइमेट-प्रेरित प्लुरिपोटेंट स्टेम सेल (आईपीएससी) से सेरेब्रल ऑर्गेनोइड्स का उत्पादन

शुरू करने के लिए, सुसंस्कृत आईपीएससी को डलबेकको के पीबीएस या डीपीबीएस के साथ धो लें। और एक मिलीलीटर प्रोटियोलिटिक और कोलेजनोलिटिक मिश्रण जोड़ें। कोशिकाओं को अलग करने के लिए पकवान को दो मिनट के लिए 37 डिग्री सेल्सियस पर इनक्यूबेट करें।इसके बाद, प्रतिक्रिया को रोकने के लिए 1.5 मिलीलीटर प्रीवार्म्ड आईपीएससी कल्चर माध्यम जोड़ें। एकल-सेल निलंबन प्राप्त करने के लिए सात से 10 बार ऊपर और नीचे पाइप करके सेल कल्चर डिश से कोशिकाओं को अलग करें। सेल निलंबन को 15-मिलीलीटर शंक्वाकार सेंट्रीफ्यूज ट्यूब में स्थानांतरित करें।कमरे के तापमान पर पांच मिनट के लिए कोशिकाओं को 200xg पर गोली दें। और सतह पर तैरने वाला जानवर है। 50 माइक्रोमोलर वाई 27632 के साथ पूरक आईपीएससी कल्चर माध्यम के दो मिलीलीटर में गोली को फिर से निलंबित करें।अब, न्यूबॉयर कक्ष का उपयोग करके कोशिकाओं की गणना करने के लिए सेल निलंबन के 10 माइक्रोलीटर का उपयोग करें। आईपीएससी कल्चर माध्यम का उपयोग करके सेल निलंबन की एकाग्रता को 9,000 कोशिकाओं प्रति 150 माइक्रोलीटर में समायोजित करें, जो 50 माइक्रोमोलर वाई 27632 के साथ पूरक है। भ्रूण के शरीर या ईबी उत्पन्न करने के लिए, सेल अवसादन को रोकने के लिए सेल निलंबन ट्यूब को हिलाएं और फिर सेल निलंबन के 150 माइक्रोलीटर को अल्ट्रा-लो अटैचमेंट 96-वेल प्लेट के प्रत्येक कुएं में डालें।ईबी को 48 घंटे के लिए एक ह्यूमाइज्ड वातावरण में कल्चर करें। इनक्यूबेशन के अंत में, प्रति अच्छी तरह से 100 माइक्रोलीटर मध्यम निकालें। और वाई 27632 के बिना 150 माइक्रोलीटर प्रीवार्म्ड ताजा माध्यम जोड़ें।पैराफिल्म पर एक चार बाई चार डिंपल ग्रिड बनाएं, पैराफिल्म ग्रिड को 0.2 मिलीलीटर ट्यूब रैक पर पेपर-लिफाफा साइड के साथ रखें। और तहखाने झिल्ली मैट्रिक्स में ईबी को एम्बेड करने के लिए प्रत्येक रैक छेद के खिलाफ धीरे से एक उंगली दबाएं। फिर कागज को हटा दें और 60-मिलीलीटर सेल कल्चर डिश में फिट होने के लिए इसके आकार को समायोजित करने के लिए कैंची का उपयोग करके पैराफिल्म स्क्वायर से डिंपल ग्रिड को काट लें।बेसमेंट झिल्ली मैट्रिक्स ड्रॉपलेट पीढ़ी के लिए आधार प्रदान करने के लिए डिंपल पैराफिल्म को 0.2 मिलीलीटर ट्यूब रैक पर वापस रखें। 200 माइक्रोलीटर पिपेट टिप के साथ एक पिपेट का उपयोग करके संस्कृति डिश के कुएं से पैराफिल्म डिंपल में एक के बाद एक ईबी को सावधानीपूर्वक स्थानांतरित करें। 16 ईबी को ग्रिड में ले जाने के बाद, एक नया 200 माइक्रोलीटर पिपेट टिप लें और शेष माध्यम को डिंपल से हटा दें।अब, एक ईबी वाले प्रत्येक डिंपल पर तहखाने झिल्ली मैट्रिक्स की एक बूंद डालें। 10-माइक्रोलीटर पिपेट टिप लें और जल्दी से बूंद सीमाओं को परेशान किए बिना ईबी को प्रत्येक बूंद के केंद्र में ले जाएं। 60 मिलीमीटर सेल कल्चर डिश में बेसमेंट मेम्ब्रेन मैट्रिक्स ड्रॉप्स के साथ डिंपल ्ड पैराफिल्म रखें। और मैट्रिक्स को पॉलीमराइज करने की अनुमति देने के लिए 37 डिग्री सेल्सियस पर 15 से 30 मिनट के लिए इनक्यूबेट करें।इसके अलावा, पैराफिल्म से मैट्रिक्स-एम्बेडेड ईबी को अलग करने के लिए, डिश में विटामिन ए के बिना पांच मिलीलीटर भेदभाव माध्यम जोड़ें। और पैराफिल्म स्क्वायर को फोर्सप्स का उपयोग करके उल्टा कर दें ताकि ईबी के साथ साइड डिश के निचले हिस्से का सामना कर सके। पैराफिल्म से ईबी युक्त तहखाने झिल्ली मैट्रिक्स बूंदों को अलग करने के लिए पकवान को ध्यान से हिलाएं।यदि उनमें से कुछ अभी भी जुड़े हुए हैं, तो फोर्सप्स का उपयोग करके पैराफिल्म स्क्वायर का एक किनारा लें और इसे कई बार पकवान के केंद्र की ओर तेजी से रोल करें। फिर 5% कार्बन डाइऑक्साइड और 37 डिग्री सेल्सियस पर 95% हवा के साथ एक ह्यूमिडिफ़ायर वातावरण में 55 आरपीएम पर ऑर्बिटल शेकर पर सेरेब्रल ऑर्गेनोइड्स को कल्चर करें। हर दूसरे दिन मध्यम परिवर्तन के साथ विटामिन ए के बिना डीएम में ऑर्गेनोइड ्स रखें।परिधि पर वेंट्रिकल जैसी संरचनाओं और एक कॉम्पैक्ट, स्वस्थ आकृति विज्ञान के साथ 32-दिवसीय पोस्ट-सीडिंग मानव सेरेब्रल ऑर्गनॉइड की एक उज्ज्वल क्षेत्र छवि दिखाई गई है।

Research

JoVE Journal

Developmental Biology

Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells (iPSCs)

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...