Name: Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier
Uploaded: 2023-10-20T12:30:00
Description: Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

constructing the 3d human-stem-cell-derived in vitro blood brain barrier

एक 3 डी रक्त-मस्तिष्क बाधा मॉडल मानव प्लुरिपोटेंट स्टेम सेल से प्राप्त

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

लेखक स्पॉटलाइट: इन विट्रो ब्लड-ब्रेन बैरियर मॉडल का उपयोग करके अल्जाइमर रोग की जांच के लिए एक व्यक्तिगत दृष्टिकोण 

मॉडल और चिकित्सीय रूप से न्यूरोलॉजिकल रोग को लक्षित करने के लिए इन विट्रो में रक्त-मस्तिष्क बाधा का पुनर्निर्माण

Our goal is to determine the molecular and cellular mechanisms that mediate Alzheimer's disease pathogenesis to uncover therapeutic and diagnostic opportunities. Currently, the molecular pathways and mechanisms involved in the onset and development of Alzheimer's disease are poorly understood. This is due to mouse models and other in vitro Alzheimer's disease models failing to recapitulate unique human biology that is crucial to the development of the disease.We have developed an in vitro model of the blood-brain barrier that can be applied to study cerebrovascular pathologies associated with Alzheimer's disease, dementia, and other forms of neurodegeneration. This model has been employed to show that APOE4, the strongest risk factor for Alzheimer's disease, acts in part through a parasite-specific pathway to increase pathogenic amyloid burden Using induced pluripotent stem cells, we can construct human brain tissue in vitro that can be used to investigate disease pathology and drug screenings. Such an in vitro blood-brain barrier model enables personalized therapeutic and diagnostic approaches.Our 3D in vitro blood-brain barrier model demonstrates physiologically relevant interactions, including vascular tube formation and localization of astrocyte endfeet with vasculature. It is also capable of modeling disease-relevant phenotypes, including amyloid pathology. Finally, the use of patient-derived induced pluripotent stem cells allows the study of any desired genetic background.Using the in vitro blood-brain barrier model, we discovered pathways that mediate pathological cerebrovascular amyloid accumulation. This led us to explore therapeutic strategies that can reverse this pathology in aged APOE4 mice, which we are currently following up on.

Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier  

3 डी मानव-स्टेम-सेल-व्युत्पन्न इन विट्रो रक्त मस्तिष्क बाधा का निर्माण

मानव pluripotent स्टेम सेल व्युत्पन्न रक्त-मस्तिष्क बाधा, या iBBB इकट्ठा, dissociated endothelial कोशिकाओं, परजीवी, और astrocytes का उपयोग कर, एक शंक्वाकार ट्यूब में कोशिकाओं के तीन प्रकार को गठबंधन जबकि सहित 10% कोशिकाओं की गणना की संख्या से अधिक pippting त्रुटियों के लिए खाते के लिए. सेल मिश्रण को तीन मिनट के लिए 300 ग्राम पर स्पिन करें। सतह पर तैरनेवाला महाप्राण, माध्यम के लगभग 50 microliters के साथ सेल गोली छोड़ने.एक P200 विंदुक का प्रयोग, धीरे एक एकल सेल घोल बनाने के लिए अवशिष्ट माध्यम में सेल गोली resuspended है. बर्फ पर परिणामी सेल घोल युक्त ट्यूब रखें और iBBBs की वांछित संख्या के अनुसार इसे कम GF बेसमेंट मेम्ब्रेन मैट्रिक्स की पर्याप्त मात्रा में जोड़ें। किसी भी हवाई बुलबुले का परिचय नहीं करते हुए कोशिकाओं को सजातीय रूप से मिलाएं।परिणामी सेल मैट्रिक्स मिश्रण के पिपेट 50 माइक्रोलीटर को 48-अच्छी तरह से या 96-अच्छी तरह से ग्लास-बॉटम कल्चर डिश के कुएं में मिलाएं और इसे डिश के नीचे समान रूप से वितरित करें। कोशिकाओं को घेरते हुए मैट्रिक्स को पोलीमराइज़ करने के लिए 30 से 40 मिनट के लिए 37 डिग्री सेल्सियस पर डिश को इनक्यूबेट करें। अंत में, कुएं में पूरक एस्ट्रोसाइट माध्यम के 500 माइक्रोलीटर जोड़ें और संस्कृति में आईबीबीबी को बनाए रखें।96-वेल प्लेट के लिए, मध्यम के 100 से 150 माइक्रोलीटर को अच्छी तरह से तब तक जोड़ें जब तक कि वे डाउनस्ट्रीम परख के लिए पर्याप्त रूप से विकसित न हो जाएं, जिसमें आमतौर पर दो सप्ताह लगते हैं। ठीक से गठित iBBB एक ब्राइटफील्ड माइक्रोस्कोप के नीचे एक ठोस, एकल पारभासी डिस्क के रूप में दिखाई दिया। 24 घंटे के बाद, समान रूप से वितरित एकल कोशिकाओं की पहचान की गई।दो हफ्तों के बाद, अधिक विशिष्ट संरचनाएं, हालांकि परिभाषित करना मुश्किल था, दिखाई दे रहे थे।

Research

JoVE Journal

Neuroscience

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...