Profiling human brain vascular cells using single-cell transcriptomics and organoids
© 2023. Springer Nature Limited..
Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:19 |
|---|---|
| Enthalten in: |
Nature protocols - 19(2024), 3 vom: 07. März, Seite 603-628 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Crouch, Elizabeth E [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
|---|
| Anmerkungen: |
Date Completed 11.03.2024 Date Revised 11.03.2024 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1038/s41596-023-00929-1 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM365930563 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM365930563 | ||
| 003 | DE-627 | ||
| 005 | 20240311232029.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231227s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1038/s41596-023-00929-1 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1323.xml |
| 035 | |a (DE-627)NLM365930563 | ||
| 035 | |a (NLM)38102365 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Crouch, Elizabeth E |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Profiling human brain vascular cells using single-cell transcriptomics and organoids |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 11.03.2024 | ||
| 500 | |a Date Revised 11.03.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2023. Springer Nature Limited. | ||
| 520 | |a Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Review | |
| 700 | 1 | |a Diafos, Loukas N |e verfasserin |4 aut | |
| 700 | 1 | |a Valenzuela, Edward J |e verfasserin |4 aut | |
| 700 | 1 | |a Wedderburn-Pugh, Kaylee |e verfasserin |4 aut | |
| 700 | 1 | |a Birrueta, Janeth Ochoa |e verfasserin |4 aut | |
| 700 | 1 | |a Caston, Jaela |e verfasserin |4 aut | |
| 700 | 1 | |a Joseph, Tara |e verfasserin |4 aut | |
| 700 | 1 | |a Andrews, Madeline G |e verfasserin |4 aut | |
| 700 | 1 | |a Bhaduri, Aparna |e verfasserin |4 aut | |
| 700 | 1 | |a Huang, Eric J |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Nature protocols |d 2006 |g 19(2024), 3 vom: 07. März, Seite 603-628 |w (DE-627)NLM167398601 |x 1750-2799 |7 nnns |
| 773 | 1 | 8 | |g volume:19 |g year:2024 |g number:3 |g day:07 |g month:03 |g pages:603-628 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1038/s41596-023-00929-1 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 19 |j 2024 |e 3 |b 07 |c 03 |h 603-628 | ||