|
| Status |
Public on Dec 20, 2019 |
| Title |
Early lineage commitment defines alveolar epithelial ontogeny in the lung (E13.5, E15.5 and E17.5) |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Multipotent Nkx2-1-positive lung epithelial primordial progenitors of the foregut endoderm are thought to be the developmental precursors to all adult lung epithelial lineages. However, little is known about the global transcriptomic programs or gene networks that regulate these gateway progenitors in vivo due to their rarity and transient presence during a narrow developmental window, embryonic day E9.0 in mice. Here we describe the unique genetic program of in vivo lung primordial progenitors and computationally identify the signaling pathways that are involved in their cell-fate determination from pre-specified embryonic foregut. We integrate this information in computational models to generate in vitro engineered lung primordial progenitors from mouse pluripotent stem cells, improving the fidelity of the resulting cells through unbiased, easy-to-interpret similarity scores and modulation of cell culture conditions, including biomechanical cues. As the genetic characterization of early in vivo embryonic progenitors is rapidly expanding, the methodology proposed here can have wide applicability to the in vitro derivation of bona fide tissue progenitors of all germ layers.
|
| |
|
| Overall design |
Lungs from Nkx2-1GFP embryos of the respective embryonic day (E13.5, E15.5 and E17.5) were harvested and processed into single cell suspensions using an enzyme solution containing dispase (Collaborative Biosciences), collagenase I (Life Technologies), and RNAse-free DNAse (Promega). Nkx2-1GFP positive cells stained with DAPI were sorted for GFP and also DAPI to exclude dead cells using a FACSJazz (BD Biosciences) flow cytometer using wild type embryonic lung single cells suspensions as a negative control. Cells were collected into FACS tubes containing cell growth media composed of 10% fetal bovine serum in DMEM/F12 media. Cells were loaded onto a GemCode instrument (10X Genomics) to produce single-cell bar-coded droplets using 10X Single Cell 3’ v2 chemistry. Libraries generated were sequenced across at least one lane on the Illumina HiSeq2500 instrument with the HiSeq Rapid SBS kit. The resulting reads were aligned and gene level unique molecular identifier (UMI) counts obtain using Cell Ranger version 2.0.1 (10x Genomics).
|
| |
|
| Contributor(s) |
Frank DB, Morley MP, Morrisey EE |
| Citation(s) |
32005814 |
| |
| Submission date |
Oct 21, 2019 |
| Last update date |
Feb 10, 2020 |
| Contact name |
Michael Patrick Morley |
| E-mail(s) |
mmorley@pennmedicine.upenn.edu
|
| Phone |
215-898-2026
|
| Organization name |
Perelman School of Medicine at the University of Pennsylvania
|
| Department |
Penn Cardiovascular Institute
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
|
| Samples (3) |
|
| Relations |
| BioProject |
PRJNA578717 |
| SRA |
SRP226498 |
