3D culture of multipotent Sox9 + mouse embryonic lung progenitors: Isolation, Expansion and Cryopreservation CURRENT STATUS: POSTED

This protocol describes the isolation and long-term culture of Sox9 + distal tip lung progenitors from mouse embryonic lung tissue. We have developed a serum-free, feeder-free 3D system to stably expand a 95% pure Sox9 + mouse embryonic lung progenitor population in vitro . These lung progenitors retain their multipotency and can differentiate into the full spectrum of lung epithelial lineages—i.e., airway and alveolar lineages— in vitro as well as in vivo upon transplantation into the injured mouse lung. Sox9 + progenitors can be isolated from mouse embryonic lungs within 4h and maintained for at least 6 months (expanding ~10 20 fold in number). Cultured Sox9 + progenitors can be also cryopreserved and resurrected without losing their properties. The ability to expand multipotent Sox9 + lung progenitors should avail multiple future applications including the generation of lung organoids and gene editing, with ramifications for disease modeling, drug screening and regenerative medicine. This protocol accompanies Nichane et al (Nature Methods, 10.1038/nmeth.4498, published online November 6, 2017). serum-free (named Progenitor Medium-3D; “LPM-3D” hereafter), a 95% pure Sox9 + lung progenitor population can be maintained for at least 6 months, transcriptionally resemble the in vivo native distal tip embryonic lung progenitors and retain a normal karyotype. Moreover, the expanded mouse lung progenitors can differentiate into both alveolar and airway lineages in vitro and in vivo , validating their multipotency. A single investigator with expertise in developmental biology and cell culture can execute this protocol—initial cell isolation from embryonic lungs takes == 4 hours


Introduction
The lung is a ramified three-dimensional tree of epithelial tubes comprising two major subdivisionsthe proximally-located conducting airways and the distally-located alveoli (where gas exchange occurs). Currently, in the adult lung, only airway-restricted and alveolar-restricted stem cells are known to exist [1][2][3][4][5][6] . Access to systems in which multipotent lung stem or progenitor cells with the ability to generate both airway and alveolar cell-types, can be cultured and expanded in vitro would be an advantageous starting point to study lung developmental biology, organ function and lung diseases. Although methods to generate 3D aggregates ("organoids") from adult lung restricted-stem cells or differentiating pluripotent stem cells have already been described 3,4,7-10 , a true system that recapitulate lung native architecture and functionalities has yet to be achieved. Establishing lung organoid from cells with full lung multipotency could be an alternative to generate such in vitro model. While only airway-restricted and alveolar-restricted stem cells subsist in the adult lung, multipotent Id2 + and Sox9 + distal tip lung progenitors that have the ability to generate both airway and alveolar lineages transiently exist during mouse embryonic development (~E11.5-E15.5) [11][12][13] .
Here we describe in detail the steps necessary to isolate and expand multipotent Sox9 + lung progenitors from lung tissue at day 12.5 (E12.5) of mouse embryonic development. Despite their transient nature in vivo, we show here that these multipotent lung progenitors can be captured and stably expanded in vitro, while maintaining their undifferentiated, proliferative state and their competence to differentiate into both airway and alveolar cell-types. In these feeder-free and serumfree conditions (named Lung Progenitor Medium-3D; "LPM-3D" hereafter), a 95% pure Sox9 + lung progenitor population can be maintained for at least 6 months, transcriptionally resemble the in vivo native distal tip embryonic lung progenitors and retain a normal karyotype. Moreover, the expanded mouse lung progenitors can differentiate into both alveolar and airway lineages in vitro and in vivo, validating their multipotency. A single investigator with expertise in developmental biology and cell culture can execute this protocol-initial cell isolation from embryonic lungs takes == 4 hours and the isolated progenitors can be long-term expanded in vitro. Procedures for passaging (==~2 hours) and the cryopreservation of Sox9 + lung multipotent progenitors are also provided.
The remit of this protocol is to enable the derivation of a highly-pure multipotent Sox9 + lung progenitor population. However, it is important to note that methodologies to enable their rapid and efficient differentiation into specific airway and alveolar cell-types or 3D organoids with complete recapitulation of lung native architecture and functionalities have yet to be developed. In the future, we believe that these in vitro-expanded Sox9 + multipotent lung progenitors could be used as building blocks to assemble such sophisticated organoids comprising both airway and alveolar lineages with

4.
Place the culture plate in the 37 o C incubator for 20 minutes to allow the Matrigel to solidify.

5.
Add gently 500 µL of LPM3D medium to each well. Dispense medium on the inner wall of the well and not directly on the Matrigel droplet.

6.
Replace medium every 2 days. Colonies should be visible from day 3-4 onwards (Fig. 2  4. and 5. See 2. and 3. from "plating the cells after sorting" section Anticipated Results Using this protocol, Sox9 + embryonic lung progenitors colonies will start to be visible after 3-4 days.
LPM-3D culture selects for, and preferentially stimulates the proliferation of, Sox9 + distal tip lung embryonic progenitors by contrast to Sox9-epithelial cells or non-epithelial cells. This protocol can be used on wild-type mouse strains and prior enrichment for Sox9 GFP cells is therefore dispensable.
Cultures can be cryopreserved even at very early passage to generate a robust, stably-frozen cell bank for future resurrection and expansion when required. FACS gating strategy used to isolate Sox9+ progenitors from E12.5 Sox9GFP embryonic lungs.