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JSRM Code: 016020300009    

Optimizing the in vitro colony-forming assay for more efficient delineation of the interaction between lung epithelial stem cells and their niche
Ozaki M1#, Kagawa S1, Ishii M1, Hegab AE1#

Author Names in full: Mari Ozaki1#, Shizuko Kagawa1, Makoto Ishii1, Ahmed E Hegab1#

1Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, Japan.

#These authors contributed equally

Supplementary Information

Supplementary Table S1: Effect of various lung digestion protocols on the number of the different cell types retrieved and their viability


Dispase alone

Elastase alone



Total number/one mouse lungs

7.83x106 ± 1.57x106

4.69x106 ± 0.51x106

11.68x106 ± 2.82x106

9.21x106 ± 0.83x106

AT-II number

0.13x106 ± 0.11x106

0.06x106 ± 0.07x106

0.15x106 ± 0.15x106

0.11x106 ± 0.08x106

AT-II vitality
(% confluence on day 7)

95 ± 5

70 ± 10

25 ± 10

40 ± 15

Other epithelial cells

0.08x106 ± 0.02x106

0.02x106 ± 0.01x106

0.05x106 ± 0.03x106

0.03x106 ± 0.01x106


0.007x106 ± 0.2x104

0.32x106 ± 0.08x106

1.57x106 ± 0.84x106

1.35x106 ± 0.72x106

Endothelial cells

0.18x106 ± 0.03x106

0.39x106 ± 0.08x106

0.67x106 ± 0.18x106

0.51x106 ± 0.12x106

All-cell viability %

93.3 ± 2.5

64.5 ± 4.7

53.9 ± 3.2

76.7 ± 3.1

Actual numbers/percentages retrieved from each protocol, from at least 3 biological replicates, using at least 3 mice/protocol in each repeat. Data are expressed as means ± standard error of the mean.

Supplementary Table S2: Changes in the percentage of expression of various fibroblast markers between freshly-collected, and 7 day-propagated fibroblasts (related to Figure 2)

















No data

No data






No data



No data



No data



No data

No data

No data


-Upper right half shows the double positive expressions on day 0, while the lower left half shows the expressions on day 7.
-Expression level is written as (Negative/low +medium +high).

Supplementary Figure S1: Summary of the processing of WLCs into the various cell types, and representative images for examination of AT-II cell vitality

Figure S1 : WLC solutions collected using the various enzyme digestion protocols are stained with PI and EpCAM. Live cells are gated (upper left, viability), then AT-II cells and other non-AT-II epithelial cells are sorted based on GFP and EpCAM expression (center). To examine vitality of collected AT-II cells, cells are seeded on laminin-coated plates, and examined by fluorescent microscope on D7 to assess percentage of confluence (right side photomicrographs). As most AT-II cells differentiate in culture into AT-I cells, only a few GFP+ cells are still visible at D7. Non-epithelial cells are also sorted then CD45+/CD31+ cells are depleted using AutoMACS. The CD45-/CD31- cells are cultured on uncoated plastic plates in complete medium for 2 h to isolate the fibroblasts (lower left).




Supplementary Figure S2: Effect of culture medium on the differentiation of the various types of colonies formed by lung epithelial stem cells (related to Figure 1A-D)

Figure S2 : Whole lung epithelial cells from Sftpc-GFP mice were co-cultured with lung fibroblasts in the in vitro colony forming assay in either "MTEC/Plus" or "lung epithelial" culture medium. The growing colonies were collected at 2 wks, immune-stained, and examined for the effect on their differentiation profile. A, B) Representative images showing staining with the AT-II and AT-I cell markers, Sftpc and Aquaporin5, in type C colonies. C, D) Representative images showing staining of the A/B colonies with the markers of secretory and basal cells, MUC5AC and Keratin-5. E) Representative image showing staining of the A/B colonies with the markers of ciliated and basal cells, acetylated a-tubulin and Keratin-5. Scale bars A, B = 50 m, C-E: 100 m.










Supplementary Figure S3: Effect of mild hyperoxia on lung stem cells proliferation in vitro (related to Figure 4F-I)

Figure S3 : Whole lung epithelial cells and MTECs were co-cultured with lung fibroblasts in the in vitro colony forming assay in normoxia, hyperoxia of 50%, or hyperoxia followed by normoxia, as described in the results. The colonies and cells were collected, and immune-stained for the proliferation marker PCNA. Representative images showing one colony from each condition, except WLCs hyperoxia where no colonies were detected. Only solitary cells, which were negative for PCNA were detectable. Scale bars = 50 m.








Supplementary Figure S4: Expression of the SARS-COV2 virus-related receptors on lung stem cell organoids and other control tissues from young and old mice

Figure S4 : RNA was collected from freshly-collected whole lung epithelial cells, WLC organoids, MTEC organoids, freshly collected liver tissue, and freshly collected fibroblasts, from young (8-12 wks) and old mice (1 year old), to detect the expression of these receptors, and to determine the influence of aging on the expression. Y-axis is in logarithmic scale for better comparison of the samples.