The previous studies reveal a pivotal role of ATIIC-derived signaling in promoting re-epithelialization of injured alveoli [

1

,

2

]. Here we aimed to test whether normal ATIICs may express an active signaling in response to injury via exosome miR-371b-5p-mediated cell-to-cell communication. Initially, we did not observe miR-371b-5p appearance in the cultures of hiPSC-ATIICs or hATIICs treated with BLM (Additional file

1

: Figure S4). Considering that ATIC-derived factors might be required to stimulate ATIICs’ function in response to injury, we founded an ATIC/ATIIC co-culture model using transwell plates in order to mimic the injured alveolar environment (Fig.  

5a

). ATICs and ATIICs (hiPSC-ATIICs or hATIICs) in the co-culture program were exposed to various doses of BLM for 24  hours, and then the cells and exosomes were separately remote from ATIIC chambers for miRNA preparation. Like A549-derived exosomes, the hiPSC-ATIIC- and hATIIC-derived exosomes expressed exosome-specific markers TSG101, CD63, and HSP70 (Fig.  

5b

). QRT-PCR was performed to analyze miR-371b-5p expression (as described within “ Methods” ), and the expression levels of cellular plus exosome miR-371b-5p derived from A549 cells were used because control index. As shown in Fig.  

5c

, neither mobile nor exosome miR-371b-5p were derived from control hiPSC-ATIICs or even hATIICs. In contrast, hiPSC-ATIICs and hATIICs were induced to convey cellular and exosome miR-371b-5p to significantly detectable amounts (Fig.  

5c

, represented as percentages of those derived from A549 cells) after treated with BLM at 10, 20, or 30  μ g/ml. Both cellular and exosome expressions associated with miR-371b-5p were consistently induced in these two ATIIC phenotypes, with more robust induction at lower BLM concentrations: around 43% (hiPSC-ATIICs) or approximately 35% (hATIICs) at 10  μ g/ml BLM; 20% (hiPSC-ATIICs) or approximately 17% (hATIICs) at 20  μ g/ml BLM, and; around 8% (hiPSC-ATIICs) or approximately 6% (hATIICs) at 30  μ g/ml BLM. However , miR-371b-5p expression by hiPSC-ATIICs and hATIICs was barely detected after treatment along with 40  μ g/ml BLM, possibly due to seriously broken ATIICs so that they failed to respond to injury in the cultures.

Fig. five

ATIICs express exosome miR-371b-5p in response to injury. a Schematic strategy of co-cultures of hiPSC-ATIICs or hATIICs with hiPSC-ATICs in six-well transwell plates using exosome-depleted DM with and without BLM. w Western blotting of TSG101, CD63 plus HSP70 in exosomes derived from cultured A549 (1), hiPSC-ATIICs (2) and hATIICs (3). QRT-PCR analysis of manifestation levels of cellular and exosome miR-371b-5p in the cultures associated with ATIIC phenotypes treated with various doses of BLM ( c ) as well as the level of exosome miR-371b-5p in the lavage samples isolated from BLM-treated computer mouse lungs transplanted with hiPSC-ATIICs or hmonos ( d ) by using ABM primer. The particular expression levels were normalized to 18  s plus presented as percentage of the cellular or exosome miR-371b-5p level of A549 cells. e Schematic structures to show the sequences of miR-371b-5p ( top ), in which 2 underlined nucleotides are not identical in miR-292b-5p ( bottom ), as well as the sequences of miR-371b-5p-specific reverse primer (R-primer-I) and miR-292b-5p-specific reverse primer (R-primer-II). f QRT-PCR evaluation of expression levels of exosome miR-371b-5p and miR-292b-5p within BLM-treated lungs with or without transplantation by using R-primer-I or R-primer-II. The expression levels were normalized to 18  h and presented as a ratio to the ABM primer discovered ‘ miR-371b-5p’ expression level as indicated. g Immunofluorescent staining of consultant lung sections from BLM-challenged mice with or without transplanted hiPSC-ATIICs or hmonos by using a mouse anti-human nuclei antibody plus rabbit anti-human proSPC antibody with DAPI counterstaining. A persons nuclei antibody recognizes human cells ( red ) in mouse lungs; the anti-human proSPC stains both mouse and human SPC-expressing ATIICs ( green ). Many nuclei SPC + mouse ATIICs (indicated by white arrows ) were identified in control plus BLM-treated lungs transplanted with hiPSC-ATIICs, but not in the BLM-treated lungs receiving hmonos or saline. The nuclei + SPC + human ATIICs (indicated by green arrows ) were found only within the BLM-treated lungs transplanted with hiPSC-ATIICs. Some nuclei + cells were SPC harmful (indicated by red arrows ), suggesting that they were ATICs that had been differentiated in the transplanted hiPSC-ATIICs. A magnified view of a mouse ATIIC (indicated by the white arrowhead ), a human ATIIC (indicated by the green arrowhead ), or an ATIC (indicated by red arrowhead ) is shown in the corresponding image. BLM bleomycin, DAPI 4′, 6-diamidino-2-phenylindole, DM differentiation medium, hATIICs human primary ATIICs, hiPSC-ATIICs human induced pluripotent come cell-derived ATIICs, hmonos human peripheral blood monocytes, SPC surfactant protein C

We also examined the capacity of ATIICs to obtain exosome miR-371b-5p in vivo in injured microenvironment simply by transplanting hiPSC-ATIICs into a BLM-induced acute lung alveolar damage model, using immune-deficient SCID mice to avoid graft being rejected [

40

,

41

]. As previously reported by our lab [

1

,

2

], 8– 10-week-old female SCID mice had been exposed to BLM (3. 5 units/kg) 2  days prior to transplantation with hiPSC-ATIICs (1  ×   10

6

in 50  μ l of saline). After exposure to this fairly high dose of BLM, injured alveolar ATIICs hardly survive [

1

,

2

]. Thus, transplanted hiPSC-ATIIC-derived signaling along with its capacity to promote survival of endogenous ATIICs could be clearly evaluated. To demonstrate the specificity of hiPSC-ATIIC-derived signaling, BLM-challenged mice were also transplanted with same variety of hmonos. Lavage exosomes, which expressed exosome-specific markers TSG101, CD63, and HSP70 (data not shown), were remote from BLM-challenged lungs with and without transplantation on times 2, 4, 6, 8, and 10 for miRNA preparation. Initially, miR-371b-5p primers (ABM Inc. ) had been used to analyze hiPSC-ATIIC-derived exosome signaling. Our results demonstrated that no exosome miR-371b-5p was expressed in control lung area as well as BLM-challenged lungs at each time point (Fig.  

5d

). In comparison, a significant high level of exosome miR-371b-5p was discovered in BLM-challenged lungs 2  days after transplantation along with hiPSC-ATIICs (on day 4). As no exosome miR-371b-5p was expressed in BLM-challenged lungs transplanted with hmonos, our data demonstrated an induced expression of hiPSC-ATIIC-specific miR-371b-5p in response to alveolar injury (Fig.  

5d

). However , as much miRNAs and miRNA recognition (target) motifs are very well conserved in many animal species [

42

45

], it is rather likely which the ABM primers used for QRT-PCR analysis of the human miR-371b-5p expression can also detect its mouse homolog [

46

]. We therefore compared the sequence of miR-371b-5p with this of its mouse homolog. As shown in Fig.  

5e

, miR-371b-5p and its mouse homologous miR-292b-5p do share approximately 91% sequence homology. For clarification, we designed two invert primers, R-primer-I and R-primer-II (Fig.  

5e

), to particularly analyze miR-371b-5p and miR-292b-5p expression, respectively. As demonstrated in Fig.  

5e

, the isolated Exo-miRs were ligated for an RNA 5′ adaptor (Qiagen, Hilden, Germany) before QRT-PCR analysis by using an universal forward primer (Qiagen) and a particular reverse primer (R-primer-I or R-primer-II). The ABM special primer detected ‘ miR-371b-5p’ expression level was used since control index because the ABM primers are expected to identify both miR-371b-5p and miR-292b-5p (also demonstrated by our own preliminary experiments, data not shown). Consistent with the result proven in Fig.  

5d

, the exosomes isolated from control lung area, BLM-challenged lungs as well as BLM-challenged lungs transplanted with hmonos did not express miR-371b-5p and miR-292b-5p at each time stage (Fig.  

5f

). The fact that no miR-371b-5p/miR-292b-5p expression was recognized in control lungs could be because its expression is only limited to early developmental stage [

46

]. Interestingly, BLM-challenged lung area expressed significant levels of exosome miR-371b-5p and miR-292b-5p 2  days after transplantation with hiPSC-ATIICs (on day four, Fig.  

5f

, represented as a ratio to the ABM primer recognized ‘ miR-371b-5p’ expression level). These data indicated how the transplanted hiPSC-ATIICs were induced to express miR-371b-5p

+

Exo-miRs in response to alveolar injuries in vivo just as in the in vitro experiments. Considering that mATIICs can also be induced to proliferate by miR-371b-5p

+

Exo-miRs (A549-Exo-miRs, Fig.  

2a

), the expression of mouse exosome miR-292b-5p within the BLM-challenged lungs transplanted with hiPSC-ATIICs, but not in control lung area, BLM-challenged lungs or BLM-challenged lungs transplanted with hmonos, suggests that the hiPSC-ATIIC-derived miR-371b-5p

+

Exo-miRs may promote survival/recovery of endogenous injured ATIICs, which subsequently release miR-292b-5p

+

Exo-miRs and participate in the particular repair process. In support of the capacity of mATIICs to release the particular miR-292b-5p

+

Exo-miRs, the miR-292b-5p expression was demonstrated in BLM-treated mATIIC cultures (Additional file

one

: Figure S5). Negative expression associated with exosome miR-292b-5p in the BLM-treated lungs (without ATIIC transplantation) may reflect the severity of endogenous ATIIC injuries. As indicated in Fig.  

5d and f

, expression associated with hiPSC-ATIIC-derived exosome miR-371b-5p as well as endogenous exosome miR-292b-5p considerably decreased in BLM-challenged lungs on day 6 plus was barely detected on day 10 when hurt alveoli were almost completely repaired (data not shown). Such decreased expression of exosome miR-371b-5p/miR-292b-5p over time towards the end point (day 10) may reflect the process of recuperation of injured alveoli after transplantation. Consistently, the endogenous ATIICs were severely damaged after exposure to this higher dose of BLM, and in comparison to control lungs, just a few mouse ATIICs (nuclei

SPC

+

) survived in the BLM-challenged lungs receiving saline or even hmonos (Table 

1

and Fig.  

5g

). Remarkably, numerous computer mouse ATIICs were observed in the BLM-challenged lungs where transplanted hiPSC-ATIICs (nuclei

+

SPC

+

) had efficiently engrafted (Table 

one

and Fig.  

5g

), differentiated into ATICs (Additional file

1

: Table S2), and launched exosome miR-371b-5p (Fig.  

5d and f

). Taken together, our own results demonstrate the capacity of ATIICs to derive miR-371b-5p

+

Exo-miRs within vitro and in vivo in response to injury, which may mediate the communication between ATIICs to promote survival and participation associated with endogenous injured ATIICs in repair of injured alveoli.

Desk 1

Relative content associated with human and mouse ATIICs in bleomycin-mouse lung tissues

Saline-SCID

1083  ±   54. 1

0

1083

0

BLM-SCID/saline

thirteen. 2  ±   3. 2

0

13

0

BLM-SCID/hmonos

14. 8  ±   5. 2

0

15

0

BLM-SCID/hiPSC-ATIICs

991  ±   39. 6

227. 6  ±   11. 8

763. 4  ±   32. 8

29. 8