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Introduction

The mesothelium, a distinctive cell type forming the pleural monolayer, envelopes

the outermost layers of the viscera and facilitates the growth of developing organs. Despite

the known fact that aberrant proliferation of adult mesothelial cells, often aggravated by

asbestos exposure, can lead to mesothelioma through the manipulation of developmental

pathways, the specific signaling processes that dictate progenitor pool expansion, embryonic

mesothelial progenitor cell (MPC) maturation, and their differentiation into smooth muscle

cells (SMC) remain poorly understood.

Anatomically, adult mature mesothelial cells of the parietal and visceral pleura

encase the inner layer of the thorax and the outer layer of the lungs, respectively. Mouse

lineage-tracing analyses showed that visceral mesothelial cells in developing lung pleura

migrate inward and differentiate into vascular smooth muscle cells

, parabronchial smooth

muscle cells

, and myofibroblast

, highlighting the multipotency of developmental MPCs.

During development, the MPC arises from the exact origin, lateral plate mesoderm

, while

mesothelioma tends to originate from parietal mesothelial cells

. Since carcinogenesis often

hijacks developmental programs

, studying parietal mesothelial development could

significantly advance mesothelioma diagnosis and treatment.

Mesothelioma, a rare and aggressive cancer often caused by carcinogens like

asbestos or tar, has a notably high mortality rate

. The prevalence is high in the countries such

as the United Kingdom, Australia, and New Zealand

. Various tumor markers were

identified, including Calretinin (CALB2), mesothelin (MSLN), type III collagen (COL3A1),

and secretory leukocyte peptidase inhibitor (SLP1)

. Despite the availability of treatments

such as surgical decertification and chemotherapy, most cases are diagnosed at advanced

stages, limiting effective intervention options

. A better understanding of the behavior of

MPCs in the parietal pleura during development could develop the prognostic markers of

mesothelioma.

In mouse embryos, Wilms Tumor Protein 1 (WT1), a representative mesothelial cell

marker, is expressed on visceral and parietal mesothelial cells from the lung and the thoracic

cavity

1,11

WT1

knockout mice showed hypoplastic lung phenotype

11,12

and the defects of

human mesothelial cells by Congenital Diaphragmatic Hernia (CDH), also known to develop

lung hypoplasia

Previous in vitro studies have shown that Fibroblast growth factor 2 (FGF2) and

platelet-derived growth factor (PDGF) are required for the proliferation of adult mesothelial

cells

. Notably, high expression of FGF2 in mesothelioma correlated with poor prognosis

Bone morphogenic protein 4 (BMP4) is expressed in the human adult peritoneal

mesothelium and plays a pivotal role in mesothelial-to-mesenchyme transition (MMT),

attenuating the TGF-beta-mediated MMT phenotype

. BMP4 is expressed ventral to the

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distal lung bud mesenchyme and at the distal lung bud tips of the endoderm

17,18

, but the

association with the behavior of WT1

MPC is unknown.

Additionally, Sonic hedgehog (SHH) and Retinoic acid (RA) are implicated in MPC

migration and epithelial morphology transformation, respectively

However, how these signaling pathways intertwine and distinctively regulate MPC

pool expansion, differentiation, and maturation during development has yet to be determined,

necessitating robust culture methods for detailed study.

This study successfully allowed us to establish the method to isolate and culture

embryonic parietal MPC from developing pig and mouse thorax. By culturing these cells with

a range of small molecules and growth factors, we aimed to elucidate the signaling pathways

crucial for mesothelial cell development.

Results

Establishment of Cell Culture Protocol for the Expansion of Developing Pig Mesothelial

Cells

The development of pig lungs undergoes embryonic, pseudo glandular, canalicular,

and alveolar stages around embryonic day 19 (E19), E25, 60, and E90, respectively

20,21

. The

developmental stage at which pig parietal mesothelial progenitor cells (MPCs) could be

efficiently harvested was unknown. We harvested the parietal MPCs from the E80 canalicular

stage thorax to have enough cell numbers.

To harvest a WT1

developing MPC efficiently, we compared several methods

100

previously reported

22–25

, including collecting pleural fluid, pinching porcine thoracic walls

101

with tweezers, scaring it with scrapers, or trypsinizing the porcine thoracic wall. Among

102

those methods, trypsinization with a 0.05% trypsin inside the E80 thoracic walls showed the

103

highest yield of MPC collection (

Figure 1A

). Interestingly, 0.25% trypsin treatment to the

104

thorax did not expand the MPC (

Figure S1A, B

). Previous papers showed the requirement of

105

EGF for culturing EGF

23,25

. Contrary to expectations, MPC culture with EGF didn’t offer an

106

apparent effect on MPC colony expansion (

Figure S1C

). To expand MPC efficiently, we

107

coated the cell culture dish with extracellular matrix (ECM) molecules (type I collagen (Col

108

I) and hyaluronic acid (HA)), given their expression in adult mature mesothelial cells

26,27

. We

109

found that the isolated MPC showed the sustained expression of

Col I

expression and its

110

receptor,

integrin beta 1

(

ITGB1

), but a relatively low expression of HA receptor (

CD44

)

111

(

Figure 1B

). Indeed, Col I coating significantly enhanced MPC expansion compared to HA

112

coating (HA) and an uncoated control (

Figure 1C, D

). Since the gelatin and Col1 share the

113

integrin-binding motif, RGD sequence

, we cultured the MPC on the gelatin-coated dish and

114

confirmed its efficacy in expanding MPCs

(

Figure 1E

). Based on this, we performed all

115

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downstream analyses on the gelatin-coated dish. Additionally, we confirmed that mouse

116

MPC can be collected and expanded well after the trypsinization directly on the E17.5 mouse

117

canalicular ~ sacculation stage thorax, noting that 0.25% trypsin was more effective for

118

mouse MPCs than 0.05% (

Figure S1D-F

). These results underscore the robustness and

119

effectiveness of our trypsinization-based protocol for isolating parietal MPCs in

120

development.

121

122

FGF2 Promotes Expansion of Pig Mesothelial Progenitor Cells (MPCs)

123

While the role of FGF2 and PDGF in adult mesothelial cell proliferation is known,

124

their impact during development is little known

. To confirm each molecule’s effect on

125

developing MPCs, we cultured MPC with FGF2 and PDGF-BB for 3 days (

Figure 2

126

PDGF-BB was chosen as the signaling molecule for the PDGF signaling pathway due to its

127

binding potential to all PDGF receptors

. We found that FGF2 and PDGF-BB treatment

128

increased total cell number as well as the WT1

cell numbers compared to the basal condition

129

control (

Figure 2A-D

). Ki67 immunostaining confirmed that FGF2 and PDGF-BB

130

significantly increased proliferating cell numbers (

Figure 2A, B, E

). Notably, FGF2 and

131

PDGF-BB induced a more than four times increase in proliferating Ki67

WT1

MPC

132

proportion compared with the control in the short-term culture (

Figure 2F

). In contrast, the

133

treatment with SU5402, a FGFR inhibitor, and CP 673451, a PDGFR inhibitor, significantly

134

decreased both total and WT1 cell numbers (

Figure 2C, D

) by inducing 30~40% of cell

135

death, labeled by Cleaved Caspase3 (CASP3) 1-day post-treatment (

Figure S2

). These

136

results suggested that the effect of endogenous FGF2 and PDGF activation cultured in the

137

basal medium impacts ~40% of MPC survival and that FGF2 and PDGF signaling may be

138

essential for WT1

MPC maintenance. To investigate the effect of FGF2 and PDGF on MPC

139

pool expansion in the long term, we cultured the MPCs with FGF2 or PDGF-BB for 14 days

140

and analyzed

WT1

mRNA expression by qPCR (

Figure 2G-I

). We found that FGF2

141

maintained

WT1

mRNA expression more than 5 times fold change compared to the control

142

during long-term culture (

Figure 2H

), while the effect of PDGF-BB pool expansion did not

143

significantly influence the

WT1

expression compared to the control over time (

Figure 2I

144

These results suggest that FGF2 efficiently expands the MPC pools, but the PDGF-BB effect

145

on the expansion is temporally and limited.

146

147

BMP4 Drives Differentiation of MPCs into SMC

148

During the MPC control culture condition, WT1

α-SMA

cells were observed (5.8

149

3.3 %) (

Figure 2B

). We speculated that WT1

MPCs could spontaneously differentiate

150

into smooth muscle cells (SMCs), given that mouse visceral lung mesothelial cells

151

differentiate into smooth muscle cells during mouse lung development

1,2

. To find which

152

signaling molecules induce MPC differentiation into SMC, we cultured MPC with various

153

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small molecules and inhibitors with different concentrations and screened

α-SMA

mRNA

154

expression

qPCR analysis (

Figure S3A

). We discovered that the BMP4 and ascorbic acid

155

(AA) condition enhanced

α-SMA

mRNA expression compared to control among the tested

156

conditions. Since BMP4 more dramatically induced SMC differentiation than AA, we

157

focused on further analyses of BMP signaling. qPCR analyses found that BMP4 treatment

158

showed significantly higher

α-SMA

mRNA induction both in short-term and long-term

159

cultures, while BMP4 treatment had a transient effect on

WT1

mRNA increase only in the

160

short term but did not sustain its impact in the long term (

Figure 3B, C

). In contrast,

161

Dorsomorphin, a BMP4 inhibitor, significantly reduced

α-SMA

mRNA expression with no

162

significant change in

WT1

mRNA expression (

Figure 3B

). Since the kinetics of

WT1

and

α-

163

SMA

mRNA by BMP4 treatment indicated the MPC differentiation into SMC, we

164

investigated the detailed cell fate change from MPC to SMC by immunostainings in short-

165

term culture (

Figure 3A

). Consistent with the qPCR observations, immunostaining analysis

166

showed a significantly increased α-SMA

cell proportion (Control: 7.8 ± 1.7 % vs. BMP4:

167

31.4 ± 1.4 %) and the number by BMP4 treatment (

Figure 3A, D-F

), while dorsomorphin

168

significantly reduced the α-SMA

SMC proportion (6.7 ± 3.0 %). Unlike FGF2 and PDGF-

169

BB (

Figure 2

), BMP4 treatment did not alter the total cell number, WT1

MCP numbers, or

170

WT1

proportion but significantly increased Ki67

cells (

Figure 3F-H

) while inducing about

171

20% of CASP3

cell death, which might be the cell selection step (

Figure S2

). Indeed, BMP4

172

selectively eliminates the WT1

Ki67

α-SMA

unknown cell type while dorsomorphin

173

significantly increased it (

Figure 3D

). Intriguingly, we observed a significantly increased

174

proportion of WT1

α-SMA

cells in WT1

MPC (control: 9.9

1.9 % vs. BMP4 group: 46.4

175

6.4 %) by BMP4 treatment (

Figure 3I

), but proportion of WT1

α-SMA

in SMC (control:

176

30.7

15.4 % vs. BMP4 group: 43.7

8.6 %) (

Figure 3J

) was not significantly changed.

177

On the other hand, we did not observe any change in the proportion of WT1

α-SMA

in α-

178

SMA

cells (

Figure 3K)

. These results indicate that BMP4 treatment primes the mesothelial

179

progenitor pools to co-express WT1 and α-SMA, facilitating MPC differentiation into SMCs.

180

Based on these results, including long-term culture, we concluded that the pivotal role of

181

BMP4 is to

induce parietal MPC differentiation into α-SMA

SMC with losing WT1

182

expression.

183

184

FGF2 and PDGF-BB Suppressed MPC Differentiation into SMCs

185

We observed MPC progenitor pool regulation by FGF2 and PDGF-BB (

Figure 2

)

186

and differentiation into α-SMA

SMC by BMP4 (

Figure 3

), but it was unclear whether FGF2

187

and PDGF-BB influence the SMC pools. To address this, we performed qPCR analyses. We

188

found that the decreased

α-SMA

mRNA expression by the FGF2 or PDGF-BB over time

189

(

Figure 4A, B, S3

), and the further analysis of IF data showed that the proportion of α-SMA

190

cells was significantly reduced by the FGF2 or PDGF-BB treatment (Control vs. FGF2 vs.

191

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PDGF-BB groups: 7.8

1.7 % vs. 2.5

0.5 % vs. 3.2

0.4 %), while BMP4 significantly

192

induced α-SMA

cells (31.4

1.4 %) (

Figure 4C

). In particular, PDGF-BB showed a

193

dramatic decrease of

α-SMA

mRNA than FGF2 (

Figure 4B

). While there were no significant

194

changes in the proportion of proliferating α-SMA

cells, the proportion of WT1

α-SMA

195

cells was significantly decreased by the FGF2 or PDGF treatment (Control vs. FGF2 vs.

196

PDGF-BB groups: 9.9

1.9 % vs. 3.8

0.9 % vs. 3.4

1.3 %) (

Figure 4D, E

). These

197

results indicate that FGF2 and PDGF play a central role in MPC progenitor pool expansion

198

by inhibiting the induction of WT1

α-SMA

primed cells, leading to α-SMA

smooth muscle

199

cells (

Figure 4F

200

201

Dominance of FGF2 Effect Over BMP Signaling in MPC Pool Regulation

202

Since we found FGF2 and PDGF suppressed BMP4-mediated MPC differentiation

203

into SMC (

Figure 2-4

), we cultured MPC with the combination of FGF2 and BMP4 (FGF2 +

204

BMP4) or PDGF-BB and BMP4 (PDGF-BB + BMP4) to investigate the potential counter

205

effect. We found that MPC culture with FGF2 + BMP4 and PDGF-BB + BMP4 significantly

206

suppressed the BMP4-mediated MPC differentiation into SMC with lower

α-SMA

mRNA

207

expression than the BMP4 group (

Figure 5A)

. This mRNA expression trend was the same in

208

the long-term culture (

Figure 5B

). Although the short-term treatment with FGF2 + BMP4

209

and PDGF-BB + BMP4 showed a decrease in

WT1

mRNA expression (

Figure 5A

), the long-

210

term effect with FGF2 + BMP4 exhibited an increase in the

WT1

mRNA expression

211

compared to controls (

Figure 5B

), consistent with the FGF2 effect (

Figure 2

). The long-term

212

effect of PDGF-BB + BMP4 did not impact the

WT1

mRNA expression. Interestingly, the

213

FGF2+BMP4 or PDGF-BB+BMP4 condition induced more cell proliferation with a higher

214

total cell number than the BMP4 group in the short term (

Figure 5C-G

). In contrast, FGF2 +

215

PDGF-BB and PDGF-BB + BMP4 conditions significantly increased WT1

MPCs and

216

proliferating cell numbers than the control condition in the short-term but could not sustain

217

WT1

mRNA expression in the long-term (

Figure 5A, E, F

). FGF2 + PDGF-BB and PDGF-

218

BB + BMP4 conditions treatment significantly decreased α-SMA+ cells and showed no

219

increase of primed WT1

α-SMA

cells in WT1

cells (

Figure 5G, H

). As we expected, there

220

was no significant change in WT1

α-SMA

cells in α-SMA

cells (

Figure 5I

). These results

221

suggest the critical role of FGF2 in maintaining the MPC pool and its self-renewal that

222

counteracts the BMP signaling effects on MPC differentiation into SMC.

223

224

Wnt Signaling Facilitates MPC Maturation

225

During development, mesenchymal β-catenin signaling controls parabronchial

226

smooth muscle cell (PSMC) progenitors in the sub-mesothelial mesenchyme

. Wnt signaling

227

is involved in the outer mesothelial pool size of the zebrafish swimbladder during

228

development

. However, the molecular characterization of MPCs and their maturation

229

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during pig lung development have been little studied. To address this issue, we performed

230

immunostaining of WT1 and CALB2 in pig and mouse lung development (

Figure S4

231

Developing porcine pleural mesothelial cells expressed high levels of WT1 in the E26 early

232

pseudoglandular stage of porcine lungs, but the relative expression level in the peripheral

233

layer of the lungs was decreased in the later stage (

Figure S4A, B

). In contrast, CALB2

234

expression was not detected in the peripheral layer of the lungs in the E26 and E40 early

235

pseudoglandular stage but appeared in the canalicular stage and afterward (

Figure S4D, E

236

These results indicate that CALB2 is the marker for mesothelial cell maturation during

237

porcine lung development. We also confirmed that the WT1 expression pattern was also

238

similar during mouse lung development, supported by previous studies

1,19

(Figure S4C)

239

while CALB2 started to be expressed in the sub-peripheral layer from the E14.5

240

pseudoglandular stage in mouse lung development (

Figure S4F

241

To investigate the common MPC maturation markers across the species, we revisited

242

the deposit single-cell RNA-seq (scRNA-seq) database of developing human

and mouse

243

lung mesenchyme (

Figure S5

). We found that

WT1

was highly expressed in the early

244

pseudoglandular stage but decreased its expression in the late pseudoglandular and

245

canalicular stages of human and mouse-developing lungs.

CALB2

, a mature mesothelial cell

246

marker, was slightly observed but not abundant in human lung development. During mouse

247

lung development,

CALB2

was observed in non-mesothelial cells. In contrast, mesothelin

248

(

MSLN

) expression was observed in the late pseudoglandular stage of developing human

249

lungs to the canalicular stage while around the E18 sacculation stage and afterward in the

250

mouse lungs. These results suggest that decreased expression of

WT1

and increased

MSLN

251

are the evolutionarily conserved markers for MPC maturation, but

CALB2

is a pig-specific

252

unique marker for MPC maturation. Based on these results, we examined pig MPC

253

maturation in an in vitro study using

WT1

CALB2,

and

MSLN

254

We performed qPCR to screen the most potent signaling molecules regulating pig

255

MPC maturation to CALB2

and MSLN

mature mesothelial cells (

Figure S3B, C

)

Among

256

them, we found that most signaling molecules induced the upregulation of

CALB2

and

MSLN

257

mRNA. In particular, the GSK3β inhibitor that acts as a Wnt activator (CHIR) showed the

258

most dramatic increase in

CALB2

mRNA expression. Thus, we focused on analyzing Wnt

259

signaling using CHIR in the MPC maturation. Three days of short-term CHIR treatment

260

increased

WT1

CALB2

, and

MSLN

mRNA expressions, while the long-term CHIR treatment

261

lost WT1

MPC pools but relatively sustained

CALB2

expression (

Figure 6A

). Since high

262

WT1

mRNA expression is the landmark for immature MPC pool expansion, these results

263

indicate that the MPC maturation by CHIR occurred as a long-term effect (

Figure 6A

264

Interestingly, we also found that long-term treatment with FGF2 or BMP4 significantly

265

increased

MSLN

mRNA expression compared to the control (

Figure 6B

). However, FGF2

266

did not increase the mRNA expression of

MSLN

and

CALB2

in a dose-dependent manner in

267

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short-term culture, while BMP4 induced

CALB2

mRNA expression in a dose-dependent

268

manner (

Figure S3C

). Furthermore, the

CALB2

mRNA upregulation by FGF2 or BMP4 was

269

transient and relatively limited in the long-term treatment compared to the CHIR treatment

270

(

Figure 6B

). Consistent with the qPCR results, the CALB2 immunostaining exhibited a

271

consistent trend with qPCR results, indicating the increased CALB2

cells by CHIR

272

treatment (

Figure 6C, D

). As shown in the PDGF-BB effect, CHIR induced Ki67

273

proliferative WT1

cells and significantly increased total cell numbers compared to control

274

(

Figure S6A-C

), while no WT1

cell number or proportional change and reduced α-SMA

275

cell number (

Figure S6D, E

). These results indicate that Wnt signaling activation induces

276

MPC maturation into MSLN

CALB2

cells, corresponding to the expression pattern of

277

CALB2 in porcine lung development.

278

279

Discussion

280

Previous studies showed the markers of adult mesothelial cells or in mesothelioma,

281

but it has been unclear how developing mesothelial progenitors shift the marker expressions

282

and their association with cellular behaviors. We established an MPC expansion protocol that

283

allows us to find the foundation of signaling pathways involved in MPC pool expansion,

284

differentiation, and maturation. Technically, we could not expand the cells from the E40 or

285

earlier time point’s thoracic wall in either method due to the low effectiveness of isolating

286

MPCs even using swine specimens larger than mice (data not shown). Harvesting MPC

287

exclusively from the lungs was also challenging because it contained various other cell types

288

after the culture (data not shown). Based on these technical limitations, we focused on the

289

MPC cellular analysis derived from the E80 thoracic walls. Of note, we also expand mouse

290

MPC, in this culture condition, from the thorax at E17.0 ~ E17.5 canalicular ~ sacculation

291

stage, corresponding to E80 pig developmental time points, indicating the robustness of our

292

culture protocol to harvest and expand MPC (

Figure S1

293

FGF signaling pathways have been classically known as critical mitogens for both

294

epithelium and mesenchyme

32–34

. Interestingly, mesothelial cells and mesothelioma have

295

been characterized as epithelial-like and mesenchymal-like features

35,36

. We found that FGF2

296

has the most potent effect on MPC self-renewal in the long-term culture among tested

297

conditions and inhibits BMP4-mediated SMC differentiation. Given that FGF2 high

298

expression in mesothelioma is one of the critical prognosis factors and carcinogenesis often

299

renders developmental program

37–39

, we speculate that targeting therapy for the FGF2 and its

300

downstream, such as Spry2

, Ras

, or Sos

, may be critical for controlling FGF2

high+

301

mesothelioma expansion and metastasis.

302

We found BMP4 signaling was critical for inducing MPC differentiation into SMC

303

with an increase of α-SMA

cells,

including primed, transitioning WT1

α-SMA

cells and

304

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differentiated WT1

α-SMA

cells (

Figure 4

). The molecular mechanism of how BMP4

305

converts MPC to SMC needs to be determined in the future. Interestingly, our

306

immunostaining analyses revealed that proliferating Ki67

α-SMA

cells were never observed

307

without tuning on WT1 (

Figure 4

). BMP4 initially induced WT1

Ki67

α-SMA

transitioning

308

cells but later lost the

WT1

mRNA expression (

Figure 3B

), suggesting that the critical role of

309

BMP4 in MPC cell fate change to post-mitotic terminally differentiated SMC. Since retinoic

310

acid treatment for acute leukemia patients induces terminally differentiated cells and is an

311

effective therapy for those patients

, how BMP4 signaling activation would influence

312

mesothelioma would be an attractive question.

313

Parietal MPC and lung peripheral MPC showed distinct morphology and function

314

Our study showed that potential CALB2 descendants of MPC appeared around the

315

neighboring WT1

mesothelium (

Figure S4D, E

), supported by previous studies of mouse

316

lung development

. There are remaining exciting questions regarding MPC maturation:

317

about the role of CALB2 in porcine parietal MPC, its developmental distributions, how the

318

parietal and lung-peripheral MPC distinctively mature, and how these MPC pools

319

communicate during development. Interestingly, we did not observe CALB2

cells on the

320

parietal mesothelium during mouse development (

Figure S4F

). We examined three different

321

antibodies against MSLN to investigate the maturation of MPC during development.

322

However, MSLN expression was not detected in developing lungs and thorax, as in the

323

previous study

, which is inconsistent with the scRNA-seq result (

Figure S5B

). This

324

indicates that protein expression may be regulated at post-translational levels or require

325

further technical advancements.

326

Interestingly, the WT1

MPC showed α-SMA expression, reminiscent of porcine

327

parietal mesothelial cells in the E26 early pseudoglandular stage (

Figure 1E, Figure S4A

328

while it is uncommon in peripheral lung MPC. In our culture model, we used MPC at the

329

canalicular ~ sacculation stage. Our results indicate that porcine parietal MPCs may be a

330

source of SMCs around the developing ribs.

331

We summarized MPC fate change by signaling molecules (

Figure 7

). Interestingly,

332

FGF2 promoted the expansion of both WT1

MPC and WT1

α-SMA

pool compared to the

333

control (

Figure 2B

). The WT1

α-SMA

pool would involve CALB2

mature mesothelial

334

cells. However, BMP4 suppressed the WT1

α-SMA

pool expansion (

Figure 3D

), while

335

BMP4 also increased CALB2 expression in short-term culture (

Figure 6B, D

). This

336

discrepancy suggests the existence of WT1

α-SMA

CALB2

unknown

pool, which may have

337

a role in the MPC regulation (

Figure 7

). Further analysis using genetic lineage tracing or

338

single cell level bioinformatics analysis may reveal the lineage hierarchy, parietal MPC vs.

339

peripheral lung MPC vs. WT1

α-SMA

niche interactions, and association with

340

mesothelioma, which will lead to further understanding of mesothelial development and

341

pathogenesis.

342

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343

Acknowledgments

344

We thank Zurab Ninish for his technical assistance. We sincerely appreciate scientific

345

input from Dr. Jianwen Que and Dr. Wellington Cardoso at the Columbia Center for Human

346

Development (CCHD) and the members of Cardoso’s lab and CCHD. We acknowledge the

347

support from the CCHD Medicine Microscopy Core (MMC)

(NIH S10 OD032447-01)

. This

348

work was funded by NIH-NHLBI 1R01 HL148223-01, DoD PR190557, PR191133 to M. M..

349

350

Author contributions

351

Youngmin Hwang, Validation, Investigation, Visualization, Methodology, Writing – original

352

draft; Yuko Shimamura, Junichi Tanaka, Akihiro Miura, Anri Sawada, Hemanta Sarmah, Dai

353

Shimizu, Yuri Kondo, Investigation, Validation; Zurab Ninish, Kazuhiko Yamada,

354

Methodology; Munemasa Mori, Conceptualization, Data curation, Supervision, Funding

355

acquisition, Validation, Investigation, Methodology, Project administration, Writing – review

356

and editing.

357

358

Declaration of interests

359

The authors declare no competing interests.

360

361

362

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

References

363

Que, J., Wilm, B., Hasegawa, H., Wang, F., Bader, D., and Hogan, B.L.M. (2008).

364

Mesothelium contributes to vascular smooth muscle and mesenchyme during lung

365

development. Proc Natl Acad Sci U S A

105

. 10.1073/pnas.0808649105.

366

De Langhe, S.P., Carraro, G., Tefft, D., Li, C., Xu, X., Chai, Y., Minoo, P.,

367

Hajihosseini, M.K., Drouin, J., Kaartinen, V., et al. (2008). Formation and

368

differentiation of multiple mesenchymal lineages during lung development is regulated

369

by β-catenin signaling. PLoS One

. 10.1371/journal.pone.0001516.

370

Choo, Y.Y., Sakai, T., Komatsu, S., Ikebe, R., Jeffers, A., Singh, K.P., Idell, S.,

371

Tucker, T.A., and Ikebe, M. (2022). Calponin 1 contributes to myofibroblast

372

differentiation of human pleural mesothelial cells. Am J Physiol Lung Cell Mol

373

Physiol

322

. 10.1152/AJPLUNG.00289.2021.

374

Obacz, J., Yung, H., Shamseddin, M., Linnane, E., Liu, X., Azad, A.A., Rassl, D.M.,

375

Fairen-Jimenez, D., Rintoul, R.C., Nikolić, M.Z., et al. (2021). Biological basis for

376

novel mesothelioma therapies. Preprint, 10.1038/s41416-021-01462-2

377

10.1038/s41416-021-01462-2.

378

Boutin, C., Schlesser, M., Frenay, C., and Astoul, P. (1998). Malignant pleural

379

mesothelioma. European Respiratory Journal

. 10.1183/09031936.98.12040972.

380

Manzo, G. (2019). Similarities between embryo development and cancer process

381

suggest new strategies for research and therapy of tumors: A new point of view. Front

382

Cell Dev Biol

. 10.3389/fcell.2019.00020.

383

Rehrauer, H., Wu, L., Blum, W., Pecze, L., Henzi, T., Serre-Beinier, V., Aquino, C.,

384

Vrugt, B., De Perrot, M., Schwaller, B., et al. (2018). How asbestos drives the tissue

385

towards tumors: YAP activation, macrophage and mesothelial precursor recruitment,

386

RNA editing, and somatic mutations. Oncogene

. 10.1038/s41388-018-0153-z.

387

Huang, J., Chan, S.C., Pang, W.S., Chow, S.H., Lok, V., Zhang, L., Lin, X., Lucero-

388

Prisno, D.E., Xu, W., Zheng, Z.J., et al. (2023). Global Incidence, Risk Factors, and

389

Temporal Trends of Mesothelioma: A Population-Based Study. Journal of Thoracic

390

Oncology

. 10.1016/j.jtho.2023.01.095.

391

Gueugnon, F., Leclercq, S., Blanquart, C., Sagan, C., Cellerin, L., Padieu, M.,

392

Perigaud, C., Scherpereel, A., and Gregoire, M. (2011). Identification of novel markers

393

for the diagnosis of malignant pleural mesothelioma. American Journal of Pathology

394

178

. 10.1016/j.ajpath.2010.12.014.

395

10.

Ricciardi, S., Cardillo, G., Zirafa, C.C., Carleo, F., Facciolo, F., Fontanini, G., Mutti,

396

L., and Melfi, F. (2018). Surgery for malignant pleural mesothelioma: An international

397

guidelines review. Preprint, 10.21037/jtd.2017.10.16 10.21037/jtd.2017.10.16.

398

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

11.

Cano, E., Carmona, R., and Muñoz-Chápuli, R. (2013). Wt1-expressing progenitors

399

contribute to multiple tissues in the developing lung. Am J Physiol Lung Cell Mol

400

Physiol

305

. 10.1152/ajplung.00424.2012.

401

12.

Sontake, V., Kasam, R.K., Sinner, D., Korfhagen, T.R., Reddy, G.B., White, E.S.,

402

Jegga, A.G., and Madala, S.K. (2018). Wilms’ tumor 1 drives fibroproliferation and

403

myofibroblast transformation in severe fibrotic lung disease. JCI Insight

404

10.1172/jci.insight.121252.

405

13.

Gilbert, R.M., Schappell, L.E., and Gleghorn, J.P. (2021). Defective mesothelium and

406

limited physical space are drivers of dysregulated lung development in a genetic model

407

of congenital diaphragmatic hernia. Development (Cambridge)

148

408

10.1242/DEV.199460.

409

14.

Mutsaers, S.E., McAnulty, R.J., Laurent, G.J., Versnel, M.A., Whitaker, D., and

410

Papadimitriou, J.M. (1997). Cytokine regulation of mesothelial cell proliferation in

411

vitro and in vivo. Eur J Cell Biol

412

15.

Kumar-Singh, S., Weyler, J., Martin, M.J.H., Vermeulen, P.B., and Van Marck, E.

413

(1999). Angiogenic cytokines in mesothelioma: A study of VEGF, FGF-1 and -2, and

414

TGFβ expression. Journal of Pathology

189

. 10.1002/(SICI)1096-

415

9896(199909)189:1<72::AID-PATH401>3.0.CO;2-0.

416

16.

Namvar, S., Woolf, A.S., Zeef, L.A.H., Wilm, T., Wilm, B., and Herrick, S.E. (2018).

417

Functional molecules in mesothelial-to-mesenchymal transition revealed by

418

transcriptome analyses. Journal of Pathology

245

. 10.1002/path.5101.

419

17.

Weaver, M., Yingling, J.M., Dunn, N.R., Bellusci, S., and Hogan, B.L. (1999). Bmp

420

signaling regulates proximal-distal differentiation of endoderm in mouse lung

421

development. Development

126

, 4005–4015.

422

18.

Weaver, M., Dunn, N.R., and Hogan, B.L. (2000). Bmp4 and Fgf10 play opposing

423

roles during lung bud morphogenesis. Development

127

, 2695–2704.

424

19.

Dixit, R., Ai, X., and Fine, A. (2013). Derivation of lung mesenchymal lineages from

425

the fetal mesothelium requires hedgehog signaling for mesothelial cell entry.

426

Development

140

, 4398–4406. 10.1242/dev.098079.

427

20.

Shimamura, Y., Tanaka, J., Kakiuchi, M., Sarmah, H., Miura, A., Hwang, Y., Sawada,

428

A., Ninish, Z., Yamada, K., Cai, J.J., et al. (2022). A developmental program that

429

regulates mammalian organ size offsets evolutionary distance. bioRxiv.

430

10.1101/2022.10.19.512107.

431

21.

McGeady, T.A., Quinn, P.J., Fitzpatrick, E.S., Ryan, M.T., Kilroy, D., and Lonergan,

432

P. (2017). Veterinary Embryology.

433

22.

Kienzle, A., Servais, A.B., Ysasi, A.B., Gibney, B.C., Valenzuela, C.D., Wagner,

434

W.L., Ackermann, M., and Mentzer, S.J. (2018). Free-floating mesothelial cells in

435

pleural fluid after lung surgery. Front Med (Lausanne)

. 10.3389/fmed.2018.00089.

436

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

23.

Mierzejewski, M., Paplinska-Goryca, M., Korczynski, P., and Krenke, R. (2021).

437

Primary human mesothelial cell culture in the evaluation of the inflammatory response

438

to different sclerosing agents used for pleurodesis. Physiol Rep

439

10.14814/phy2.14846.

440

24.

Kawai, N., Ouji, Y., Sakagami, M., Tojo, T., Sawabata, N., Yoshikawa, M., and

441

Taniguchi, S. (2019). Isolation and culture of pleural mesothelial cells. Exp Lung Res

442

. 10.1080/01902148.2018.1511002.

443

25.

Pruett, N., Singh, A., Shankar, A., Schrump, D.S., and Hoang, C.D. (2020). Normal

444

mesothelial cell lines newly derived from human pleural biopsy explants. Am J

445

Physiol Lung Cell Mol Physiol

319

. 10.1152/AJPLUNG.00141.2020.

446

26.

Saed, G.M., Zhang, W., Chegini, N., Holmdahl, L., and Diamond, M.P. (1999).

447

Alteration of type I and III collagen expression in human peritoneal mesothelial cells

448

in response to hypoxia and transforming growth factor-β1. Wound Repair and

449

Regeneration

. 10.1046/j.1524-475X.1999.00504.x.

450

27.

Breborowicz, A., Korybalska, K., Grzybowski, A., Wieczorowska-Tobis, K., Martis,

451

L., and Oreopoulos, D.G. (1996). Synthesis of hyaluronic acid by human peritoneal

452

mesothelial cells: Effect of cytokines and dialysate. Peritoneal Dialysis International

453

. 10.1177/089686089601600410.

454

28.

Davidenko, N., Schuster, C.F., Bax, D. V., Farndale, R.W., Hamaia, S., Best, S.M.,

455

and Cameron, R.E. (2016). Evaluation of cell binding to collagen and gelatin: a study

456

of the effect of 2D and 3D architecture and surface chemistry. J Mater Sci Mater Med

457

. 10.1007/s10856-016-5763-9.

458

29.

Ö stman, A. (2017). PDGF receptors in tumor stroma: Biological effects and

459

associations with prognosis and response to treatment. Preprint,

460

10.1016/j.addr.2017.09.022 10.1016/j.addr.2017.09.022.

461

30.

He, P., Lim, K., Sun, D., Pett, J.P., Jeng, Q., Polanski, K., Dong, Z., Bolt, L.,

462

Richardson, L., Mamanova, L., et al. (2022). A human fetal lung cell atlas uncovers

463

proximal-distal gradients of differentiation and key regulators of epithelial fates. Cell

464

185

, 4841-4860.e25. 10.1016/J.CELL.2022.11.005.

465

31.

Negretti, N.M., Plosa, E.J., Benjamin, J.T., Schuler, B.A., Habermann, A.C., Jetter,

466

C.S., Gulleman, P., Bunn, C., Hackett, A.N., Ransom, M., et al. (2021). A single-cell

467

atlas of mouse lung development. Development

148

. 10.1242/dev.199512.

468

32.

Ornitz, D.M., and Itoh, N. (2001). Fibroblast growth factors. Preprint, 10.1007/978-3-

469

662-46875-3_2175 10.1007/978-3-662-46875-3_2175.

470

33.

Lebeche, D., Malpel, S., and Cardoso, W. V (1999). Fibroblast growth factor

471

interactions in the developing lung. Mech Dev

, 125–136.

472

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

34.

Yuan, T., Volckaert, T., Chanda, D., Thannickal, V.J., and De Langhe, S.P. (2018).

473

Fgf10 Signaling in Lung Development, Homeostasis, Disease, and Repair After Injury.

474

Preprint, 10.3389/fgene.2018.00418 10.3389/fgene.2018.00418.

475

35.

Travis WD Müller-Hermelink HK, B.E. (2004). Pathology and Genetics: Tumours of

476

the Lung, Pleura, Thymus and Heart. International agency for research on cancer

477

36.

Koopmans, T., and Rinkevich, Y. (2018). Mesothelial to mesenchyme transition as a

478

major developmental and pathological player in trunk organs and their cavities.

479

Preprint, 10.1038/s42003-018-0180-x 10.1038/s42003-018-0180-x.

480

37.

Perantoni, A.O., Dove, L.F., and Karavanova, I. (1995). Basic fibroblast growth factor

481

can mediate the early inductive events in renal development. Proc Natl Acad Sci U S A

482

. 10.1073/pnas.92.10.4696.

483

38.

Dudley, A.T., Godin, R.E., and Robertson, E.J. (1999). Interaction between FGF and

484

BMP signaling pathways regulates development of metanephric mesenchyme. Genes

485

Dev

. 10.1101/gad.13.12.1601.

486

39.

Schelch, K., Wagner, C., Hager, S., Pirker, C., Siess, K., Lang, E., Lin, R., Kirschner,

487

M.B., Mohr, T., Brcic, L., et al. (2018). FGF2 and EGF induce epithelial-mesenchymal

488

transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal.

489

Carcinogenesis

. 10.1093/carcin/bgy018.

490

40.

García-Domínguez, C.A., Martínez, N., Gragera, T., Pérez-Rodríguez, A., Retana, D.,

491

León, G., Sánchez, A., Oliva, J.L., Pérez-Sala, D., and Rojas, J.M. (2011). Sprouty2

492

and spred1-2 proteins inhibit the activation of the ERK pathway elicited by

493

cyclopentenone prostanoids. PLoS One

. 10.1371/journal.pone.0016787.

494

41.

Ichise, T., Yoshida, N., and Ichise, H. (2014). FGF2-induced Ras-MAPK signalling

495

maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific

496

gene expression and suppressing TGFβ signalling through Smad2. J Cell Sci

127

497

10.1242/jcs.137836.

498

42.

Tan, Y., Qiao, Y., Chen, Z., Liu, J., Guo, Y., Tran, T., Tan, K. Sen, Wang, D.Y., and

499

Yan, Y. (2020). FGF2, an Immunomodulatory Factor in Asthma and Chronic

500

Obstructive Pulmonary Disease (COPD). Preprint, 10.3389/fcell.2020.00223

501

10.3389/fcell.2020.00223.

502

43.

Stahl, M., and Tallman, M.S. (2019). Acute promyelocytic leukemia (APL): remaining

503

challenges towards a cure for all. Preprint, 10.1080/10428194.2019.1613540

504

10.1080/10428194.2019.1613540.

505

44.

Shelton, E.L., Galindo, C.L., Williams, C.H., Pfaltzgraff, E., Hong, C.C., and Bader,

506

D.M. (2013). Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and

507

Parietal Mesothelia. PLoS One

. 10.1371/journal.pone.0069712.

508

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

Figure Legends

515

Figure 1. Isolation of Mesothelial cell progenitors (MPCs) from pig fetuses.

(A)

516

Schematic illustration of pig MPC isolation: The embryonic thorax (middle panel in A) was

517

isolated from E80 pig fetuses (left panel in A) and treated with the following procedures. (i)

518

scraping MPCs followed by trypsinization with 0.05% trypsin in the tube: (ii) trypsinization

519

with 0.05% trypsin directly on the thorax. In both methods, the mesothelial cell was

520

neutralized with DMEM + 10% FBS, followed by PBS washing and filtration with a cell

521

strainer to remove the residual connective tissue. The trypsinization on the porcine thorax (ii)

522

method showed a higher yield of MPC expansion than the scraping method (i) (right panels

523

in A). (B) Graphs: quantitative qRT-PCR (RT-qPCR) analysis of type I collagen (

COL1A1

524

integrin beta-1 (

ITGB1

), and

CD44

cultured in a basal culture medium.

Error bars represent

525

mean

SD.

Each plot showed different biological replicates (n = 3).

Each gene expression

526

was normalized with the housekeeping gene (

GAPDH

) expression. (C) Representative phase

527

contrast images of MPCs isolated from E80 pig thorax cultured on different cell culture dish

528

coating conditions. Col I: type I collagen coating, HA: hyaluronic acid coating, Non: non-

529

coating. (D) Graphs: Quantification of the isolated pig MPC number per each field.

Each plot

530

showed different biological replicates (n = 3).

(E) Representative immunofluorescence (IF)

531

image of MPC after 3 days of culture. Red: WT1, Green: α-SMA, Blue: DAPI. Scale bars:

532

(A) 1 cm, (C) 100 μm, (E) 20μm. *p<0.05, ****p<0.0001, ns: no significant difference by

533

one-way ANOVA test and t-test in (D).

534

535

Figure 2. MPC self-renewal by FGF2, PDGF-BB stimulation.

(A) Representative IF

536

images of MPCs after 3 days of treatment with FGF2, PDGF, SU5404 (FGF signaling

537

inhibitor, SU), a CP673451 (PDGF signaling inhibitor, CP), or Control (no treatment). FGF2

538

and PDGF-BB showed more cell numbers per field. WT1 (red), Ki67 (blue), DAPI (grey).

539

Arrows (white): WT1

Ki67

cells. (B) Graph: Quantification of cell numbers per field with

540

each marker from IF images in (A). (n = 4) (C-F) Graphs: quantification of cell number from

541

IF images with total cell number (C), WT1

cell number (D), Ki67

proliferative cell number

542

(E), and proportion of WT1

Ki67

proliferative MPCs (F).

Error bars represent mean

SD.

543

Each plot showed different biological replicates

(n = 4). (G-I) Graphs: RT-qPCR analysis of

544

WT1

mRNA expression after 3 days of culture with FGF2, PDGF-BB, SU, and CP (G).

WT1

545

mRNA expression during long-term culture by FGF2 (H) and PDGF-BB treatment (I).

Error

546

bars represent mean

SD.

Each plot showed different biological replicates (n = 3).

Relative

547

mRNA expression of each gene was normalized with the control basal culture condition.

548

Scale bars = 20 μm. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant

549

difference by one-way ANOVA test and t-test in (C-F).

550

551

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

552

Figure 3. MPC differentiation into α-SMA

smooth muscle cell by BMP4 stimulation.

553

(A) Representative IF images of MPC after 3 days of treatment with BMP4, dorsomorphin

554

(BMP signaling inhibitor, Dor), or Control (no treatment). BMP4 induced α-SMA expression,

555

while a Dor reduced its expression. WT1 (red), α-SMA (green), Ki67 (blue), and DAPI

556

(grey). Arrows (white): WT1

α-SMA

cells, asterisks: WT1

Ki67

α-SMA

cells, arrowhead

557

(white): WT1

α-SMA

cells. (B-C) Graphs: RT-qPCR analysis of

WT1

and

α-SMA

mRNA

558

expression for 3 days of MPCs culture with BMP4, Dor, or Control (B) and long-term culture

559

(C).

Error bars represent mean

SD.

Each plot showed different biological replicates (n = 3).

560

Relative mRNA expression of each gene was normalized with the control basal culture

561

condition. (D) Quantification of cell numbers per field with each marker from IF images in

562

(A). (E-I) Quantification of cell number from IF with α-SMA

cell proportion (E), total cell

563

number(F), WT1

cell proportion (G), Ki67

proliferating cell number (H), the proportion of

564

WT1

α-SMA

primed cells in WT1

cells (I), WT1

α-SMA

cells in SMA

cells (J), and

565

WT1

α-SMA

cells in α-SMA

cells (K).

Error bars represent mean

SD.

Each plot showed

566

different biological replicates

(n = 4). Scale bars = 20 μm. *p< 0.05, **p<0.01, ***p<0.001,

567

****p<0.0001, ns: no significant difference by one-way ANOVA test and t-test in (B, C, E-

568

K).

569

570

Figure 4. FGF2 and PDGF suppressed MPC differentiation into smooth muscle cells.

571

(A-B) Graphs: RT-qPCR analysis of α-SMA.

α-SMA

mRNA expression after 3 days of MPCs

572

culture with FGF2, PDGF-BB, BMP4, and its inhibitors (SU, CP, Dor) (A) and long-term

573

culture of MPCs with FGF2, PDGF-BB (B).

Error bars represent mean

SD.

Each plot

574

showed different biological replicates (n = 3).

Relative mRNA expression of each gene was

575

normalized with the control basal culture condition. (C-E) Graphs: Quantification of cell

576

proportion from IF of MPCs (from

Figure 2, 3

) with α-SMA

cell proportion (C), proportion

577

of WT1

α-SMA

cells in WT1

cells (D), and proportion of Ki67

α-SMA

cells in α-SMA

578

cells (E).

Error bars represent mean

SD.

Each plot showed different biological replicates

579

= 4) (F) Schematic summary of MPC self-renewal and differentiation into SMC by FGF2,

580

PDGF-BB, and BMP4. **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference

581

by one-way ANOVA test and t-test in (A-E)

582

583

Figure 5. The dominance of FGF2 effect over BMP signaling in MPC pool regulation.

584

(A-B) Graphs: RT-qPCR analysis of WT1 and

α-SMA

mRNA expression of MPC culture

585

with signaling molecules and its combination during 3 days of culture (A) and long-term

586

culture (B). (C) Graph: Quantification of cell numbers per field with each marker from IF

587

images. (n = 4) (D-G) Graphs: quantification of cell number from IF with total cell number

588

(D), WT1

cells (E), Ki67

cells (F), and α-SMA

cells (G). (n = 4) (H-I) Graphs: proportion

589

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

of WT1

α-SMA

cells in WT1

cells (H), proportion of WT1

α-SMA

cells in α-SMA

cells

590

(I).

Error bars represent mean

SD.

Each plot showed different biological replicates

(n = 4)

591

Scale bars = 20 μm. *p<0.05, **p<0.01, ****p<0.0001, ns: no significant difference by one-

592

way ANOVA test and t-test in (A-I).

593

594

Figure 6.



-catenin (wnt) activation induced the maturation of MPCs to CALB2

595

mature mesothelial cells.

(A-B) Graphs: RT-qPCR analysis of

WT1

α-SMA

CALB2

, and

596

MSLN

mRNA expression for long-term culture of MPC treatment with CHIR99021 (CHIR)

597

(A), and FGF2, BMP4 (B).

Error bars represent mean

SD.

Each plot showed different

598

biological replicates (n = 3).

Relative mRNA expression of each gene was normalized with

599

the control basal culture condition. (C) Representative IF images of MPCs after 3 days of

600

treatment with BMP4 and CHIR. CALB2 (red), DAPI (blue). (C) Graph: quantification of

601

CALB2

cell number from IF.

Error bars represent mean

SD.

Each plot showed different

602

biological replicates

(n = 4). Scale bars = 20 μm. *p<0.05, **p<0.01, ***p<0.001,

603

****p<0.0001, ns: no significant difference by one-way ANOVA test and t-test in (A, B, D).

604

605

Figure 7.

Schematic model of embryonic pig MPC cell behavior control by intertwined

606

signaling.

FGF2 induces self-renewal of WT1

MPC. MPC differentiates into α-SMA

SMC

607

through primed WT1

α-SMA

cells by BMP4 stimulation. FGF and PDGF signaling

608

suppresses the BMP4-mediated SMC differentiation. Developing mesothelium shows stage-

609

specific markers: high WT1 expression in the early pseudoglandular stage of porcine lung

610

development and low WT1 expression and CALB2 expression in the calanlicular~alveolar

611

stage. Wnt activation by CHIR facilitates the MPC maturation process. The role of WT1

α-

612

SMA

unknown pools in MPC proliferation and differentiation is unclear.

613

614

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

STAR

★

Methods

636

Key resources table

637

638

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit anti-WT1

Proteintech

Cat#12609-1-AP

RRID:AB_2216225

Mouse anti-α-SMA

Bio-Rad

Cat#MCA5781GA

RRID:AB_3076452

Chicken anti-Ki-67

Novus Biologicals

Cat#NBP3-05538

RRID: AB_3076453

Mouse anti-calretinin (2D7A9)

Thermo Fisher Scientific

Cat#66496

RRID:AB_2664066

Chicken anti-calretinin

EnCor Biotechnology

Cat#CPCA-Calret

RRID:AB_2572241

Rabbit anti-cleaved caspase-3 (Asp175)

Cell Signaling

Cat#9661

RRID:AB_2341188

Rabbit anti-mesothelin (D9R5G)

Cell Signaling

Cat#99966

RRID:AB_2800323

Rabbit anti-mesothelin (SP74)

Abcam

Cat#93620

RRID:AB_10563844

Mouse anti-mesothelin (MSLN/2131)

Novus Biologicals

Cat#NBP2-79724

RRID: AB_3076454

Donkey anti-mouse Alexa 488

Invitrogen

Cat#A21202

RRID:AB_141607

Donkey anti-mouse Alexa 647

Invitrogen

Cat#A10042

RRID:AB_2534017

Donkey anti-rabbit Alexa 568

Invitrogen

Cat#A31571

RRID:AB_162542

Donkey anti-chicken Alexa 488

Jackson Immunoresearch
Labs

Cat#703-545-155

RRID:AB_2340375

Goat anti-chicken HRP

Invitrogen

Cat#A16054

RRID:AB_2534727

Chemicals, peptides, and recombinant proteins

Cy3 tyramide

AAT Bioquest

Cat#11065

RBC Lysis Buffer (10x)

Biolegend

Cat#420301

NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342)

Invitrogen

Cat#R37605

rhEGF

R&D Systems

Cat#236-EG

rhFGF-basic

PeproTech

Cat#100-18B

SU5402

MedChem Express

Cat#HY-10407

rhPDGF-BB

R&D Systems

Cat#220-BB

CP673451

MedChem Express

Cat#HY-12050

rhBMP4

R&D Systems

Cat#314-BP

Dorsomorphin

Tocris

Cat#3093

CHIR99021

MedChem Express

Cat#HY-10182

Ascorbic acid

Fisher Chemical

Cat#FLA61100

Retinoic acid

Sigma-Aldrich

Cat#R2625

Purmorphamine

Tocris

Cat#4551

Critical commercial assays

PrimeScript RT Master Mix (Perfect Real time)

Takara Bio

Cat#RR036B

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

https://www.graphpad.co
m/

bioRxiv preprint

Direct-zol™ RNA Purification kit

Zymo Research

Cat#R2062

Luna Universal qPCR Master Mix

New England Biolabe
(NEB)

Cat#M3003X

Deposited data

Human RNA-seq

Mouse RNA-seq

Pig RNA-seq

Experimental models: Organisms/strains

Mouse: Crl:CD1(ICR)

Charles River
Laboratories

Strain: 022

Yucatan pig

Sinclair

BioResources

N/A

Oligonucleotides

qPCR primers, see Table S1

This paper

N/A

Software and algorithms

GraphPad Prism 10.0

N/A

Cellpose

https://www.cellpose.org
/

N/A

ImageJ

https://imagej.net/ij/

N/A

Leica Application Suite X (LAS X)

https://www.leica-
microsystems.com/

N/A

Other

Fetal Bovine Serum

Cytiva

Cat#SH30088.03HI

Trypsin-EDTA (0.05%)

Gibco

Cat#25300054

Trypsin-EDTA (0.25%)

Gibco

Cat#15050065

DMEM medium, high glucose

Cytiva

Cat#SH30243.02

639

Resource availability

640

Lead contact

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Further information and requests for resources and reagents should be directed to and will

642

be fulfilled by the lead contact, Munemasa Mori (

mm4452@cumc.columbia.edu

643

Materials availability

644

All biological materials used in this study are available from the

lead contact

upon request.

645

Data and code availability

646

•

This paper does not report original code.

647

•

Any additional information required to reanalyze the data reported in this paper is

648

available from the

lead contact

upon request.

649

650

Experimental model and study participant details

651

Animals

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All surgical procedures were conducted under the approval of the Columbia University

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Institutional Animal Care and Use Committee and USAMRMC Animal Care and Use Review

654

Office (ACURO). For pig experiment, Timed-pregnant Yucatan miniature sows were

655

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

obtained from Sinclair BioResources. For mouse experiment, CD-1 mice (male (8 weeks),

656

female (8 weeks)) were purchased from Charles River Laboratories.

657

658

Parietal pig mesothelial progenitor cell (MPC) isolation

659

E80 Yucatan pig embryo was surgically collected from the Yucatan pig mother. After

660

euthanization, the thorax was collected. For MPC isolation, we performed 2 methods; 1) the

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mesothelial tissue was isolated from the E80 pig thoracic wall with a cell scraper (Fisher

662

Scientific), by following incubation in 0.25% trypsin-EDTA solution for 20 min at 37

C and

663

2) 0.25% trypsin treatment on the thoracic wall, by following 20 min incubation at 37

664

After trypsin-EDTA treatment, the dissociated cell was washed with PBS by centrifuge and

665

replacement of the PBS (350 x g, 5 min, 4

C). The cell pellet was incubated in RBC lysis

666

buffer solution for 10 min at 4

C for RBC lysis (Biolegend), following PBS wash by

667

centrifuge (350 x g, 5 min, 4

C). After washing with PBS, the cell pellet was filtered with a

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cell strainer (40um pore size, MTC Bio) and seeded on a type I collagen (from rat tail,

669

Sigma-Aldrich)- coated 6-well tissue culture plate. The MPCs (P0) were cultured in MPC

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culture medium (DMEM (high glucose, Gibco) + 10% FBS (Cytiva) + 1% pen/strep (Gibco))

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for 7 days. For passage, MPCs were washed with PBS and dissociated with 0.05% trypsin-

672

EDTA (Gibco) for 5min at 37

C). For MPC culture and its analysis for the experiments,

673

passages 6-8 MPC were cultured on gelatin-coated tissue culture plates.

674

675

Parietal mouse mesothelial progenitor cell (MPC) isolation

676

Mouse MPC was isolated from E17.5 embryonic thorax by treatment of 0.05 % or 0.25 %

677

trypsin-EDTA (Gibco) solution for 20 min at 37

C. The isolation procedure was the same as

678

pig MPC isolation. The mouse MPC was cultured in an MPC culture medium with the

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replacement of the cell culture media every other day.

680

681

Parietal pig mesothelial progenitor cell (MPC) culture

682

To investigate the MPC cell fate by signaling molecules, MPCs were cultured in the MPC

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culture medium with various signaling molecules (FGF2 (Peprotech), PDGF-BB, BMP4

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(R&D systems), retinoic acid (RA, Sigma-Aldrich), CHIR99021 (MedChem Express),

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ascorbic acid (AA, Fisher Chemical), purmorphamine (Shh, Tocris)) and the inhibitors

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(SU5402 as FGFR inhibitor (MedChem Express), CP673451 as PDGFR inhibitor (MedChem

687

Express), and dorsomorphin (Tocris) for 3, 10, or 14 days. During MPC culture, the MPC

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culture medium, including signaling molecules, was replaced every other day and passaged

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at day 3, 6, and 10 to avoid full confluency.

690

691

RT-qPCR

692

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

bioRxiv preprint

mRNA was isolated from MPCs with Direct-zol RNA Microprep isolation kit (Zymo

693

Research) after lysis of MPCs with IBI isolate total reagent (IBI Scientific). For cDNA

694

synthesis, the isolated mRNA was mixed with PrimeScript RT Master Mix (Takara bio),

695

followed by cDNA synthesis protocol. For RT-qPCR analysis, the synthesized cDNA was

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mixed with qPCR primers and Luna Universal qPCR Master Mix (New England Biolabs

697

(NEB). RT-qPCR was conducted with Quantstudio (Applied Biosystems). mRNA expression

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of each gene was normalized with the housekeeping gene (GAPDH). The relative mRNA

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expression of the genes was normalized with the control group (MPC culture in DMEM +

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10% FBS + 1% pen/strep).

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702

Immunofluorescence (IF)

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For cell sample preparation, MPCs were fixed with 3.7% paraformaldehyde (PFA) for 10

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min at room temperature. For tissue sample preparation, 10um-frozen sectioned tissue

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samples were washed with PBS 3 times, followed by antigen retrieval with citrate-based

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buffer (Vector Laboratories) in the microwave for 8 min. After washing the cells and the

707

tissue samples with PBS 3 times, the primary antibodies in dilution solution (0.25% triton X-

708

100 + 0.75% BSA in PBS) were treated to the samples and incubated at 4

C for overnight.

709

After 3 times PBS wash on the following day, the secondary antibodies and DAPI were

710

treated (0.75% BSA in PBS) for 1 hour at room temperature. Then, the sample was mounted

711

with a coverglass, anti-fade reagent (Invitrogen). For pig cell/tissue CALB2 staining,

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primary antibody-treated samples were treated with HRP conjugated anti-chicken antibody

713

(in PBS) and incubated for 30 min at room temperature. After PBS wash, Cy3 tyramide

714

(1:1000 diluted in 100 mM borate + 0.1% Tween-20 + 0.003 % H

solution (pH 8.5)) was

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treated in the samples and incubated for 15 min at room temperature in the dark. After PBS

716

wash, the samples were mounted with a coverglass and an anti-fade reagent (Invitrogen).

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The cell samples were visualized with a Leica DMI microscope (Leica). The tissue samples

718

were visualized with a Zeiss confocal microscope (Zeiss).

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RNA-seq data analysis

721

For human and mouse RNA-seq data analysis, we utilized the database from the previous

722

studies.

30,31

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724

Quantification and statistical analysis

725

Quantification of cell number in the phase contrast images was conducted by ImageJ. For

726

immunostained cell (single-immunostained and co-immunostained cell population) and

727

DAPI-stained cell counting from IF images, Cellpose software was used. The mean

728

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.

https://cellxgene.cziscience.com/e/f9846bb4-784d-4582-92c1-

bioRxiv preprint

fluorescence intensity (MFI) of each IF sample was measured in the non-overlapping random

729

fields using ImageJ software. Data analysis was performed using Prism 10. Data acquired by

730

performing biological replicas ((n = 3) for RT-qPCR and phase contrast images, (n = 4) for

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IF images) of three or four independent experiments are presented as the mean

standard

732

derivation (SD). Statistical significance was determined using a one-way ANOVA or a two-

733

tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant.

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Additional resources

737

Human scRNA-seq:

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3f279e4c6f0c.cxg/

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Mouse sdRNA-seq:

https://lungcells.app.vumc.org/

740

741

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted February 2, 2024.