YJPSU-59460; No of Pages 4

Journal of Pediatric Surgery xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Pediatric Surgery

journal homepage: www.elsevier.com/locate/jpedsurg

Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic

Stem Cell Therapy
Alexander V. Chalphin, Sarah A. Tracy, Stefanie P Lazow, Ina Kycia, David Zurakowski, Dario O. Fauza ⁎
Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: We sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH).
Received 5 October 2019 Methods: Twelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = 22 days) to induce fetal
Accepted 26 October 2019 CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched
Available online xxxx intraamniotic injections of either saline (n = 37) or a suspension of 2 × 106 cells/mL of amniotic fluid-derived
mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of fibroblast
Key words:
growth factor-10 (FGF-10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC)
Congenital diaphragmatic hernia
Transamniotic stem cell therapy
was quantified by qRT-PCR. Statistical analysis was by the Mann–Whitney U test with Bonferroni adjusted crite-
TRASCET rion (p ≤ 0.01).
Amniotic mesenchymal stem cells Results: Among survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of
Fetal stem cells FGF-10 and VEGF-A gene expressions compared to the untreated (p b 0.001 for both) and saline groups
Fetal therapy (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to
the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact
on these comparisons.
Conclusions: Transamniotic stem cell therapy affects select processes of lung development in experimental
congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the
prenatal management of this disease is warranted.
Level of evidence: N/A (animal and laboratory study).
© 2019 Elsevier Inc. All rights reserved.

To date, transamniotic stem cell therapy (TRASCET) has been stud-
ied experimentally exclusively in models of congenital anomalies in
which the defect is directly exposed to the amniotic fluid, such as
spina bifida and gastroschisis [1]. The rationale behind that initial devel-
opment was the idea that the fetal-derived mesenchymal stem cells
(MSCs) used in TRASCET would reach the diseased areas of the fetus
through direct seeding within the amniotic cavity, after the
intraamniotic injection. Although this type of direct cell trafficking
does seem to occur, more recently it has been demonstrated that hema-
togenous donor cell routing is a germane component of TRASCET,
apparently owing to active migration of donor MSCs through the gesta-
tional membranes into the placenta, from which site cells can reach
both the fetal and maternal circulations, including the fetal bone
marrow [2–5]. This realization has led us to hypothesize that birth
defects not exposed to the amniotic cavity could possibly also benefit
from TRASCET. This study is the first examination of that hypothesis,
in which we aimed at determining whether TRASCET could impact
⁎ Corresponding author at: Boston Children's Hospital - Department of Surgery, 300
Longwood Avenue - Fegan 3, Boston, MA 02115. Tel.: +1 617 919 2966; fax: +1 617
730 0910.
E-mail address: dario.fauza@childrens.harvard.edu (D.O. Fauza).
any aspect at all of pulmonary development in the setting of experimen-
tal congenital diaphragmatic hernia (CDH).
1. Methods
This study was approved by the Boston Children's Hospital Institu-
tional Animal Care and Use Committee under protocol #18-07-3737.
1.1. Donor afMSC sourcing and phenotyping
Donor afMSCs consisted of previously banked cells procured from
normal syngeneic Lewis rat dams on gestational day 21 (E21; term =
21–22 days). A midline laparotomy was performed and the bicornuate
uterus eviscerated. Amniotic fluid samples were obtained using a 30G
needle (Becton Dickinson, Franklin Lakes, NJ) on a 1 mL syringe (Becton
Dickinson) introduced into each amniotic cavity upon the ventral aspect
of the fetus. Isolation and expansion of afMSCs were per methods as
previously described by our group [6,7]. Fluorescence-activated cell
sorting (FACS) analysis was used to confirm their mesenchymal progen-
itor identity using the Vantage SE cell sorter (Becton Dickinson) and
unconjugated mouse monoclonal antibodies previously validated for
use in rats, namely for CD29 (Becton Dickinson); CD44 (R&D Systems,
Minneapolis, MN); CD45 (Invitrogen, Grand Island, NY); and CD90

https://doi.org/10.1016/j.jpedsurg.2019.10.033
0022-3468/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: A.V. Chalphin, S.A. Tracy, S.P. Lazow, et al., Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic Stem
Cell Therapy, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2019.10.033
2 A.V. Chalphin et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
(Becton Dickinson). A purified CD73 (Becton Dickinson) conjugated
with an anti-mouse IgG1 against purified CD73 (Biolegend, San Diego,
CA) was also used. Nonspecific cell staining was excluded using
mouse isotype immunoglobulin controls.
1.2. Donor afMSC labeling
Proliferating donor afMSCs were labeled with the Cignal™ Lenti
Reporter firefly luciferase reporter gene (Qiagen, Germantown, MD)
based on methods as previously described [8,9]. Briefly, cells were
transduced with puromycin-resistant Cignal™ Lenti Reporter lentivirus
(multiplicity of Infection = 50; Qiagen) in high-glucose Dulbecco's
Modified Eagle medium with L-glutamine (DMEM; Corning, Corning,
NY) supplemented with 10% fetal bovine serum (FBS; Life Technologies,
Grand Island, NY) and 5 ng/mL basic fibroblast growth factor (bFGF;
Promega, Madison, WI). Infected cells were then spinoculated at room
temperature for 30 min at 2000 rpm, followed by overnight incubation
at 37 °C, 5% CO2, and 90% humidity. The next day, cells were washed
twice with Dulbecco's Phosphate Buffered Saline without Ca2 + and
Mg2+ (PBS; Sigma-Aldrich, St. Louis, MO) and allowed to incubate in
standard afMSC growth medium consisting of DMEM (Corning) supple-
mented with 10% FBS (Life Technologies) and 5 ng/mL bFGF (Promega).
At 72 h post transduction, virally infected cells were positively selected
in 60 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO) supplemented
medium.
1.3. Congenital diaphragmatic hernia creation and intraamniotic injections
Twelve Time-dated pregnant Sprague–Dawley dams (Charles River
Laboratories, Inc., Wilmington, MA) were exposed to nitrofen on E9.5
for the induction of fetal CDH based on methods as previously described
[10–12]. Briefly, they were anesthetized with isoflurane (Abbot Labora-
tories, North Chicago, IL), chamber inhaled at 2–4% in 100% oxygen and
gavage fed 100 mg of nitrofen (2,4-dichlorophenyl p-nitrophenyl-ether;
Sigma-Aldrich) dissolved in 1 ml of olive oil.
All their fetuses were then divided into three groups. An untreated
group (n = 31) underwent no further manipulation. The other two
groups received intraamniotic injections on E17, volume-matched at
50 μL of either saline (n = 37), or a suspension of labeled afMSCs at 2
× 10 6 cells/mL in PBS (n = 65). Injections were performed under direct
vision as we have previously described [13]. Briefly, general anesthesia
was induced and maintained with isoflurane (Abbot Laboratories),
chamber inhaled at 2–4% in 100% oxygen. Using sterile technique, a
large midline laparotomy was performed and the bicornuate uterus
exposed. A 33G noncoring needle on a 100 μL syringe (both from Ham-
ilton Company, Reno, NV) was introduced into each and every amniotic
cavity by the ventral aspect of the fetus, carefully avoiding it, the pla-
centa and the umbilical cord. The uterus was then returned to the abdo-
men and the incision closed in two layers with 3-0 Vicryl (Ethicon,
Somerville, NJ) and 5-0 Monocryl (Ethicon) simple running sutures. An-
imals were allowed to recover receiving only postoperative analgesia
with buprenorphine (Reckitt and Colman Pharmaceuticals, Richmond,
VA) and Meloxicam (Norbrook, Lenexa, KS) as needed.
1.4. Lung procurement and quantitative gene expression analysis
Dams were euthanized with chamber-inhaled carbon dioxide at
term on E21.5. A midline laparotomy was again made, the uterus was
eviscerated, the amniotic cavities were opened and the deceased fetuses
were retrieved. All fetuses with CDH had both lungs excised en bloc and
immediately fresh frozen.
In all three groups, relative gene expressions of surrogate markers
for select constituent processes of pulmonary development, specifically:
alveolar growth and development (fibroblast growth factor-10; FGF-
10), lung vascularization (vascular endothelial growth factor-A; VEGF-
A), and alveolar maturation/surfactant production (surfactant protein-
Please cite this article as: A.V. Chalphin, S.A. Tracy, S.P. Lazow, et al., Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic Stem
Cell Therapy, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2019.10.033
C; SPC) were analyzed by quantitative reverse transcription polymerase
chain reaction (qRT-PCR) in lungs both ipsi- and contralateral to the
CDH. Primers were designed using Primer-BLAST (http://www.ncbi.
nlm.nih.gov/tools/primer-blast/), with sequences shown in Table 1.
Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA). First-
strand cDNA was prepared from 5 μg of total RNA with a Superscript
IV Reverse Transcriptase reagent kit using oligo dT primers
(ThermoFisher Scientific, Waltham, MA). Samples without reverse tran-
scriptase were processed in parallel as negative controls. Quantitative
RT-PCR was performed on a CFX96 Touch System real-time PCR detec-
tion system (Bio-Rad Laboratories, Hercules, CA) using Fast SYBR
Green Master Mix (Applied Biosystems, Foster City, CA) under the
following thermal cycling conditions: enzyme activation at 95 °C for
15 s, followed by 42 cycles of denaturation at 94 °C for 15 s, annealing
at 55 °C for 30 s, and extension at 72 °C for 30 s. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) was used as a reference gene for
normalization of target gene expression using the 2 −ΔΔCtmethod, also
controlled by normal lungs from healthy fetuses (n = 10).
1.5. Statistical analysis
Quantitative data were presented as median and interquartile range
and analyzed by the Mann–Whitney U-test with Bonferroni adjusted
criterion for multiple comparisons with significance set at p ≤ 0.01
using IBM SPSS software (SPSS version 21.0, IBM, Armonk, NY).
2. Results
Total fetal survival to term with a diaphragmatic defect was 20% (27/
133), yielding n = 10 in the untreated group, n = 9 in the saline group,
and n = 8 in the TRASCET group. There was no significant difference in
overall survival rate with a CDH between the groups (p = 0.107).
The qRT-PCR data are summarized in Fig. 1. The TRASCET group
showed significant downregulation of FGF-10 and VEGF-A expressions
compared to the untreated (p b 0.001 for both) and saline groups
(p = 0.005 and p = 0.004, respectively). SPC expression was narrowly
significantly higher among TRASCET fetuses compared to untreated an-
imals (p = 0.01), but not compared to the saline group (p = 0.043).
Comparing the expression data in lungs ipsilateral or contralateral to
the hernia had minimal impact to these results (Fig. 2). There were no
significant differences in the expression of FGF-10, VEGF-A, or SPC
between normal fetuses and either control group.
3. Discussion
Therapies based on MSCs for pediatric lung disease have been re-
ported experimentally and even clinically, albeit only postnatally, thus
not involving the unique donor cell routing and timely effects afforded
by the TRASCET principle [14–17]. In a first exploratory study such as
this one, we chose to probe different fundamental processes contained
in pulmonary development, so as to determine whether which one(s),
if any, should deserve further examination in future experiments. This
was the basis for our selection of surrogate markers analyzed.
Perhaps more important than the simplistic determination of
whether a given marker is up or downregulated, the somewhat
Table 1
Primer sequences for quantitative real-time polymerase chain reaction.
Forward Primer Reverse Primer
GAPDH 5′-CTGAACGGGAAGCTCACTGG 5′-ATACTTGGCAGGTTTCTCCAGG-3′
−3′
SPC 5′-TCAAGAACTTCCAGGCCAAGT-3′ 5′-CAGGTCTCGCCCAGAAGAATC-3′
FGF-10 5′-GTGGAAATCGGAGTTGTTGC-3′ 5′-ATTCTTTTGAGCCATAGAGTTTCCC-3′
VEGF-A 5′-TTGTCCAAGATCCGCAGACG-3′ 5′-GCTTGTCACATCTGCAAGTACG-3′
GAPDH: glyceraldehyde 3-phosphate dehydrogenase; SPC: surfactant protein-C; FGF-10,:
fibroblast growth factor-10; VEGF-A: vascular endothelial growth factor-A.
 A.V. Chalphin et al. / Journal of Pediatric Surgery xxx (xxxx) xxx 3

Fig. 1. Relative mRNA expression of fibroblast growth factor-10 (FGF10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC), in the lungs of normal fetuses and
of fetuses with nitrofen induced CDH that were either untreated, or received intraamniotic injections of either saline (NS), or of a concentrated suspension afMSCs (TRASCET). * p ≤ 0.01 vs.
normal, ◊ p ≤ 0.01 vs. nitrofen, □ p ≤ 0.01 vs. saline, ○ p ≤ 0.01 vs. TRASCET.

surprising observation that TRASCET can impact fetal lung develop- indicate a deleterious effect, but possibly the opposite, for example
ment, in whichever way it may be, is the chief outcome of this work. In- reflecting less need of a response from the host to defined harmful stim-
deed, the interpretation of our findings is far from simple. For example, uli. An illustrative example of that is VEGF-A. Previous studies on VEGF-
the downregulation of certain genes' expressions does not necessarily A expression in the setting of nitrofen induced CDH have had conflicting

Fig. 2. Relative mRNA expression of fibroblast growth factor-10 (FGF10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC), in the lungs ipsilateral and
contralateral to the diaphragmatic defect in fetuses with nitrofen induced CDH that were either untreated, or received intraamniotic injections of either saline (NS), or of a
concentrated suspension of afMSCs (TRASCET). ◊ p ≤ 0.017 vs. nitrofen, □ p ≤ 0.017 vs. saline, ○ p ≤ 0.017 vs. TRASCET.

Please cite this article as: A.V. Chalphin, S.A. Tracy, S.P. Lazow, et al., Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic Stem
Cell Therapy, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2019.10.033
4 A.V. Chalphin et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
results [18–20]. Further, molecules of the VEGF-A family are capable of
both inducing and inhibiting angiogenesis [21]. Yet another layer of
this particular analysis is the fact that VEGF-A is subject to extensive al-
ternative splicing, which must be taken into account in the PCR method-
ology used. While we saw significant differences in VEGF-A expression
between TRASCET and both untreated and saline treated fetuses, given
the complexity of VEGF-A activity and regulation, the meaning of this
observation is not yet clear.
Both VEGF-A and FGF-10 play key roles in pulmonary develop-
ment, including pulmonary angiogenesis and airway branching, re-
spectively [22]. A previous study reported that FGF-10 expression
decreases in the setting of nitrofen induced CDH [23]. That report
also showed no difference in FGF-10 expression between nitrofen
exposed fetuses without CDH and healthy controls. It would thus ap-
pear that the degree of FGF-10 inhibition is more directly related to
the diaphragmatic hernia. Much like with VEGF-A, we saw signifi-
cant downregulation of FGF-10 expression in TRASCET animals
when compared with both untreated and saline treated fetuses. At
this point, it is also too early to infer what clinical impact such down-
regulation of FGF-10 may have. Still, overexpression of FGF-10 has
been linked with progenitor state arrest, goblet cell metaplasia, and
overall impaired lung bud development [24].
SPC is a marker of type-II pneumocyte maturation, which begins
in the pseudoglandular phase of lung development [25]. Surfactant
deficiency has been reported in late term nitrofen-exposed rat fe-
tuses and in human neonates with CDH [26]. Our results showed
that TRASCET significantly upregulated SPC expression in compari-
son to untreated fetuses, but not saline treated controls. Upon close
examination of the data shown in Fig. 1, we cannot rule out the pos-
sibility that this may have simply been because of the data being
underpowered.
This study must of course be considered in the context of its lim-
itations. While the nitrofen model is widely regarded as the most
representative of human CDH, it does not perfectly recapitulate the
clinical disease [27]. It is also not easily conducive to controlling for
the size of the diaphragmatic defect, which is a known predictor of
variations in the clinical course. Additionally, and expectedly, it
does not allow for conclusive interpretations in the context of the
complex signaling network directing fetal pulmonary development,
until more data on different fronts can be generated. Nevertheless,
it does show that TRASCET can affect fundamental aspects of lung de-
velopment in experimental CDH, which was a necessary first step in
determining whether further scrutiny into this new strategy was at
all warranted. Our results show that it certainly is. We are currently
pursuing it. Pending the aforementioned further scrutiny,
transamniotic stem cell therapy might become a new addition to
the armamentarium for the prenatal management of CDH.
Acknowledgments
This work was supported by the Kevin and Kate McCarey Fund for
Surgical Research at Boston Children's Hospital. AVC was supported by
the Chairman's Research Fellowship Fund from the Department of
Surgery.
Please cite this article as: A.V. Chalphin, S.A. Tracy, S.P. Lazow, et al., Congenital Diaphragmatic Hernia as a Potential Target for Transamniotic Stem
Cell Therapy, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2019.10.033
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