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Transamniotic stem cell therapy (TRASCET) mitigates bowel damage in a model of gastroschisis
Journal of Pediatric Surgery 51 (2016) 56–61
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Transamniotic stem cell therapy (TRASCET) mitigates bowel damage in a model of gastroschisis☆ Christina Feng, Christopher D. Graham, John Patrick Connors, Joseph Brazzo III, Amy H.S. Pan, James R. Hamilton, David Zurakowski, Dario O. Fauza ⁎ Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA
a r t i c l e
i n f o
Article history: Received 19 September 2015 Accepted 6 October 2015 Key words: Amniotic mesenchymal stem cells Gastroschisis Fetal therapy Fetal stem cells Regenerative medicine
a b s t r a c t Purpose: We sought to determine whether intraamniotic delivery of concentrated amniotic-derived mesenchymal stem cells (afMSCs) could reduce damage to exposed bowel in experimental gastroschisis. Methods: Rat fetuses (n = 117) with surgically created gastroschisis were divided into three groups: untreated animals (n = 62) and two groups receiving volume-matched intraamniotic injections of either saline (n = 25) or 2 × 106 cells/mL of syngeneic, labeled afMSCs (n = 30). Animals were killed before term, along with normal controls (NL). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thicknesses. Statistical comparisons were by ANOVA (P b 0.05). Results: Among survivors with gastroschisis, there were statistically significant decreases in total bowel wall, serosal, muscular, and mucosal thicknesses in the afMSC group vs. the untreated group (P = 0.001/0.035/0.001/ 0.005, respectively) and vs. the saline group (P = 0.003/0.05/b 0.001/0.026, respectively). There were no such significant differences between the untreated and saline groups. There were no differences between the afMSC group and NL, except for a significantly thicker muscular layer in the afMSC group (P = 0.014). Labeled afMSCs were scarcely identified, suggesting a paracrine effect. Conclusions: Amniotic mesenchymal stem cells mitigate bowel damage in experimental gastroschisis after concentrated intraamniotic injection. Transamniotic stem cell therapy (TRASCET) may become a practical component of the treatment of gastroschisis. © 2016 Elsevier Inc. All rights reserved.
Morbidity from gastroschisis stems primarily from bowel damage sustained during prolonged intrauterine exposure to amniotic fluid, meconium and other waste products, as well as from vascular constriction and ischemia of the eviscerated bowel. This exposure leads to intestinal wall thickening, edema, chronic inflammation, and often a fibrous peel. Functional consequences of such an insult are the norm, with lack of peristalsis and a very slow return to normal gastrointestinal function practically always present in the neonatal period and beyond. The intensity of the functional/clinical manifestations correlates with the intensity of the structural damage. A number of prenatal strategies have been proposed in order to minimize intestinal damage and improve intestinal function in gastroschisis. To date, either clinically or only experimentally, these include early delivery, amnioexchange, amniotic fluid dilution, prenatal steroid administration, induced fetal diuresis, and even intrauterine
☆ Level of Evidence: N/A (Animal and Laboratory Study). ⁎ 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: [email protected] (D.O. Fauza). http://dx.doi.org/10.1016/j.jpedsurg.2015.10.011 0022-3468/© 2016 Elsevier Inc. All rights reserved.
repair of the defect [1–4]. To our knowledge a cell-based approach had yet to be described. Mesenchymal stem cells (MSCs) have been shown to minimize tissue damage and possess anti-inflammatory effects in a variety of settings [5]. In this study, we sought to determine whether simple intraamniotic delivery of large amounts of amniotic fluid-derived MSCs (afMSCs) could lessen bowel damage in a rodent model of gastroschisis. 1. Methods This study was approved by the Boston Children's Hospital Institutional Animal Care and Use Committee under protocol #14-06-2735. 1.1. Amniotic fluid procurement and donor afMSC processing Donor afMSCs consisted of previously banked cells from normal syngeneic Lewis rat dams that served purely as amniotic fluid donors on gestational day 21 (E21; term = 21–22 days), isolated and expanded based on the methods as we have previously described [6,7]. Fluorescence-activated cell sorting analysis was used to confirm their mesenchymal progenitor identity with unconjugated mouse
C. Feng et al. / Journal of Pediatric Surgery 51 (2016) 56–61
monoclonal antibodies previously validated for use in rats, namely for CD29 (BD Pharmingen, San Diego, CA); CD73 (BD Pharmingen); CD44 (R&D Systems, Minneapolis, MN); CD90 (BD Pharmingen); and CD45 (Molecular Probes Life Technologies, Grand Island, NY), using the Vantage SE cell sorter (BD Biosciences). A mouse isotype immunoglobulin control was used to exclude nonspecific staining. Cells were labeled by fluorescent nanocrystal technology using the Qtracker® cell labeling kit 525 (Life Technologies, Chicago, IL) emitting a wavelength of 525 nm with excitation at 405–485 nm, as per the manufacturer's instructions [8]. Green fluorescence was observed in 80–90% of cells postincubation using an EVOS® FL Color Imaging System microscope fitted with an on-board computer and integrated imaging software (Life Technologies, Carlsbad, CA).
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with an on-board computer and integrated imaging software (Life Technologies). Quantitative histomorphologic measurements previously validated as surrogates of bowel damage in the rat model of gastroschisis were performed [2,3]. Specifically, the total thickness of the bowel wall as well as the thicknesses of individual intestinal layers – mucosa, muscularis, and serosa – was separately measured in pixels at 200 × magnification using Image J software (NIH, Bethesda, MD) on at least four cardinal sections of axial slides of the intestine, by two examiners blinded to the groups. One to 12 eviscerated bowel loops per fetus were individually analyzed, based on the extent of bowel exposed to the amniotic cavity, which is quite variable in this model. Screening for labeled cell engraftment was performed in unstained slides using an EVOS® FL Color Imaging System microscope fitted with an onboard computer and integrated imaging software (Life Technologies).
1.2. Gastroschisis creation and intraamniotic injections 1.4. Statistical analysis Twenty-three time-dated pregnant Sprague–Dawley dams (Charles River Laboratories, Inc., Wilmington, MA) were fed a normal diet ad libitum and housed individually under standard dark/light cycling conditions. All dams underwent surgery for creation of a gastroschisis defect on day 17.5–18.5 of gestation, using methods adapted from Correia-Pinto et al. [9]. Briefly, anesthesia was induced and maintained with isoflurane (Abbot Laboratories, North Chicago, IL), chamber inhaled at 2–4% in 100% oxygen. Using sterile technique, a large midline laparotomy was performed and the bicornuate uterus was exposed. Under 2.5× magnification, a 6–0 polypropylene purse-string suture incorporating the amniotic membranes was placed over a fetal hind limb and a 5 mm hysterotomy was made with an 11-blade within the suture. The hind limb was exteriorized, keeping the remainder of the fetus within the amniotic sac. A 2 mm full thickness abdominal defect was created on either the right or left lower abdominal quadrant using an 18G needle followed by forceps dilation. Bowel loops were then eviscerated by gently applying pressure on the lower abdomen of the fetus. The partially delivered fetus was returned to the amniotic cavity and the hysterotomy closed by simply tying the purse-string suture. A total of 117 fetuses underwent creation of such a defect. During the same procedure, fetuses were divided into three groups. One group underwent no further manipulation (untreated group, n = 62). One group received an intraamniotic injection of 100 μL of plain phosphate buffered saline (PBS-saline group, n = 25), and one group received an intraamniotic injection of 100 μL of a suspension of the labeled syngeneic afMSCs, at 2 × 10 6 cells/mL in PBS (afMSC group, n = 30). All injections were performed via a 33G noncoring needle on a 100 μL syringe (both from Hamilton Company, Reno, NV) introduced into the amniotic cavity by the ventral aspect of the manipulated fetus, carefully avoiding it and the umbilical cord. Up to 8 fetuses per dam were manipulated. Fetuses that did not undergo gastroschisis creation were used as normal controls (further details below). The uterus was returned to the abdomen and the incision closed in two layers with 3–0 Vicryl (Ethicon, Somerville, NJ) and 5–0 Monocryl (Ethicon) simple running sutures. Animals were allowed to recover with no additional manipulations other than postoperative analgesia with buprenorphine (Reckitt and Colman Pharmaceuticals, Richmond, VA) and Meloxicam (Norbrook Inc., Lenexa, KS) as needed. 1.3. Histological analysis All dams were euthanized with chamber-inhaled carbon dioxide on E21.5. A midline incision was made and the uterus was eviscerated. The amniotic cavity was incised and the fetus was examined. In fetuses with gastroschisis, the eviscerated bowel was excised en bloc, fixed in 4% formaldehyde, paraffin-embedded, and then processed for histology with hematoxylin–eosin (H&E) staining. In the normal controls, representative samples of the intestine were randomly procured and processed for histology in like manner. All histological analyses were performed using an EVOS® XL Core Imaging System microscope fitted
Total and individual intestinal wall layer thicknesses were compared among multiple bowel loops from the four groups (normal, untreated, saline, afMSC) using nested analysis of variance (ANOVA). A generalized estimating equations (GEE) approach with repeated measures was applied to account for multiple measurements within the same fetuses (i.e. correlated data) and the Wald test was used for determining statistical significance. Continuous variables followed a normal distribution closely, as assessed by the Kolmogorov–Smirnov goodness-of-fit test, and therefore results are presented as the mean with variability indicated by the standard error of the mean (SEM). Power analysis indicated that the sample sizes of the four groups provided 80% power to detect mean differences of 50 pixels or more in wall thickness of the different layers based on ANOVA (version 7.0, nQuery Advisor, Statistical Solutions, Saugus, MA). Two-tailed values of P b 0.05 were considered statistically significant. Data analysis was performed using IBM SPSS software (SPSS version 21.0, IBM, Armonk, NY). 2. Results Among the surgically manipulated fetuses, a total of 30 were still viable at euthanasia. A gastroschisis was present in 50% (15/30) of them, distributed as follows: 7/62 (11%) in the untreated group; 4/25 (16%) in the saline group; and 4/30 (13%) in the afMSC group. A total of 122 bowel loops were compared, which were not significantly different across the groups in the median number and range per fetus (Kruskal–Wallis test = 0.68, p = 0.88). ANOVA also confirmed no significant group differences in the mean and standard deviation of the number of bowel loops per fetus (F = 0.44, P = 0.72). Specifically, the mean ± SD, median and range of bowel loops per fetus for each group were distributed as follows: untreated group: n = 41, mean 4.2 ± 2.6, median 4, range 1–10; saline group: n = 26, mean 4.1 ± 2.4, median 4, range 1–9; afMSC group: n = 25, mean 4.9 ± 3.2, median 4, range 1–12; normal control group: n = 30, mean 4.4 ± 2.5, median = 4, range 1–10. On gross inspection, the eviscerated bowel loops of fetuses with gastroschisis in the untreated and saline groups seemed grossly characteristic of what had been previously described in this model, with a leathery or matted appearance, dilated, and exhibiting a thick peel. The exposed bowel loops in the afMSC group seemed to be somewhat less affected grossly (Fig. 1). There were statistically significant decreases in the thickness of the total bowel wall (P = 0.001), the serosa (P = 0.035), the muscularis (P = 0.001), and the mucosa (P = 0.005) between the afMSC group and the untreated group (Figs. 2 and 3). Total bowel wall thickness (P = 0.003), serosal (P = 0.05), muscularis (P b 0.001), and mucosal (P = 0.026) layer thicknesses were also significantly less in the afMSC group compared to the saline group (Fig. 3). There were no significant differences in mean intestinal wall thickness between the untreated and the saline groups – total bowel wall (P = 0.206), serosal layer
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Fig. 1. Representative gross views of rat fetuses with gastroschisis at euthanasia. The exposed intestine in the (A) untreated and (B) saline groups seemed heterogeneous, edematous, and thickened. The intestine in the (C) afMSC group appeared somewhat more homogenous and less affected macroscopically.
(P = 0.422), muscularis (P = 0.335), and mucosa (P = 0.093). The serosal (P = 0.188), mucosal (P = 0.382), and total intestinal (P = 0.064) layer thicknesses were similar between the afMSC and the normal control groups, but the muscularis was thicker in the afMSC group than in the normal controls (P = 0.014) (Fig. 3). Labeled cells were identified in the afMSC group on fluorescence microscopy, albeit scarcely and seemingly occupying different layers of the intestinal wall (Fig. 4). 3. Discussion Although the mortality of newborns with gastroschisis has long decreased considerably, morbidity rates remain high. Protracted and
costly hospitalizations, long-term parenteral nutrition, infectious and other complications are still commonplace. It is well established that the intestinal inflammation and damage in gastroschisis are largely derived from direct, prolonged exposure of the bowel to the amniotic fluid, particularly during the third trimester of pregnancy. Few strategies currently exist in clinical practice that can reliably prevent prenatal intestinal damage, or have shown to significantly reduce postnatal morbidity in gastroschisis. The premise that preterm delivery may prevent further intestinal injury seen late in gestation has not proven more effective than spontaneous delivery close to term [10]. Additionally, amnioinfusion in the third trimester seems to be of limited efficacy in improving perinatal outcomes clinically [11].
Fig. 2. Representative histological views of (A) normal rat fetus intestine and of the exposed intestine in (B) untreated, (C) saline, and (D) afMSC groups. The total bowel wall, serosal, muscular, and mucosal thicknesses appeared different in the afMSC group when compared with the untreated and saline groups, which were virtually indistinguishable between each other. H&E, 200× magnification.
C. Feng et al. / Journal of Pediatric Surgery 51 (2016) 56–61 250
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Fig. 3. The mean total and segmental thicknesses of the intestinal wall were compared across groups. There were significant decreases in serosal, muscular, and mucosal layer thicknesses and in total bowel wall thickness in the afMSC group vs. the untreated and saline groups. There were no such differences between the untreated and saline groups. There were no differences between the afMSC group and normal controls, except for a significantly thicker muscular layer in the afMSC group.
Several experimental studies have focused on alternative strategies for decreasing the intestinal injury resulting from the inflammatory process, perhaps most notably intraamniotic and intraperitoneal steroid administration. Prenatal administration of steroids, however, is not without risks, as prolonged administration may lead to deterioration in maternal cardiovascular and metabolic status, as well as have deleterious effects on the fetus and the placenta. In this study, we start to explore a different, as of yet untested strategy, based on (feasibly autologous) cells induced to amplify a role that they already play in nature. More specifically, we have previously shown that afMSCs play a central role in normal fetal wound healing, a process which is more akin to regeneration than to basic repair [12]. By boosting afMSC numbers in the amniotic fluid via the cell injections performed, we could arguably be simply enhancing an activity already present in the setting of gastroschisis and potentially in the setting of other exposed fetal defects as well. In point of fact, we have recently shown that the concept of afMSC-based transamniotic stem cell therapy (TRASCET) can also be of therapeutic benefit in a model of spina bifida [13,14].
It has been widely shown that MSCs possess anti-inflammatory and immunomodulatory capabilities. While the TRASCET paradigm is novel, numerous experimental and even a few clinical studies have previously uncovered the benefits of MSCs from different sources, administered locally and/or systemically, in lung injury, articular damage, cardiac repair, inflammatory bowel disease, graft-versus-host-disease, and a number of other pathological processes [15,16]. At the same time, despite a few isolated insights, much remains to be learned about the mechanisms underlying MSC homing and effects in tissue repair and inflammation [5]. Interestingly, unlike our findings in the spina bifida model of TRASCET, we did not document sustained, robust engraftment of nanocrystal-labeled afMSCs within the intestinal wall layers in this gastroschisis model. Rather, cell engraftment seemed scarce, suggesting a paracrine effect. This finding is in accordance with that from a study on the effects of intraperitoneal administration of these cells in a model of necrotizing enterocolitis [17]. Importantly, in the present study, cells necessarily had to be injected concomitantly with the creation of the defect (one of the limitations of this rodent model), therefore there was no bowel damage at that time, which may explain such sparse engraftment
Fig. 4. Labeled donor cells were identified in the afMSC group on fluorescence microscopy sparsely occupying different, mostly outer layers of the intestinal wall.
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given that MSCs tend to home preferably to areas of tissue damage. Perhaps understandably, an in depth mechanistic analysis of how afMSCs may promote mitigation of intestinal damage in the setting of gastroschisis is beyond the scope of a first study such as the present one, in that the very presence of such an effect would first have to be scrutinized, as we have done. From a translational perspective, the use of afMSCs is ideally suited to prenatal therapeutic strategies such as TRASCET. The procurement of these cells requires only a simple amniocentesis, which is not only much less invasive than other forms of fetal cell procurement, it is also routinely indicated in the presence of fetal anomalies for diagnostic purposes, therefore bypassing the ethical limitations of using most other (mesenchymal or not) stem cells. In addition, afMSCs have been shown to proliferate faster and respond better to hypoxic conditions than other stem cell lines in vitro [18,19]. This would allow for procurement, expansion in vitro and intraamniotic administration one or more times beginning fairly early in gestation, possibly enhancing outcomes. Different animal models of gastroschisis have been described [3,9]. The rat model has been validated and appears to mimic the pathophysiology of human gastroschisis [9]. It has benefits over large animal models, including minimal expense, short gestation, and higher number of fetuses per litter, thus being conducive to an initial attempt at a novel therapeutic approach, as it was the case here. Limitations of this model include high fetal mortality, which can be somewhat minimized after a steep learning curve, the inability to conduct detailed motility and most functional studies postnatally, along with difficulties in repeating interventions, such as performing intraamniotic injections sometime after the defect has been created, or more than one intraamniotic cell delivery on a given animal. Still, it is an ideal model for a proof of principle study such as this one. Next steps from our group will include large animal models in parallel with continued work on the rat model, which remains particularly helpful for mechanistic analyses. Experimental limitations notwithstanding, our results show that afMSCs can mitigate bowel damage in experimental gastroschisis after simple concentrated intraamniotic injection. TRASCET may become a practical component of the treatment of gastroschisis. Author contributions CF: data collection, provision of study material, data analysis, manuscript writing. CDG: data collection, provision of study material, data analysis. JPC: data collection. JB: provision of study material. AHSP: provision of study material. JRH: data collection. DZ: data analysis, manuscript writing. DOF: conception, design, data collection, data analysis, manuscript writing. Appendix A. Discussions
the exposure to amniotic fluid really also shows this histological difference in intestinal damage and the presence of this fibrous peel which is really what we were targeting with our assessment. SHAUN KUNISAKI (Ann Arbor, MI) I have two questions. Very nice study.
First question is, were there any differences in mortality across the three groups? I know the overall mortality was pretty high. The second question was, I wanted to get your thoughts in terms of whether you envision this ultimately being an autologous or an allogeneic type of application because at least in theory one may be concerned that using this autologous that the amniotic fluid mesenchymal stem cells may be primed or somehow different in a gastroschisis baby as opposed to a normal child. CHRISTINA FENG Thank you for your questions. In terms of the mortality, we did not see any difference in mortality across the three groups. Of course because of the overall low survival, our numbers were fairly low, but we did compare that and we did not see a difference. Now I argue if we did see a difference it may be more directly related to the actual procedure of creating gastroschisis.
In terms of envisioning further down the line how this would translate into clinical practice, we do envision the use of autologous mesenchymal stem cells mainly for the reason that there could be perhaps a difference in the cells that are present in the fetuses with gastroschisis, but these cells could be procured as early as whenever a routine amniocentesis is performed. With very limited quantity of fluid that is procured, we can isolate these cells and either bank them or grow them up in culture in parallel with gestation. Diagnosis of gastroschisis could be made later on but we can still use the previously banked cells or procured cells in this therapy. ALLAN GOLDSTEIN (Boston, MA) I was just curious. Your control group was saline injected as opposed to a cell injection. Is it possible that the mesenchymal stem cells lead to either local or systemic immune reaction and therefore you are seeing an indirect benefit? CHRISTINA FENG Absolutely. That is actually part of some our ongoing studies looking at the mechanism of what these cells are doing within that amniotic fluid environment. We know a few unique characteristics of these cells is that they do home to areas of injury and that the presence of these cells, they are well known to have immunomodulatory and antiinflammatory capabilities. One of our hypotheses for some of our future studies is that because of the presence of these cells they may be creating a more anti-inflammatory environment and really reducing the inflammation and intestinal damage that we see from the proinflammatory states. ALLAN GOLDSTEIN I might encourage you to use a cell type rather than saline as a more reasonable control for the experiment. CHRISTINA FENG Absolutely. That could be a potential future avenue of study.
Presented by Christina Feng, Boston, MA GEORGE MYCHALISKA (Ann Arbor, MI) Christina, that was an excellent study. It is very interesting. I have one question for you. You showed in your introductory slide Dr. Langer's paper where he suggested that the pathophysiology is not just exposure to fluid but constriction and I think we've all seen the problems with constriction. Your model does not really address that. Is that correct? CHRISTINA FENG Definitely. In this animal model, because of the rodent model and the size of the fetuses, we do not have a component of the constriction to contribute to the damage seen in our model of gastroschisis. What they have shown in that study is that also the present – or in subsequent studies that
References [1] Yu J, Gonzalez-Reyes S, Diez-Pardo JA, et al. Effects of prenatal dexamethasone on the intestine of rats with gastroschisis. J Pediatr Surg 2003;38(7):1032–5. [2] Hakguder G, Olguner M, Gurel D, et al. Induction of fetal diuresis with intraamniotic furosemide injection reduces intestinal damage in a rat model of gastroschisis. Eur J Pediatr Surg 2011;21(3):183–7. [3] Bittencourt DG, Barreto MW, Franca WM, et al. Impact of corticosteroid on intestinal injury in a gastroschisis rat model: morphometric analysis. J Pediatr Surg 2006; 41(3):547–53. [4] Till H, Muensterer O, Mueller M, et al. Intrauterine repair of gastroschisis in fetal rabbits. Fetal Diagn Ther 2003;18(5):297–300. [5] Hong HS, Lee J, Lee E, et al. A new role of substance P as an injury-inducible messenger for mobilization of CD29(+) stromal-like cells. Nat Med 2009;15(4):425–35. [6] Kaviani A, Perry TE, Dzakovic A, et al. The amniotic fluid as a source of cells for fetal tissue engineering. J Pediatr Surg 2001;36(11):1662–5.
C. Feng et al. / Journal of Pediatric Surgery 51 (2016) 56–61 [7] Klein JD, Fauza DO. Amniotic and placental mesenchymal stem cell isolation and culture. Methods Mol Biol 2011;698:75–88. [8] Muller-Borer BJ, Collins MC, Gunst PR, et al. Quantum dot labeling of mesenchymal stem cells. J Nanobiotechnol 2007;5:1–9. [9] Correia-Pinto J, Tavares ML, Baptista MJ, et al. A new fetal rat model of gastroschisis: development and early characterization. J Pediatr Surg 2001;36(1):213–6. [10] Logghe HL, Mason GC, Thornton JG, et al. A randomized controlled trial of elective preterm delivery of fetuses with gastroschisis. J Pediatr Surg 2005;40(11): 1726–31. [11] Luton D, de Lagausie P, Guibourdenche J, et al. Effect of amnioinfusion on the outcome of prenatally diagnosed gastroschisis. Fetal Diagn Ther 1999;14(3): 152–5. [12] Klein JD, Turner CG, Steigman SA, et al. Amniotic mesenchymal stem cells enhance normal fetal wound healing. Stem Cells Dev 2011;20(6):969–76. [13] Dionigi B, Ahmed A, Brazzo III J, et al. Partial or complete coverage of experimental spina bifida by simple intra-amniotic injection of concentrated amniotic mesenchymal stem cells. J Pediatr Surg 2015;50(1):69–73.
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[14] Dionigi B, Brazzo III J, Ahmed A, et al. Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida. J Pediatr Surg 2015;50(6): 1037–41. [15] Barry F, Murphy M. Mesenchymal stem cells in joint disease and repair. Nat Rev Rheumatol 2013;9(10):584–94. [16] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 2007;25(11):2896–902. [17] Zani A, Cananzi M, Fascetti-Leon F, et al. Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism. Gut 2014;63(2):300–9. [18] Dionigi B, Ahmed A, Pennington EC, et al. A comparative analysis of human mesenchymal stem cell response to hypoxia in vitro: implications to translational strategies. J Pediatr Surg 2014;49(6):915–8. [19] Kunisaki SM, Fuchs JR, Steigman SA, et al. A comparative analysis of cartilage engineered from different perinatal mesenchymal progenitor cells. Tissue Eng 2007;13(11):2633–44.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Transamniotic stem cell therapy (TRASCET) mitigates bowel damage in a model of gastroschisis☆ Christina Feng, Christopher D. Graham, John Patrick Connors, Joseph Brazzo III, Amy H.S. Pan, James R. Hamilton, David Zurakowski, Dario O. Fauza ⁎ Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA
a r t i c l e
i n f o
Article history: Received 19 September 2015 Accepted 6 October 2015 Key words: Amniotic mesenchymal stem cells Gastroschisis Fetal therapy Fetal stem cells Regenerative medicine
a b s t r a c t Purpose: We sought to determine whether intraamniotic delivery of concentrated amniotic-derived mesenchymal stem cells (afMSCs) could reduce damage to exposed bowel in experimental gastroschisis. Methods: Rat fetuses (n = 117) with surgically created gastroschisis were divided into three groups: untreated animals (n = 62) and two groups receiving volume-matched intraamniotic injections of either saline (n = 25) or 2 × 106 cells/mL of syngeneic, labeled afMSCs (n = 30). Animals were killed before term, along with normal controls (NL). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thicknesses. Statistical comparisons were by ANOVA (P b 0.05). Results: Among survivors with gastroschisis, there were statistically significant decreases in total bowel wall, serosal, muscular, and mucosal thicknesses in the afMSC group vs. the untreated group (P = 0.001/0.035/0.001/ 0.005, respectively) and vs. the saline group (P = 0.003/0.05/b 0.001/0.026, respectively). There were no such significant differences between the untreated and saline groups. There were no differences between the afMSC group and NL, except for a significantly thicker muscular layer in the afMSC group (P = 0.014). Labeled afMSCs were scarcely identified, suggesting a paracrine effect. Conclusions: Amniotic mesenchymal stem cells mitigate bowel damage in experimental gastroschisis after concentrated intraamniotic injection. Transamniotic stem cell therapy (TRASCET) may become a practical component of the treatment of gastroschisis. © 2016 Elsevier Inc. All rights reserved.
Morbidity from gastroschisis stems primarily from bowel damage sustained during prolonged intrauterine exposure to amniotic fluid, meconium and other waste products, as well as from vascular constriction and ischemia of the eviscerated bowel. This exposure leads to intestinal wall thickening, edema, chronic inflammation, and often a fibrous peel. Functional consequences of such an insult are the norm, with lack of peristalsis and a very slow return to normal gastrointestinal function practically always present in the neonatal period and beyond. The intensity of the functional/clinical manifestations correlates with the intensity of the structural damage. A number of prenatal strategies have been proposed in order to minimize intestinal damage and improve intestinal function in gastroschisis. To date, either clinically or only experimentally, these include early delivery, amnioexchange, amniotic fluid dilution, prenatal steroid administration, induced fetal diuresis, and even intrauterine
☆ Level of Evidence: N/A (Animal and Laboratory Study). ⁎ 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: [email protected] (D.O. Fauza). http://dx.doi.org/10.1016/j.jpedsurg.2015.10.011 0022-3468/© 2016 Elsevier Inc. All rights reserved.
repair of the defect [1–4]. To our knowledge a cell-based approach had yet to be described. Mesenchymal stem cells (MSCs) have been shown to minimize tissue damage and possess anti-inflammatory effects in a variety of settings [5]. In this study, we sought to determine whether simple intraamniotic delivery of large amounts of amniotic fluid-derived MSCs (afMSCs) could lessen bowel damage in a rodent model of gastroschisis. 1. Methods This study was approved by the Boston Children's Hospital Institutional Animal Care and Use Committee under protocol #14-06-2735. 1.1. Amniotic fluid procurement and donor afMSC processing Donor afMSCs consisted of previously banked cells from normal syngeneic Lewis rat dams that served purely as amniotic fluid donors on gestational day 21 (E21; term = 21–22 days), isolated and expanded based on the methods as we have previously described [6,7]. Fluorescence-activated cell sorting analysis was used to confirm their mesenchymal progenitor identity with unconjugated mouse
C. Feng et al. / Journal of Pediatric Surgery 51 (2016) 56–61
monoclonal antibodies previously validated for use in rats, namely for CD29 (BD Pharmingen, San Diego, CA); CD73 (BD Pharmingen); CD44 (R&D Systems, Minneapolis, MN); CD90 (BD Pharmingen); and CD45 (Molecular Probes Life Technologies, Grand Island, NY), using the Vantage SE cell sorter (BD Biosciences). A mouse isotype immunoglobulin control was used to exclude nonspecific staining. Cells were labeled by fluorescent nanocrystal technology using the Qtracker® cell labeling kit 525 (Life Technologies, Chicago, IL) emitting a wavelength of 525 nm with excitation at 405–485 nm, as per the manufacturer's instructions [8]. Green fluorescence was observed in 80–90% of cells postincubation using an EVOS® FL Color Imaging System microscope fitted with an on-board computer and integrated imaging software (Life Technologies, Carlsbad, CA).
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with an on-board computer and integrated imaging software (Life Technologies). Quantitative histomorphologic measurements previously validated as surrogates of bowel damage in the rat model of gastroschisis were performed [2,3]. Specifically, the total thickness of the bowel wall as well as the thicknesses of individual intestinal layers – mucosa, muscularis, and serosa – was separately measured in pixels at 200 × magnification using Image J software (NIH, Bethesda, MD) on at least four cardinal sections of axial slides of the intestine, by two examiners blinded to the groups. One to 12 eviscerated bowel loops per fetus were individually analyzed, based on the extent of bowel exposed to the amniotic cavity, which is quite variable in this model. Screening for labeled cell engraftment was performed in unstained slides using an EVOS® FL Color Imaging System microscope fitted with an onboard computer and integrated imaging software (Life Technologies).
1.2. Gastroschisis creation and intraamniotic injections 1.4. Statistical analysis Twenty-three time-dated pregnant Sprague–Dawley dams (Charles River Laboratories, Inc., Wilmington, MA) were fed a normal diet ad libitum and housed individually under standard dark/light cycling conditions. All dams underwent surgery for creation of a gastroschisis defect on day 17.5–18.5 of gestation, using methods adapted from Correia-Pinto et al. [9]. Briefly, anesthesia was induced and maintained with isoflurane (Abbot Laboratories, North Chicago, IL), chamber inhaled at 2–4% in 100% oxygen. Using sterile technique, a large midline laparotomy was performed and the bicornuate uterus was exposed. Under 2.5× magnification, a 6–0 polypropylene purse-string suture incorporating the amniotic membranes was placed over a fetal hind limb and a 5 mm hysterotomy was made with an 11-blade within the suture. The hind limb was exteriorized, keeping the remainder of the fetus within the amniotic sac. A 2 mm full thickness abdominal defect was created on either the right or left lower abdominal quadrant using an 18G needle followed by forceps dilation. Bowel loops were then eviscerated by gently applying pressure on the lower abdomen of the fetus. The partially delivered fetus was returned to the amniotic cavity and the hysterotomy closed by simply tying the purse-string suture. A total of 117 fetuses underwent creation of such a defect. During the same procedure, fetuses were divided into three groups. One group underwent no further manipulation (untreated group, n = 62). One group received an intraamniotic injection of 100 μL of plain phosphate buffered saline (PBS-saline group, n = 25), and one group received an intraamniotic injection of 100 μL of a suspension of the labeled syngeneic afMSCs, at 2 × 10 6 cells/mL in PBS (afMSC group, n = 30). All injections were performed via a 33G noncoring needle on a 100 μL syringe (both from Hamilton Company, Reno, NV) introduced into the amniotic cavity by the ventral aspect of the manipulated fetus, carefully avoiding it and the umbilical cord. Up to 8 fetuses per dam were manipulated. Fetuses that did not undergo gastroschisis creation were used as normal controls (further details below). The uterus was returned to the abdomen and the incision closed in two layers with 3–0 Vicryl (Ethicon, Somerville, NJ) and 5–0 Monocryl (Ethicon) simple running sutures. Animals were allowed to recover with no additional manipulations other than postoperative analgesia with buprenorphine (Reckitt and Colman Pharmaceuticals, Richmond, VA) and Meloxicam (Norbrook Inc., Lenexa, KS) as needed. 1.3. Histological analysis All dams were euthanized with chamber-inhaled carbon dioxide on E21.5. A midline incision was made and the uterus was eviscerated. The amniotic cavity was incised and the fetus was examined. In fetuses with gastroschisis, the eviscerated bowel was excised en bloc, fixed in 4% formaldehyde, paraffin-embedded, and then processed for histology with hematoxylin–eosin (H&E) staining. In the normal controls, representative samples of the intestine were randomly procured and processed for histology in like manner. All histological analyses were performed using an EVOS® XL Core Imaging System microscope fitted
Total and individual intestinal wall layer thicknesses were compared among multiple bowel loops from the four groups (normal, untreated, saline, afMSC) using nested analysis of variance (ANOVA). A generalized estimating equations (GEE) approach with repeated measures was applied to account for multiple measurements within the same fetuses (i.e. correlated data) and the Wald test was used for determining statistical significance. Continuous variables followed a normal distribution closely, as assessed by the Kolmogorov–Smirnov goodness-of-fit test, and therefore results are presented as the mean with variability indicated by the standard error of the mean (SEM). Power analysis indicated that the sample sizes of the four groups provided 80% power to detect mean differences of 50 pixels or more in wall thickness of the different layers based on ANOVA (version 7.0, nQuery Advisor, Statistical Solutions, Saugus, MA). Two-tailed values of P b 0.05 were considered statistically significant. Data analysis was performed using IBM SPSS software (SPSS version 21.0, IBM, Armonk, NY). 2. Results Among the surgically manipulated fetuses, a total of 30 were still viable at euthanasia. A gastroschisis was present in 50% (15/30) of them, distributed as follows: 7/62 (11%) in the untreated group; 4/25 (16%) in the saline group; and 4/30 (13%) in the afMSC group. A total of 122 bowel loops were compared, which were not significantly different across the groups in the median number and range per fetus (Kruskal–Wallis test = 0.68, p = 0.88). ANOVA also confirmed no significant group differences in the mean and standard deviation of the number of bowel loops per fetus (F = 0.44, P = 0.72). Specifically, the mean ± SD, median and range of bowel loops per fetus for each group were distributed as follows: untreated group: n = 41, mean 4.2 ± 2.6, median 4, range 1–10; saline group: n = 26, mean 4.1 ± 2.4, median 4, range 1–9; afMSC group: n = 25, mean 4.9 ± 3.2, median 4, range 1–12; normal control group: n = 30, mean 4.4 ± 2.5, median = 4, range 1–10. On gross inspection, the eviscerated bowel loops of fetuses with gastroschisis in the untreated and saline groups seemed grossly characteristic of what had been previously described in this model, with a leathery or matted appearance, dilated, and exhibiting a thick peel. The exposed bowel loops in the afMSC group seemed to be somewhat less affected grossly (Fig. 1). There were statistically significant decreases in the thickness of the total bowel wall (P = 0.001), the serosa (P = 0.035), the muscularis (P = 0.001), and the mucosa (P = 0.005) between the afMSC group and the untreated group (Figs. 2 and 3). Total bowel wall thickness (P = 0.003), serosal (P = 0.05), muscularis (P b 0.001), and mucosal (P = 0.026) layer thicknesses were also significantly less in the afMSC group compared to the saline group (Fig. 3). There were no significant differences in mean intestinal wall thickness between the untreated and the saline groups – total bowel wall (P = 0.206), serosal layer
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Fig. 1. Representative gross views of rat fetuses with gastroschisis at euthanasia. The exposed intestine in the (A) untreated and (B) saline groups seemed heterogeneous, edematous, and thickened. The intestine in the (C) afMSC group appeared somewhat more homogenous and less affected macroscopically.
(P = 0.422), muscularis (P = 0.335), and mucosa (P = 0.093). The serosal (P = 0.188), mucosal (P = 0.382), and total intestinal (P = 0.064) layer thicknesses were similar between the afMSC and the normal control groups, but the muscularis was thicker in the afMSC group than in the normal controls (P = 0.014) (Fig. 3). Labeled cells were identified in the afMSC group on fluorescence microscopy, albeit scarcely and seemingly occupying different layers of the intestinal wall (Fig. 4). 3. Discussion Although the mortality of newborns with gastroschisis has long decreased considerably, morbidity rates remain high. Protracted and
costly hospitalizations, long-term parenteral nutrition, infectious and other complications are still commonplace. It is well established that the intestinal inflammation and damage in gastroschisis are largely derived from direct, prolonged exposure of the bowel to the amniotic fluid, particularly during the third trimester of pregnancy. Few strategies currently exist in clinical practice that can reliably prevent prenatal intestinal damage, or have shown to significantly reduce postnatal morbidity in gastroschisis. The premise that preterm delivery may prevent further intestinal injury seen late in gestation has not proven more effective than spontaneous delivery close to term [10]. Additionally, amnioinfusion in the third trimester seems to be of limited efficacy in improving perinatal outcomes clinically [11].
Fig. 2. Representative histological views of (A) normal rat fetus intestine and of the exposed intestine in (B) untreated, (C) saline, and (D) afMSC groups. The total bowel wall, serosal, muscular, and mucosal thicknesses appeared different in the afMSC group when compared with the untreated and saline groups, which were virtually indistinguishable between each other. H&E, 200× magnification.
C. Feng et al. / Journal of Pediatric Surgery 51 (2016) 56–61 250
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Fig. 3. The mean total and segmental thicknesses of the intestinal wall were compared across groups. There were significant decreases in serosal, muscular, and mucosal layer thicknesses and in total bowel wall thickness in the afMSC group vs. the untreated and saline groups. There were no such differences between the untreated and saline groups. There were no differences between the afMSC group and normal controls, except for a significantly thicker muscular layer in the afMSC group.
Several experimental studies have focused on alternative strategies for decreasing the intestinal injury resulting from the inflammatory process, perhaps most notably intraamniotic and intraperitoneal steroid administration. Prenatal administration of steroids, however, is not without risks, as prolonged administration may lead to deterioration in maternal cardiovascular and metabolic status, as well as have deleterious effects on the fetus and the placenta. In this study, we start to explore a different, as of yet untested strategy, based on (feasibly autologous) cells induced to amplify a role that they already play in nature. More specifically, we have previously shown that afMSCs play a central role in normal fetal wound healing, a process which is more akin to regeneration than to basic repair [12]. By boosting afMSC numbers in the amniotic fluid via the cell injections performed, we could arguably be simply enhancing an activity already present in the setting of gastroschisis and potentially in the setting of other exposed fetal defects as well. In point of fact, we have recently shown that the concept of afMSC-based transamniotic stem cell therapy (TRASCET) can also be of therapeutic benefit in a model of spina bifida [13,14].
It has been widely shown that MSCs possess anti-inflammatory and immunomodulatory capabilities. While the TRASCET paradigm is novel, numerous experimental and even a few clinical studies have previously uncovered the benefits of MSCs from different sources, administered locally and/or systemically, in lung injury, articular damage, cardiac repair, inflammatory bowel disease, graft-versus-host-disease, and a number of other pathological processes [15,16]. At the same time, despite a few isolated insights, much remains to be learned about the mechanisms underlying MSC homing and effects in tissue repair and inflammation [5]. Interestingly, unlike our findings in the spina bifida model of TRASCET, we did not document sustained, robust engraftment of nanocrystal-labeled afMSCs within the intestinal wall layers in this gastroschisis model. Rather, cell engraftment seemed scarce, suggesting a paracrine effect. This finding is in accordance with that from a study on the effects of intraperitoneal administration of these cells in a model of necrotizing enterocolitis [17]. Importantly, in the present study, cells necessarily had to be injected concomitantly with the creation of the defect (one of the limitations of this rodent model), therefore there was no bowel damage at that time, which may explain such sparse engraftment
Fig. 4. Labeled donor cells were identified in the afMSC group on fluorescence microscopy sparsely occupying different, mostly outer layers of the intestinal wall.
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given that MSCs tend to home preferably to areas of tissue damage. Perhaps understandably, an in depth mechanistic analysis of how afMSCs may promote mitigation of intestinal damage in the setting of gastroschisis is beyond the scope of a first study such as the present one, in that the very presence of such an effect would first have to be scrutinized, as we have done. From a translational perspective, the use of afMSCs is ideally suited to prenatal therapeutic strategies such as TRASCET. The procurement of these cells requires only a simple amniocentesis, which is not only much less invasive than other forms of fetal cell procurement, it is also routinely indicated in the presence of fetal anomalies for diagnostic purposes, therefore bypassing the ethical limitations of using most other (mesenchymal or not) stem cells. In addition, afMSCs have been shown to proliferate faster and respond better to hypoxic conditions than other stem cell lines in vitro [18,19]. This would allow for procurement, expansion in vitro and intraamniotic administration one or more times beginning fairly early in gestation, possibly enhancing outcomes. Different animal models of gastroschisis have been described [3,9]. The rat model has been validated and appears to mimic the pathophysiology of human gastroschisis [9]. It has benefits over large animal models, including minimal expense, short gestation, and higher number of fetuses per litter, thus being conducive to an initial attempt at a novel therapeutic approach, as it was the case here. Limitations of this model include high fetal mortality, which can be somewhat minimized after a steep learning curve, the inability to conduct detailed motility and most functional studies postnatally, along with difficulties in repeating interventions, such as performing intraamniotic injections sometime after the defect has been created, or more than one intraamniotic cell delivery on a given animal. Still, it is an ideal model for a proof of principle study such as this one. Next steps from our group will include large animal models in parallel with continued work on the rat model, which remains particularly helpful for mechanistic analyses. Experimental limitations notwithstanding, our results show that afMSCs can mitigate bowel damage in experimental gastroschisis after simple concentrated intraamniotic injection. TRASCET may become a practical component of the treatment of gastroschisis. Author contributions CF: data collection, provision of study material, data analysis, manuscript writing. CDG: data collection, provision of study material, data analysis. JPC: data collection. JB: provision of study material. AHSP: provision of study material. JRH: data collection. DZ: data analysis, manuscript writing. DOF: conception, design, data collection, data analysis, manuscript writing. Appendix A. Discussions
the exposure to amniotic fluid really also shows this histological difference in intestinal damage and the presence of this fibrous peel which is really what we were targeting with our assessment. SHAUN KUNISAKI (Ann Arbor, MI) I have two questions. Very nice study.
First question is, were there any differences in mortality across the three groups? I know the overall mortality was pretty high. The second question was, I wanted to get your thoughts in terms of whether you envision this ultimately being an autologous or an allogeneic type of application because at least in theory one may be concerned that using this autologous that the amniotic fluid mesenchymal stem cells may be primed or somehow different in a gastroschisis baby as opposed to a normal child. CHRISTINA FENG Thank you for your questions. In terms of the mortality, we did not see any difference in mortality across the three groups. Of course because of the overall low survival, our numbers were fairly low, but we did compare that and we did not see a difference. Now I argue if we did see a difference it may be more directly related to the actual procedure of creating gastroschisis.
In terms of envisioning further down the line how this would translate into clinical practice, we do envision the use of autologous mesenchymal stem cells mainly for the reason that there could be perhaps a difference in the cells that are present in the fetuses with gastroschisis, but these cells could be procured as early as whenever a routine amniocentesis is performed. With very limited quantity of fluid that is procured, we can isolate these cells and either bank them or grow them up in culture in parallel with gestation. Diagnosis of gastroschisis could be made later on but we can still use the previously banked cells or procured cells in this therapy. ALLAN GOLDSTEIN (Boston, MA) I was just curious. Your control group was saline injected as opposed to a cell injection. Is it possible that the mesenchymal stem cells lead to either local or systemic immune reaction and therefore you are seeing an indirect benefit? CHRISTINA FENG Absolutely. That is actually part of some our ongoing studies looking at the mechanism of what these cells are doing within that amniotic fluid environment. We know a few unique characteristics of these cells is that they do home to areas of injury and that the presence of these cells, they are well known to have immunomodulatory and antiinflammatory capabilities. One of our hypotheses for some of our future studies is that because of the presence of these cells they may be creating a more anti-inflammatory environment and really reducing the inflammation and intestinal damage that we see from the proinflammatory states. ALLAN GOLDSTEIN I might encourage you to use a cell type rather than saline as a more reasonable control for the experiment. CHRISTINA FENG Absolutely. That could be a potential future avenue of study.
Presented by Christina Feng, Boston, MA GEORGE MYCHALISKA (Ann Arbor, MI) Christina, that was an excellent study. It is very interesting. I have one question for you. You showed in your introductory slide Dr. Langer's paper where he suggested that the pathophysiology is not just exposure to fluid but constriction and I think we've all seen the problems with constriction. Your model does not really address that. Is that correct? CHRISTINA FENG Definitely. In this animal model, because of the rodent model and the size of the fetuses, we do not have a component of the constriction to contribute to the damage seen in our model of gastroschisis. What they have shown in that study is that also the present – or in subsequent studies that
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