Background

Despite advances in prenatal repair, myelomeningocele (MMC) still produces devastating neurologic deficits. The amniotic membranes (AM) are a biologically active tissue that has been used anecdotally for human fetal MMC repair. This study evaluated the use of autologous AM compared to skin closure in an established fetal MMC model.

Methods

Seven fetal lambs underwent surgical creation of MMC at gestational age of 75days followed by in utero repair at gestational age of 100days. Lambs were repaired with an autologous AM patch followed by skin closure (n=4) or skin closure alone (n=3). Gross necropsy and histopathology of the spinal cords were performed at term to assess neuronal preservation at the lesion.

Results

An increase in preserved motor neurons and a larger area of spinal cord tissue were seen in AM-repaired lambs, as was decreased wound healing of the overlying skin. Loss of nearly all spinal cord tissue with limited motor neuron preservation was seen in skin only-repaired lambs.

Conclusions

AM-repaired lambs showed increased protection of spinal cord tissue compared to skin only-repaired lambs, but the overlying skin failed to close in AM-repaired lambs. These results suggest a potential role for AM in fetal MMC repair that warrants further study.

Introduction

Fetal amniotic membranes (FM) have been shown to preserve spinal cord histology in the fetal sheep model of myelomeningocele (MMC). This study compares the effectiveness of placenta-derived mesenchymal stromal cells (PMSCs) from early-gestation versus term-gestation placenta to augment FM repair to improve distal motor function in a sheep model.

Methods

Fetal lambs (n = 4) underwent surgical MMC creation followed by repair with FM patch with term-gestation PMSCs (n = 1), FM with early-gestation PMSCs (n = 1), FM only (n = 1), and skin closure only (n = 1). Histopathology and motor assessment was performed.

Results

Histopathologic analysis demonstrated increased preservation of spinal cord architecture and large neurons in the lamb repaired with early-gestation cells compared to all others. Lambs repaired with skin closure only, FM alone, and term-gestation PMSCs exhibited extremely limited distal motor function; the lamb repaired with early-gestation PMSCs was capable of normal ambulation.

Discussion

This pilot study is the first in vivo comparison of different gestational-age placenta-derived stromal cells for repair in the fetal sheep MMC model. The preservation of large neurons and markedly improved motor function in the lamb repaired with early-gestation cells suggest that early-gestation placental stromal cells may exhibit unique properties that augment in utero MMC repair to improve paralysis.

Hirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

Unlabelled

Myelomeningocele (MMC)-commonly known as spina bifida-is a congenital birth defect that causes lifelong paralysis, incontinence, musculoskeletal deformities, and severe cognitive disabilities. The recent landmark Management of Myelomeningocele Study (MOMS) demonstrated for the first time in humans that in utero surgical repair of the MMC defect improves lower limb motor function, suggesting a capacity for improved neurologic outcomes in this disorder. However, functional recovery was incomplete, and 58% of the treated children were unable to walk independently at 30 months of age. In the present study, we demonstrate that using early gestation human placenta-derived mesenchymal stromal cells (PMSCs) to augment in utero repair of MMC results in significant and consistent improvement in neurologic function at birth in the rigorous fetal ovine model of MMC. In vitro, human PMSCs express characteristic MSC markers and trilineage differentiation potential. Protein array assays and enzyme-linked immunosorbent assay show that PMSCs secrete a variety of immunomodulatory and angiogenic cytokines. Compared with adult bone marrow MSCs, PMSCs secrete significantly higher levels of brain-derived neurotrophic factor and hepatocyte growth factor, both of which have known neuroprotective capabilities. In vivo, functional and histopathologic analysis demonstrated that human PMSCs mediate a significant, clinically relevant improvement in motor function in MMC lambs and increase the preservation of large neurons within the spinal cord. These preclinical results in the well-established fetal ovine model of MMC provide promising early support for translating in utero stem cell therapy for MMC into clinical application for patients.

Significance

This study presents placenta-derived mesenchymal stromal cell (PMSC) treatment as a potential therapy for myelomeningocele (MMC). Application of PMSCs can augment current in utero surgical repair in the well-established and rigorously applied fetal lamb model of MMC. Treatment with human PMSCs significantly and dramatically improved neurologic function and preserved spinal cord neuron density in experimental animals. Sixty-seven percent of the PMSC-treated lambs were able to ambulate independently, with two exhibiting no motor deficits whatsoever. In contrast, none of the lambs treated with the vehicle alone were capable of ambulation. The locomotor rescue demonstrated in PMSC-treated lambs indicates great promise for future clinical trials to improve paralysis in children afflicted with MMC.

Introduction

The ovine model is the gold standard large animal model of myelomeningocele (MMC); however, it has a high rate of fetal loss. We reviewed our experience with the model to determine risk factors for fetal loss.

Methods

We performed a retrospective review from 2009 to 2018 to identify operative factors associated with fetal loss (early fetal demise, abortion, or stillbirth). Operative risk factors included gestational age at operation, operative time, reduction of multiple gestations, amount of replaced amniotic fluid, ambient temperature, and method of delivery.

Results

MMC defects were created in 232 lambs with an overall survival rate of 43%. Of the 128 fetuses that died, 53 (42%) had demise prior to repair, 61 (48%) aborted, and 14 (11%) were stillborn. Selective reduction of multiple gestations in the same uterine horn was associated with increased fetal demise (OR 3.03 [95% CI 1.29-7.05], p = 0.01). Later gestational age at MMC repair and Cesarean delivery were associated with decreased abortion/stillbirth (OR 0.90 [95% CI 0.83-0.90], p = 0.03, and OR 0.37 [95% CI 0.16-0.31], p = 0.02), respectively.

Conclusion

Avoiding selective reduction, repairing MMC later in gestation, and performing Cesarean delivery decreases the rate of fetal loss in the ovine MMC model.

Background

The inherent morbidity associated with fetal ovine models of myelomeningocele (MMC) has created challenges for long-term survival of lambs. We aimed to develop a fetal ovine surgical spinal exposure model which could be used to evaluate long-term safety after direct spinal cord application of novel therapeutics for augmentation of in utero MMC repair.

Methods

At gestational age (GA) 100-106, fetal lambs underwent surgical intervention. Laminectomy of L5-L6 was performed, dura was removed, and an experimental product was directly applied to the spinal cord. Paraspinal muscles and skin were closed and the fetus was returned to the uterus. Lambs were delivered via cesarean section at GA 140-142. Lambs were survived for 3 months with regular evaluation of motor function by the sheep locomotor rating scale. Spinal angulation was evaluated by magnetic resonance imaging at 2 weeks and 3 months.

Results

Five fetal surgical intervention lambs and 6 control lambs who did not undergo surgical intervention were included. All lambs survived to the study endpoint of 3 months. No lambs had motor function abnormalities or increased spinal angulation.

Conclusion

This model allows for long-term survival after fetal spinal cord exposure with product application directly onto the spinal cord.

Introduction

Translational models of myelomeningocele (MMC) are needed to test novel in utero interventions. An ideal animal model for MMC has locomotor function at birth and is low cost enough to allow for high throughput. The rat MMC model is limited by immature locomotor function at birth. The ovine MMC model is a costly surgical model. Guinea pigs are uniquely suited for an MMC model being a small animal model with locomotor function at birth. We aimed to develop a retinoic acid (RA) model of MMC in the guinea pig and to evaluate if pregnant guinea pigs could tolerate uterine manipulation.

Methods

Time-mated Dunkin Hartley guinea pig dams were dosed with 60 mg/kg of RA between gestation age (GA) 12 and 15 days in the development of an RA model. Fetuses were grossly evaluated for MMC lesions at Cesarean section after GA 31 days. Evaluation of the ability of pregnant guinea pig dams to tolerate uterine surgical intervention was performed by hysterotomy of a separated group of time-mated guinea pigs at GA 45, 50, and 55.

Results

Forty-two pregnant guinea pigs were dosed with RA, with a total of 189 fetuses. The fetal demise rate was 38% (n = 71). A total of 118 fetuses were viable, 83% (n = 98) were normal fetuses, 8% (n = 10) had a neural tube defect, and 8% (n = 10) had a hematoma or other anomalies. No fetuses developed an MMC defect. None of the fetuses that underwent hysterotomy survived to term.

Conclusion

RA dosed at 60 mg/kg in guinea pigs between GA 12 and 15 did not result in MMC. Dunkin Hartley guinea pigs did not tolerate a hysterotomy near term in our surgical model. Further work is needed to determine if MMC can be induced in guinea pigs with alternate RA dosing.

Background

We determined whether in vitro potency assays inform which placental mesenchymal stromal cell (PMSC) lines produce high rates of ambulation following in utero treatment of myelomeningocele in an ovine model.

Methods

PMSC lines were created following explant culture of three early-gestation human placentas. In vitro neuroprotection was assessed with a neuronal apoptosis model. In vivo, myelomeningocele defects were created in 28 fetuses and repaired with PMSCs at 3 × 10⁵ cells/cm² of scaffold from Line A (n = 6), Line B (n = 7) and Line C (n = 5) and compared to no PMSCs (n = 10). Ambulation was scored as ≥13 on the Sheep Locomotor Rating Scale.

Results

In vitro, Line A and B had higher neuroprotective capability than no PMSCs (1.7 and 1.8 respectively vs 1, p = 0.02, ANOVA). In vivo, Line A and B had higher large neuron densities than no PMSCs (25.2 and 27.9 respectively vs 4.8, p = 0.03, ANOVA). Line C did not have higher neuroprotection or larger neuron density than no PMSCs. In vivo, Line A and B had ambulation rates of 83% and 71%, respectively, compared to 60% with Line C and 20% with no PMSCs.

Conclusion

The in vitro neuroprotection assay will facilitate selection of optimal PMSC lines for clinical use.

Level of evidence

n/a.

Type of study

Basic science.

Introduction

Congenital diaphragmatic hernia (CDH) repair is an area of active research. Large defects requiring patches have a hernia recurrence rate of up to 50%. We designed a biodegradable polyurethane (PU)-based elastic patch that matches the mechanical properties of native diaphragm muscle. We compared the PU patch to a non-biodegradable Gore-Tex™ (polytetrafluoroethylene) patch.

Methods

The biodegradable polyurethane was synthesized from polycaprolactone, hexadiisocyanate and putrescine, and then processed into fibrous PU patches by electrospinning. Rats underwent 4 mm diaphragmatic hernia (DH) creation via laparotomy followed by immediate repair with Gore-Tex™ (n = 6) or PU (n = 6) patches. Six rats underwent sham laparotomy without DH creation/repair. Diaphragm function was evaluated by fluoroscopy at 1 and 4 weeks. At 4 weeks, animals underwent gross inspection for recurrence and histologic evaluation for inflammatory reaction to the patch materials.

Results

There were no hernia recurrences in either cohort. Gore-Tex™ had limited diaphragm rise compared to sham at 4 weeks (1.3 mm vs 2.9 mm, p = 0.003), but no difference was found between PU and sham (1.7 mm vs 2.9 mm, p = 0.09). There were no differences between PU and Gore-Tex™ at any time point. Both patches formed an inflammatory capsule, with similar thicknesses between cohorts on the abdominal (Gore-Tex™ 0.07 mm vs. PU 0.13 mm, p = 0.39) and thoracic (Gore-Tex™ 0.3 mm vs. PU 0.6 mm, p = 0.09) sides.

Conclusion

The biodegradable PU patch allowed for similar diaphragmatic excursion compared to control animals. There were similar inflammatory responses to both patches. Further work is needed to evaluate long-term functional outcomes and further optimize the properties of the novel PU patch in vitro and in vivo.

Level of evidence

Level II, Prospective Comparative Study.

Background

While fetal repair of myelomeningocele (MMC) revolutionized management, many children are still unable to walk independently. Preclinical studies demonstrated that research-grade placental mesenchymal stromal cells (PMSCs) prevent paralysis in fetal ovine MMC, however this had not been replicated with clinical-grade cells that could be used in an upcoming human clinical trial. We tested clinical-grade PMSCs seeded on an extracellular matrix (PMSC-ECM) in the gold standard fetal ovine model of MMC.

Methods

Thirty-five ovine fetuses underwent MMC defect creation at a median of 76 days gestational age, and defect repair at 101 days gestational age with application of clinical-grade PMSC-ECM (3 × 10⁵ cells/cm², n = 12 fetuses), research-grade PMSC-ECM (3 × 10⁵ cells/cm², three cell lines with n = 6 (Group 1), n = 6 (Group 2), and n = 3 (Group 3) fetuses, respectively) or ECM without PMSCs (n = 8 fetuses). Three normal lambs underwent no surgical interventions. The primary outcome was motor function measured by the Sheep Locomotor Rating scale (SLR, range 0: complete paralysis to 15: normal ambulation) at 24 h of life. Correlation of lumbar spine large neuron density with SLR was evaluated.

Results

Clinical-grade PMSC-ECM lambs had significantly better motor function than ECM-only lambs (SLR 14.5 vs. 6.5, p = 0.04) and were similar to normal lambs (14.5 vs. 15, p = 0.2) and research-grade PMSC-ECM lambs (Group 1: 14.5 vs. 15, p = 0.63; Group 2: 14.5 vs. 14.5, p = 0.86; Group 3: 14.5 vs. 15, p = 0.50). Lumbar spine large neuron density was strongly correlated with motor function (r = 0.753, p<0.001).

Conclusions

Clinical-grade placental mesenchymal stromal cells seeded on an extracellular matrix rescued ambulation in a fetal ovine myelomeningocele model. Lumbar spine large neuron density correlated with motor function, suggesting a neuroprotective effect of the PMSC-ECM in prevention of paralysis. A first-in-human clinical trial of PMSCs in human fetal myelomeningocele repair is underway.