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. Author manuscript; available in PMC: 2023 Apr 1.
Published in final edited form as: J Pediatr Surg. 2021 Jun 5;57(4):753–758. doi: 10.1016/j.jpedsurg.2021.05.025

Efficacy of Clinical-Grade Human Placental Mesenchymal Stromal Cells in Fetal Ovine Myelomeningocele Repair

Christina M Theodorou 1,2,*, Sarah C Stokes 1,2, Jordan E Jackson 1,2, Christopher D Pivetti 2, Priyadarsini Kumar 2, Kaeli J Yamashiro 1,2, Zachary J Paxton 2, Lizette Reynaga 2, Alicia A Hyllen 2, Aijun Wang 1,2, Diana L Farmer 1,2
PMCID: PMC9365331  NIHMSID: NIHMS1825457  PMID: 34217509

Abstract

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 (3x105 cells/cm2, n=12 fetuses), research-grade PMSC-ECM (3x105 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 hours 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.

Keywords: myelomeningocele, spina bifida, cellular therapy, fetal surgery

Introduction

Myelomeningocele (MMC) is the most severe form of spina bifida and is due to incomplete closure of the neural tube over the spinal cord. The resulting spinal cord exposure causes neuronal loss and lower extremity paralysis. This is based on the two-hit hypothesis of spinal cord damage in MMC, with the first hit arising from abnormalities of spinal cord development resulting in the MMC defect. The second hit is thought to arise from ongoing spinal cord damage in utero resulting in neuronal apoptosis during gestation[1]. The landmark Management of Myelomeningocele Study (MOMS)[2] published in 2011 established that prenatal MMC repair significantly improved motor function compared to postnatal repair. By 30 months of age, 45% of prenatally repaired patients were walking independently compared to 24% of postnatally repaired patients. This shifted the paradigm of MMC treatment and illustrated the potential for improved outcomes with early coverage of the exposed spinal cord. By repairing the MMC defect in utero during a highly regenerative stage of development, further spinal cord damage is prevented, minimizing the second hit. However, despite these improvements, 55% of children with prenatally repaired MMC were still unable to walk independently, and 28% were unable to walk even with assistive devices. By school-age, only 29% of children were walking independently[3]. Although significantly higher than the rate of independent ambulation in the postnatal group (11%), there still remains room for improvement.

In the quest to improve outcomes for children with MMC, we have discovered that augmentation of fetal MMC repair with cellular therapy using placental mesenchymal stromal cells (PMSCs) seeded on extracellular matrix (ECM) showed promising results. We have demonstrated that PMSCs cultured in neurogenic medium secrete neurotrophic cytokines such as brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF) and have a neuroprotective effect in vitro which correlates with improved motor function in vivo in the gold standard fetal ovine MMC model[4,5]. However, these previous studies were done with a research-grade cell line, which was not subjected to the rigorous screening and testing requirements of the United States Food and Drug Administration (FDA) for use in a human clinical trial. Thus, we sought to show that clinical-grade PMSCs that were manufactured for future human clinical trial use would improve motor function compared to the current standard of care fetal MMC repair.

In preparation for human clinical trials, a single optimal clinical-grade PMSC line was selected based on extensive characterization of in vitro neuroprotection function and prepared in accordance with Current Good Manufacturing Practice (CGMP) standards for use in the pivotal Investigational New Drug Application (IND)-enabling ovine studies[6]. In this study, we evaluated the efficacy of the selected clinical-grade PMSCs to improve motor function in a fetal ovine MMC model compared to fetal MMC repair without the use of PMSCs. We hypothesized that lambs treated with clinical-grade PMSC-ECM in utero would have better motor function than lambs treated with routine fetal MMC repair with ECM only.

Methods

Approval to perform this study was obtained from the Institutional Animal Care and Use Committee (IACUC Protocol # 21476). Animal care was performed in accordance with the Guide for the Care and Use of Laboratory Animals. The facilities used to conduct this study were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International. All cell work was done in the UC Davis GMP Facility, which is a class 10,000 clean room facility approved for the manufacturing of clinical-grade products.

Clinical-Grade Placental Mesenchymal Stromal Cell Line Selection and Preparation

An optimal clinical-grade PMSC line was selected and prepared in accordance with CGMP standards, in the same manner as preparation of research-grade PMSC lines published previously[4], with the exception of using GMP-grade reagents. For the clinical-grade line, donors underwent extensive infectious testing per FDA guidance[7]. The PMSCs were collected from a consented donor, with infectious testing of the donated placental tissue to include cytomegalovirus, hepatitis B and C, human immunodeficiency virus, human T-cell lymphotrophic virus, Zika virus, West Nile virus, as well as syphilis, gonorrhea, and chlamydia testing. Cells banks were generated in our on-campus GMP facility. Clinical-grade PMSC cell lines underwent rigorous sterility testing in accordance with FDA guidelines, including 14-day sterility testing, and testing for mycoplasma, endotoxin, bovine virus, adventitious virus, and human infectious viral testing. PMSCs additionally underwent potency testing that included the neuroprotection assay developed in our lab previously as a screening tool for choosing the optimal cell line[4], as well as testing of secretion levels of two neuroprotective growth factors brain-derived growth factor (BDNF) and hepatocyte growth factor (HGF)[8,9]. Neuroprotection was assessed by fold-increase in total neurite branchpoints and total neurite segments as previously described[10]. Based on these tests, a single cell line from the donor pool was chosen as an optimal cell line for use in the pivotal IND-enabling ovine study described here[6].

Prior to each surgery date, one vial of the PMSC product bank at passage 4 was thawed and seeded into flasks at 2x104 cells/cm2 and cultured at 37 degrees Celsius at 5% CO2 for 48 hours. The cells were then lifted and seeded onto a clinical-grade extracellular matrix (Biodesign® Dural graft, Cook Biotech, West Lafayette, IN) (PMSC-ECM) at a seeding density of 3x105 PMSCs/cm2, a previously determined optimal dose based on prior animal studies in our laboratory[11,12]. The cells were cultured on the clinical-grade ECM for an additional 24 hours. A corner notch of the seeded ECM was reserved for PMSC viability testing by staining with CalceinAM to verify the adherence and viability of the PMSCs on the ECM. The PMSC-ECM product was delivered to study personnel for surgical administration in the Animal Care Facility, UC Davis.

Clinical-Grade PMSC Lamb Cohort

The gold standard fetal ovine model of myelomeningocele was used for this study. Lambs are an ideal model for the study of postnatal effects of fetal MMC interventions as they are ambulatory at birth and allow for early evaluation of motor function. Time-mated pregnant Dorper mix ewes were obtained. Twelve fetal lambs (7 female) underwent creation of a lumbar MMC defect at a median of 73 days gestational age (GA 73). Briefly, the defect was created by maternal laparotomy, hysterotomy, exposure of the fetal back, removal of the fetal skin, paraspinal muscles, lumbar spine lamina, and dura from L1-L6 to expose the fetal lumbar spinal cord (Figure 1A–C). The fetus was returned to the uterus and lost amniotic fluid was replaced with warmed normal saline, gentamicin (100 mg) and penicillin (1 million units). Gestation was continued to allow accrual of spinal cord damage to simulate the naturally occurring ongoing spinal cord damage in human fetuses with MMC. At a median of GA 101, the fetal lambs underwent MMC defect repair. This was performed by maternal laparotomy, hysterotomy, exposure of the fetal back and exposed spinal cord, removal of scar tissue that had formed, and placement of clinical-grade PMSC-ECM. The clinical-grade PMSC-ECM was placed with the cell-seeded side in direct contact with the exposed spinal cord. The fetal skin was closed, and the fetus was returned to the uterus to continue gestation (Figure 1D–G). The lambs were delivered via Cesarean section at GA 141 days and survived for 48 hours. This endpoint was chosen to allow for histologic comparison to the historical ECM-only cohort at nearly the same timepoint, who required early humane euthanasia due to inability to walk, which results in an inability to nurse. Longer-term survival in PMSC-ECM treated lambs is possible [13], but requires intensive physical therapy and bracing due to the inherent surgical morbidity of the MMC defect creation, as described above. All surgical procedures were done using sterile technique.

Figure 1: Representative photos of fetal ovine myelomeningocele defect creation and repair.

Figure 1:

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A) Removal of the skin and paraspinal muscles, followed by B) laminectomy, and C) removal of dura. D) At time of defect repair, MMC defect shown. E) The spinal cord is re-exposed. F) The clinical-grade PMSC-ECM is placed onto the spinal cord with cells facing down, followed by G) skin closure.

Historical Comparison Lamb Cohort – ECM-only

In accordance with the principle of reduction, one of the Three Rs of animal research [14], a historical cohort of animals who underwent fetal MMC repair with an ECM only without PMSCs was utilized for comparison. Eight fetal lambs (4 female) underwent MMC defect creation at a median of GA 78.5 as described above. At a median of GA 103.5, MMC defect repair was performed with placement of an ECM without PMSCs directly onto the spinal cord. The lambs were delivered by Cesarean section at a median of GA 146 and survived for 24 hours. The earlier time of delivery in the experimental PMSC-ECM cohort was due to accruing experience in the lamb model which found that scheduled Cesarean deliveries were associated with decreased odds of fetal demise compared to spontaneous vaginal delivery[15]. Thus, the experimental cohort was delivered a few days earlier to ensure no spontaneous vaginal deliveries would occur. Three additional normal lambs which did not undergo any surgical procedures were acquired at birth following spontaneous vaginal delivery at a median of GA 147 and survived to 48 hours of life.

Historical Comparison Lamb Cohort – Research-Grade PMSCs

Historical cohorts of lambs with surgically created MMC who underwent fetal repair with research-grade PMSCs with three difference cell lines (Group 1, n = 6; Group 2, n = 6; Group 3, n = 3) were used for comparison to the clinical-grade PMSC animals [4]. Unlike the clinical-grade PMSC preparation described above, the placental donor and placenta tissues were not subjected to the rigorous infectious and sterility testing as mandated by the FDA. Additionally, all work was conducted in the UC Davis Surgical Bioengineering Laboratory, rather than the UC Davis GMP Facility. Research-grade reagents were utilized. The PMSCs were generated from three placental donors and seeded onto the ECM at the same density, 3x105 cells/cm2. These animals underwent the same surgical procedures described above, with defect creation at a median GA 76 and defect repair at a median GA 104. Lambs from the historical research-grade PMSC cohorts were delivered at a median GA 146 by a mixture of spontaneous vaginal delivery (n = 6/15, 40%) and Cesarean section (n = 9/15, 60%). These lambs were euthanized at 24 hours of life. The postnatal motor scores of these animals were compared to those of clinical-grade PMSC-ECM animals.

Postnatal Motor Evaluations

Motor function was evaluated using the validated Sheep Locomotor Rating (SLR) scale[16]. The SLR evaluates lambs for hindlimb joint movement, ability to bear weight on their hindlimbs, standing ability, ambulation, and ability to clear an obstacle. Lambs were assigned an SLR score from 0-15, with 0 representing complete paralysis and 15 representing normal ambulation. As ECM-only lambs and research-grade PMSC lambs were euthanized at 24 hours of life, the highest SLR at 24 hours of life was compared between groups.

Histologic Evaluation of the Spinal Cord

Histological analysis of the spinal cords from all lambs was performed. Nine serial 20-μm sections were taken through each lumbar spinal cord segment (L1-L6) and stained with Cresyl Violet. Each serial section was imaged at 10x magnification with a Zeiss Observer D1 microscope. The images were analyzed using ImageJ software in order to determine the cross-sectional height and width as well as the cross-sectional area of gray matter. The MMC defect epicenter, defined as the lumbar segment with the greatest degree of deformation when normalized against normal lambs, was determined by dividing the cross-sectional height of the spinal cord by the width.

A single blinded reviewer using Zeiss Zen One software conducted large neuron counts in each serial section of every animal’s MMC epicenter and of corresponding lumbar spine segments of normal lambs. Large neurons were defined as cells found within the gray matter with a diameter of 30-70μm. Large neuron density was calculated by dividing the mean number of large neurons by the mean total cross-sectional area of gray matter.

Statistical Analysis

A sample size of ten clinical-grade PMSC-ECM lambs was determined adequate to detect a 30% difference in motor function compared to ECM-only historical animals. This 30% difference corresponds to a 4.5-point change on the SLR scale with a power of 80%. Outcomes of clinical-grade PMSC-ECM lambs were compared to research-grade PMSC-ECM lambs, ECM-only negative controls and normal lambs. Non-parametric continuous outcomes are presented as median and interquartile range (IQR) and compared by Mann Whitney U test or Kruskal-Wallis test. Categorical outcomes are presented as proportions and compared by Fisher’s exact test. Spearman’s rank-order correlation was used to test for correlation between lumbar spine large neuron density and SLR motor score. All tests were two-sided. The level of significance was set at p<0.05. All analyses were done in Prism (GraphPad Software, Inc.).

Results

Motor Function

The clinical-grade cell line selected and destined for future human use was tested in the gold standard fetal ovine model. The clinical-grade PMSC-ECM lambs displayed significantly improved motor function compared to ECM-only lambs (median SLR score 14.5 vs. 6.5, p=0.049, Figure 2) at 24 hours of life. Additionally, the motor function of clinical-grade PMSC-ECM lambs was similar to normal controls (median SLR score 14.5 vs. 15, p=0.19, Figure 2) and to the historical cohorts treated with research-grade PMSCs (median SLR and p-values for comparison to clinical-grade ECM median of 14.5: Group 1, SLR 15, p=0.63; Group 2, SLR 14.5, p=0.86; Group 3, SLR 15, p=0.50). Of the clinical-grade PMSC-EM lambs, which were survived for 48 hours rather than 24 hours for the ECM-only lambs, only one was noted to have improved motor function at 48 hours compared to 24 hours of life (SLR 8 at 24 hours to SLR 12 at 48 hours). All other clinical-grade PMSC-ECM lambs attained their maximum SLR score by 24 hours of life. The majority (75.0%) of clinical-grade PMSC-ECM lambs were able to stand spontaneously (SLR≥12) compared to 37.5% of ECM-only lambs and 66.7% of clinical-grade PMSC-ECM lambs were able to stand spontaneously and walk (SLR≥14) compared to 25.0% of ECM-only lambs, but these differences did not reach statistical significance (p=0.17 for both).

Figure 2: Comparison of motor function.

Figure 2:

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Motor function, measured by sheep locomotor rating (SLR) scores, is shown for animals repaired with clinical-grade placental mesenchymal stromal cells (cPMSC-ECM, green diamonds) compared to extracellular matrix-only (ECM) historical controls (red squares), research-grade PMSC-ECM historical controls (rPMSC-ECM, black hollow circles), and normal lambs (blue circles). * indicates p < 0.05.

Histologic Evaluation of Lumbar Spine

The MMC epicenter was most commonly at L4 in both PMSC-ECM animal and ECM-only animals. The median lumbar level of the MMC epicenter was similar between groups (L3.5 in ECM-only vs. L4 in PMSC-ECM animals, p=0.25). Level of MMC epicenter did not correlate with motor function measured by SLR (r=0.385, p=0.09). Although lumbar spine MMC epicenter large neuron (LN) density was higher in clinical-grade PMSC-ECM animals compared to ECM-only animals, this was not statistically significant (12.2 LN per mm2 of grey matter in PMSC-ECM lambs vs. 4.8 LN per mm2 in ECM-only lambs, p=0.6, Figure 3A). LN density was higher in normal lambs than in clinical-grade PMSC-ECM animals and ECM-only lambs, but this was also not statistically significant (22.7 LN per mm2 in normal lambs vs. 12.2 LN per mm2 in PMSC-ECM lambs, p=0.2, and 4.8 in ECM-only lambs, p=0.09). However, LN density in the lumbar spine epicenter of the MMC defect did significantly and positively correlate with SLR score (r=0.753, p<0.001, Figure 3B). Lambs that were able to stand spontaneously (SLR≥12) had a significantly higher LN density in the MMC epicenter than lambs with lower SLR scores (22.2 LN per mm2 vs. 2.6 LN per mm2, p = 0.0003).

Figure 3: Histologic evaluation of lumbar spine myelomeningocele epicenter large neuron density.

Figure 3:

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A) Large neuron (LN) density compared between cohorts, shown as median and interquartile range. B) Correlation between large neuron density and motor function measured by sheep locomotor rating (SLR) score. ECM: extracellular matrix; PMSC:-ECM: placental mesenchymal stromal cells seeded on extracellular matrix. ECM-only depicted as red squares, PMSC-ECM depicted as green diamonds, and normal lambs depicted as blue circles.

Discussion

In this pivotal Investigational New Drug-enabling study, we have demonstrated that a clinical-grade placental mesenchymal stromal cell line, destined for future human use, prepared in current Good Manufacturing Practice fashion and seeded on an extracellular matrix, rescued ambulation when used to augment fetal myelomeningocele repair in the gold-standard ovine model. Lambs repaired prenatally with this clinical-grade PMSC-ECM had significantly improved motor function compared to lambs repaired with an ECM alone, and were similar in motor function to normal lambs without MMC and to prior animals repaired using research-grade PMSC-ECM. Large neuron density in the MMC epicenter significantly correlated with motor function, suggesting a potential neuroprotective mechanism of the clinical-grade PMSC-ECM product in utero.

The mechanism underlying lower extremity paralysis in MMC is not entirely known. In autopsy studies of human MMC patients, the exposed fetal spinal cord displayed a distorted architecture with increased apoptotic cells[17]. This damage is thought to result from a ‘two-hit’ hypothesis, with the first due to abnormal spinal cord development, and the second due to ongoing damage from chemical and mechanical trauma sustained by the exposed spinal cord in the intrauterine environment[18]. In a rat model of MMC, spinal cord damage worsened as gestation continued, with spinal cord necrosis developing. When these MMC defects were closed in utero, however, spinal cord architecture was preserved, and was similar to normal rats[18]. Data from a naturally occurring murine MMC model also support this, with the exposed spinal cord developing normally early in gestation, and subsequently accruing significant spinal cord damage over time after the MMC defect develops[1]. These animal findings formed the basis for fetal intervention for MMC, by suggesting that fetal repair could prevent ongoing spinal cord damage and preserve distal neuronal function. The landmark MOMS showed conclusively for the first time that fetal MMC repair was superior to postnatal MMC repair, with significantly improved motor function in prenatally repaired children[2]. By 30 months old, nearly half of prenatally repaired children were able to ambulate independently, compared to only one-quarter of children who underwent postnatal repair. While this improvement was significant, there remains a critical unmet need in MMC treatment[19], which has led to the development of therapies to augment the prenatal repair with the aim of further improving motor outcomes.

We hypothesized that mesenchymal stromal cells, with their innate reparative and regenerative capacity, would hold the key to unlocking the solution for curing the paralysis associated with MMC. After evaluating several different treatment modalities, including autologous amniotic membrane[20], biodegradable nanofibrous scaffolds[21], and induced pluripotent stem cells[22], we have found that PMSCs were the ideal candidate for augmentation of fetal MMC repair. The mechanism of action of the PMSCs is thought to be due the immunomodulatory, anti-apoptotic and neuroprotective effect on local spinal cord neurons controlling distal motor function. PMSCs have a notable paracrine secretion signal, producing neuroprotective cytokines brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF)[5]. Additionally, we have previously shown that when co-cultured with neuroblastoma cell line SH-SY5Y after induction of apoptosis, PMSCs result in neuroprotection, measured by increased total neurite branch points and total neurite segments[4,23]. This neuroprotection assay played a key role in the selection of the clinical trial cell line tested in this study, which had the highest level of total branching points and neurite segments compared to other candidate cell lines[6]. Additionally, PMSCs have anti-apoptotic effects in vitro[23] and in vivo, resulting in decreased spinal cord apoptosis in a rat MMC model[11]. In the gold-standard ovine model, we have reliably found that motor function has correlated with MMC epicenter large neuron density[4,12,24]. Research-grade PMSCs grown in a specific neurogenic media, have been shown to improve postnatal motor function in lambs with MMC when compared to lambs who underwent fetal MMC repair with ECM alone[4,5,12,24] and for the first time, in this study, we have shown that these results are durable with a rigorously prepared clinical-grade PMSC-ECM product. Taken together, these data support a paracrine neuroprotective effect of the PMSCs when applied to the spinal cord during fetal MMC repair.

From Bench to Bedside

The journey from a novel treatment idea in a clinician’s mind to application to human patients is a long one. The proposed treatment must have scientific validity and must be characterized and tested extensively in vitro prior to moving on to a translational animal model. The outcome must be shown to be reproducible. Testing in an animal model is critical to determine the safety and efficacy of the treatment to satisfy the rigorous preclinical testing requirements of the FDA. In the context of our previous work[4,12,24], this study was pivotal in securing FDA approval for an Investigational New Drug application studying the use of clinical-grade PMSC-ECM in human fetuses with MMC. At the time of this manuscript, a first-in-human clinical Phase 1/2a trial is underway, in which we will evaluate the safety and preliminary efficacy of the clinical-grade PMSC-ECM product in fetal MMC repair. The ability to utilize clinical-grade PMSCs to augment the improvement in distal neurologic function afforded by fetal MMC repair has the potential to change the lives of thousands of children and their families each year[25].

Future Directions

In addition to the first-in-human clinical trial which is now underway, further experiments are planned to better elucidate the basis of the improved motor function seen in animals treated with PMSC-ECM. Our laboratory is in the process of developing a series of functional assays assessing secretion of neuroprotective factors and growth factors by the PMSCs to better characterize their mechanism of action. As there may be some inherent differences between cell lines of placental origin, selection of ideal PMSC lines going forward is a critical and ongoing avenue of research. Although we have found repeatedly that spinal cord large neuron density is significantly correlated with motor function in the lamb model, it would be ideal to develop an in vitro cellular assay that corresponds to in vivo improvement in motor function. Such an assay would allow for preclinical selection of PMSC cell lines and provide valuable information on the expected improvement in motor function in patients.

Limitations

Although we utilized the gold standard fetal ovine MMC model, animal models are inherently different from human patients with MMC. In this model, the MMC defect is surgically created, as opposed to naturally occurring as in humans. Due to the healing capacity of the fetus[26], an extensive MMC defect creation is required, including removal of the paraspinal muscles, which limits the longevity of these animals due to spinal column instability with growth of the animal[13,27]. Additionally, animals in the ECM-only cohort were survived to 24 hours compared to 48 hours for clinical-grade PMSC-ECM animals and normal controls. The historical ECM-only cohort was humanely euthanized at 24 hours due to inability to walk, and thus inability to nurse, in line with guidance from IACUC. Thus, all functional data was compared at 24 hours of life, to avoid comparing the motor function of an older and more robust clinical-grade PMSC-ECM lamb at 48 hours to a younger lamb at 24 hours. Long-term functional data would be informative of the long-term effects of the PMSCs in this model; however, this is difficult to obtain due to the morbidity of the surgical MMC defect. However, we have previously survived a PMSC-ECM treated lamb to 6 months, with the aid of extensive physical therapy and bracing, and this lamb maintained a perfect SLR score of 15 until the predetermined study endpoint of 6 months, suggesting that the improvements in motor function imparted by the PMSCs are durable [13]. Lastly, although we were adequately powered for our primary outcome, motor function, due to our small sample sizes, we were underpowered to detect differences in large neuron density in the MMC epicenter of the lambs. However, increasing large neuron density did correlate strongly with motor function, suggesting neuroprotection as a potential mechanism of action of the PMSCs.

Conclusion

In the gold standard fetal ovine myelomeningocele model, augmentation of fetal MMC repair with carefully selected clinical-grade placental mesenchymal stromal cells seeded on an extracellular matrix rescued ambulation compared to prenatal repair with an ECM only. This study, conducted under FDA guidelines, supports the initiation of a Phase 1/2a clinical trial in human patients with MMC evaluating the efficacy of clinical-grade PMSCs in fetal MMC repair.

Acknowledgments:

The authors would like to thank the extensive team of personnel involved in this study, including Linda Talken, Amy Lesneski, Kirstie Tully, Jacqueline Kowalski, and all of the staff and volunteers who provided excellent care of the animals, including the UC Davis Campus Veterinary Services. Additionally, we would like to thank Dr. Jan Nolta, Dr. Gerhard Bauer, Dr. Lisa Kadyk, and William Gruenloh for the help with this study.

Funding Information:

This work was funded by the California Institute of Regenerative Medicine late stage preclinical CLIN1 grant (CLIN1-11404) and was in part supported by NIH grants (1R01NS100761-01A1 and 1R01NS115860-01A1), and Shriners Hospitals for Children research grants (85108-NCA-19 and 85135-NCA-21). The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1 TR001860 for author CMT. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

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Declarations of Interest: None

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