Placental Mesenchymal Stromal Cells: Preclinical Safety Evaluation for Fetal Myelomeningocele Repair.

BACKGROUND: Myelomeningocele (MMC) is the congenital failure of neural tube closure in utero, for which the standard of care is prenatal surgical repair. We developed clinical-grade placental mesenchymal stromal cells seeded on a dural extracellular matrix (PMSC-ECM), which have been shown to improve motor outcomes in preclinical ovine models. To evaluate the long-term safety of this product prior to use in a clinical trial, we conducted safety testing in a murine model. METHODS: Clinical grade PMSCs obtained from donor human placentas were seeded onto a 6 mm diameter ECM at a density of 3 × 105 cells/cm2. Immunodeficient mice were randomized to receive either an ECM only or PMSC-ECM administered into a subcutaneous pocket. Mice were monitored for tumor formation until two study endpoints: 4 wk and 6 mo. Pathology and histology on all tissues was performed to evaluate for tumors. Quantitative polymerase chain reaction (qPCR) was performed to evaluate for the presence of human DNA, which would indicate persistence of PMSCs. RESULTS: Fifty-four mice were included; 13 received ECM only and 14 received PMSC-ECM in both the 4-wk and 6-mo groups. No mice had gross or microscopic evidence of tumor development. A nodular focus of mature fibrous connective tissue was identified at the subcutaneous implantation pocket in the majority of mice with no significant difference between ECM only and PMSC-ECM groups (P = 0.32 at 4 wk, P > 0.99 at 6 mo). Additionally, no human DNA was detected by qPCR in any mice at either time point. CONCLUSIONS: Subcutaneous implantation of the PMSC-ECM product did not result in tumor formation and we found no evidence that PMSCs persisted. These results support the safety of the PMSC-ECM product for use in a Phase 1/2a human clinical trial evaluating fetal MMC repair augmented with PMSC-ECM.

Spinal Angulation: A Limitation of the Fetal Lamb Model of Myelomeningocele.

INTRODUCTION: The surgically induced fetal lamb model is the most commonly used large animal model of myelomeningocele (MMC) but is subject to variation due to surgical technique during defect creation. MATERIAL AND METHODS: Thirty-one fetal lambs underwent creation of the MMC defect, followed by defect repair with either an extracellular matrix (ECM) patch (n = 10) or ECM seeded with placental mesenchymal stromal cells (n = 21). Postnatal hindlimb function was assessed using the Sheep Locomotor Rating (SLR) scale. Postmortem magnetic resonance imaging of the lumbar spine was used to measure the level and degree of spinal angulation, as well as cross-sectional area of remaining vertebral bone. RESULTS: Median level of angulation was between the 2nd and 3rd lumbar vertebrae, with a median angle of 24.3 degrees (interquartile range 16.2-35.3). There was a negative correlation between angulation degree and SLR (r = -0.44, p = 0.013). Degree of angulation also negatively correlated with the normalized cross-sectional area of remaining vertebral bone (r = -0.75, p < 0.0001). DISCUSSION: Surgical creation of fetal MMC leads to varying severity of spinal angulation in the ovine model, which affects postnatal functional outcomes. Postnatal assessment of spinal angulation aids in standardization of the surgical model of fetal MMC repair.

Long-term safety evaluation of placental mesenchymal stromal cells for in utero repair of myelomeningocele in a novel ovine model.

PURPOSE: Augmentation of in utero myelomeningocele repair with human placental mesenchymal stromal cells seeded onto extracellular matrix (PMSC-ECM) improves motor outcomes in an ovine myelomeningocele model. This study evaluated the safety of PMSC-ECM application directly onto the fetal spinal cord in preparation for a clinical trial. METHODS: Laminectomy of L5-L6 with PMSC-ECM placement directly onto the spinal cord was performed in five fetal lambs at gestational age (GA) 100-106 days. Lambs and ewes were monitored for three months following delivery. Lambs underwent magnetic resonance imaging (MRI) of the brain and spine at birth and at three months. All organs from lambs and uteri from ewes underwent histologic evaluation. Lamb spinal cords and brains and ewe placentas were evaluated for persistence of PMSCs by polymerase chain reaction for presence of human DNA. RESULTS: MRIs demonstrated no evidence of abnormal tissue growth or spinal cord tethering. Histological analysis demonstrated no evidence of abnormal tissue growth or treatment related adverse effects. No human DNA was identified in evaluated tissues. CONCLUSION: There was no evidence of abnormal tissue growth or PMSC persistence at three months following in utero application of PMSC-ECM to the spinal cord. This supports proceeding with clinical trials of PMSC-ECM for in utero myelomeningocele repair. LEVEL OF EVIDENCE: N/A TYPE OF STUDY: Basic science.

Efficacy of clinical-grade human placental mesenchymal stromal cells in fetal ovine myelomeningocele repair.

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.

Early investigations into improving bowel and bladder function in fetal ovine myelomeningocele repair.

INTRODUCTION: Fetal myelomeningocele (MMC) repair improves lower extremity motor function. We have previously demonstrated that augmentation of fetal MMC repair with placental mesenchymal stromal cells (PMSCs) seeded on extracellular matrix (PMSC-ECM) further improves motor function in the ovine model. However, little progress has been made in improving bowel and bladder function, with many patients suffering from neurogenic bowel and bladder. We hypothesized that fetal MMC repair with PMSC-ECM would also improve bowel and bladder function. METHODS: MMC defects were surgically created in twelve ovine fetuses at median gestational age (GA) 73 days, followed by defect repair at GA101 with PMSC-ECM. Fetuses were delivered at GA141. Primary bladder function outcomes were voiding posture and void volumes. Primary bowel function outcome was anorectal manometry findings including resting anal pressure and presence of rectoanal inhibitory reflex (RAIR). Secondary outcomes were anorectal and bladder detrusor muscle thickness. PMSC-ECM lambs were compared to normal lambs (n = 3). RESULTS: Eighty percent of PMSC-ECM lambs displayed normal voiding posture compared to 100% of normal lambs (p = 1). Void volumes were similar (PMSC-ECM 6.1 ml/kg vs. normal 8.8 ml/kg, p = 0.4). Resting mean anal pressures were similar between cohorts (27.0 mmHg PMSC-ECM vs. normal 23.5 mmHg, p = 0.57). RAIR was present in 3/5 PMSC-ECM lambs that underwent anorectal manometry and all normal lambs (p = 0.46). Thicknesses of anal sphincter complex, rectal wall muscles, and bladder detrusor muscles were similar between cohorts. CONCLUSION: Ovine fetal MMC repair augmented with PMSC-ECM results in near-normal bowel and bladder function. Further work is needed to evaluate these outcomes in human patients.

In utero treatment of myelomeningocele with placental mesenchymal stromal cells - Selection of an optimal cell line in preparation for clinical trials.

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.

Placental Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Myelin Regeneration in an Animal Model of Multiple Sclerosis.

Mesenchymal stem/stromal cells (MSCs) display potent immunomodulatory and regenerative capabilities through the secretion of bioactive factors, such as proteins, cytokines, chemokines as well as the release of extracellular vesicles (EVs). These functional properties of MSCs make them ideal candidates for the treatment of degenerative and inflammatory diseases, including multiple sclerosis (MS). MS is a heterogenous disease that is typically characterized by inflammation, demyelination, gliosis and axonal loss. In the current study, an induced experimental autoimmune encephalomyelitis (EAE) murine model of MS was utilized. At peak disease onset, animals were treated with saline, placenta-derived MSCs (PMSCs), as well as low and high doses of PMSC-EVs. Animals treated with PMSCs and high-dose PMSC-EVs displayed improved motor function outcomes as compared to animals treated with saline. Symptom improvement by PMSCs and PMSC-EVs led to reduced DNA damage in oligodendroglia populations and increased myelination within the spinal cord of treated mice. In vitro data demonstrate that PMSC-EVs promote myelin regeneration by inducing endogenous oligodendrocyte precursor cells to differentiate into mature myelinating oligodendrocytes. These findings support that PMSCs' mechanism of action is mediated by the secretion of EVs. Therefore, PMSC-derived EVs are a feasible alternative to cellular based therapies for MS, as demonstrated in an animal model of the disease.

High density placental mesenchymal stromal cells provide neuronal preservation and improve motor function following in utero treatment of ovine myelomeningocele.

PURPOSE: The purpose of this study was to determine whether seeding density of placental mesenchymal stromal cells (PMSCs) on extracellular matrix (ECM) during in utero repair of myelomeningocele (MMC) affects motor function and neuronal preservation in the ovine model. METHODS: MMC defects were surgically created in 33 fetuses and repaired following randomization into four treatment groups: ECM only (n = 10), PMSC-ECM (42 K cells/cm2) (n = 8), PMSC-ECM (167 K cells/cm2) (n = 7), or PMSC-ECM (250-300 K cells/cm2) (n = 8). Motor function was evaluated using the Sheep Locomotor Rating Scale (SLR). Serial sections of the lumbar spinal cord were analyzed by measuring their cross-sectional areas which were then normalized to normal lambs. Large neurons (LN, diameter 30-70 µm) were counted manually and density calculated per mm2 gray matter. RESULTS: Lambs treated with PMSCs at any density had a higher median SLR score (15 [IQR 13.5-15]) than ECM alone (6.5 [IQR 4-12.75], p = 0.036). Cross-sectional areas of spinal cord and gray matter were highest in the PMSC-ECM (167 K/cm2) group (p = 0.002 and 0.006, respectively). LN density was highest in the greatest density PMSC-ECM (250-300 K/cm2) group (p = 0.045) which positively correlated with SLR score (r = 0.807, p < 0.0001). CONCLUSIONS: Fetal repair of myelomeningocele with high density PMSC-ECM resulted in increased large neuron density, which strongly correlated with improved motor function. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A.