Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes.

We have established early-gestation chorionic villus-derived placenta mesenchymal stromal cells (PMSCs) as a potential treatment for spina bifida (SB), a neural tube defect. Our preclinical studies demonstrated that PMSCs have the potential to cure hind limb paralysis in the fetal lamb model of SB via a paracrine mechanism. PMSCs exhibit neuroprotective function by increasing cell number and neurites, as shown by indirect coculture and direct addition of PMSC-conditioned medium to the staurosporine-induced apoptotic human neuroblastoma cell line, SH-SY5Y. PMSC-conditioned medium suppressed caspase activity in apoptotic SH-SY5Y cells, suggesting that PMSC secretome contributes to neuronal survival after injury. As a part of PMSC secretome, PMSC exosomes were isolated and extensively characterized; their addition to apoptotic SH-SY5Y cells mediated an increase in neurites, suggesting that they exhibit neuroprotective function. Proteomic and RNA sequencing analysis revealed that PMSC exosomes contain several proteins and RNAs involved in neuronal survival and development. Galectin 1 was highly expressed on the surface of PMSCs and PMSC exosomes. Preincubation of exosomes with anti-galectin 1 antibody decreased their neuroprotective effect, suggesting that PMSC exosomes likely impart their effect via binding of galectin 1 to cells. Future studies will include in-depth analyses of the role of PMSC exosomes on neuroprotection and their clinical applications.-Kumar, P., Becker, J. C., Gao, K., Carney, R. P., Lankford, L., Keller, B. A., Herout, K., Lam, K. S., Farmer, D. L., Wang, A. Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes.

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.

Isolation and characterization of canine placenta-derived mesenchymal stromal cells for the treatment of neurological disorders in dogs.

Spinal cord injury (SCI) is a devastating disorder that affects humans and dogs. The prognosis of SCI depends on the severity of the injury and can include varying levels of motor and sensory deficits including devastating paraplegia and quadriplegia. Placental mesenchymal stromal cells (PMSCs) have been shown to improve wound healing and possess neuroprotective and immunomodulatory capabilities, but have not yet been clinically tested for the treatment of SCI. This study established a protocol to isolate fetal PMSCs from canine placentas and characterized their paracrine secretion profile and ability to stimulate neurons in vitro to assess their potential as a treatment option for neurological disorders in dogs. Canine PMSCs (cPMSCs) were plastic adherent and capable of trilineage differentiation. cPMSCs expressed typical MSC markers and did not express hematopoietic or endothelial cell markers. Genotyping of cPMSCs revealed fetal rather than maternal origin of the cells. cPMSCs were viable and mitotically expansive in a collagen hydrogel delivery vehicle, and they secreted the immunomodulatory and neurotrophic paracrine factors interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and vascular endothelial growth factor (VEGF). cPMSCs also stimulated the growth of complex neural networks when co-cultured with SH-SY5Y cells, a neuroblastoma cell line used to model neuron growth in vitro. cPMSCs are analogous to human PMSCs. They meet the criteria to be defined as MSCs and represent a potential regenerative therapy option for neurological disorders in dogs with their robust growth in collagen hydrogel, stimulation of neural network formation, and secretion of potent paracrine factors. © 2017 International Society for Advancement of Cytometry.

Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery.

BACKGROUND: In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). METHODS: PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). RESULTS: PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DISCUSSION: PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

Age Does Matter: A Pilot Comparison of Placenta-Derived Stromal Cells for in utero Repair of Myelomeningocele Using a Lamb Model.

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.

The key components of Schwann cell-like differentiation medium and their effects on gene expression pattern of adipose-derived stem cells.

BACKGROUND: Schwann cell-like cells differentiated from adipose-derived stem cells may have an important role in peripheral nerve regeneration. Herein, we document the individual effects of growth factors in Schwann cell-like differentiation medium. METHODS: There were 6 groups in the study. In the control group, we supplemented the rat adipose-derived stem cells with normal cell culture medium. In group 1, we fed the cells with Schwann cell-like differentiation medium (normal cell culture medium supplemented with platelet-derived growth factor, basic fibroblast growth factor, forskolin, and glial growth factor). In the other groups, we removed the components of the medium one at a time from the differentiation medium so that group 2 lacked glial growth factor, group 3 lacked forskolin, group 4 lacked basic fibroblast growth factor, and group 5 lacked platelet-derived growth factor. We examined the expression of the Schwann cell-specific genes with quantitative reverse transcription polymerase chain reaction and immunofluorescence staining in each group. RESULTS: Groups 3 and 4, lacking forskolin and basic fibroblast growth factor, respectively, had the highest expression levels of integrin-ß4, and p75. Group 1 showed a 3.2-fold increase in the expression of S100, but the expressions of integrin-ß4 and p75 were significantly lower compared to groups 3 and 4. Group 2 [glial growth factor (-)] did not express significant levels of Schwann cell-specific genes. The gene expression profile in group 4 most closely resembled Schwann cells. Immunofluorescence staining results were parallel with the quantitative real-time polymerase chain reaction results. CONCLUSIONS: Glial growth factor is a key component of Schwann cell-like differentiation medium.

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.

Hypoxic Preconditioning Enhances Survival and Proangiogenic Capacity of Human First Trimester Chorionic Villus-Derived Mesenchymal Stem Cells for Fetal Tissue Engineering.

Prenatal stem cell-based regenerative therapies have progressed substantially and have been demonstrated as effective treatment options for fetal diseases that were previously deemed untreatable. Due to immunoregulatory properties, self-renewal capacity, and multilineage potential, autologous human placental chorionic villus-derived mesenchymal stromal cells (CV-MSCs) are an attractive cell source for fetal regenerative therapies. However, as a general issue for MSC transplantation, the poor survival and engraftment is a major challenge of the application of MSCs. Particularly for the fetal transplantation of CV-MSCs in the naturally hypoxic fetal environment, improving the survival and engraftment of CV-MSCs is critically important. Hypoxic preconditioning (HP) is an effective priming approach to protect stem cells from ischemic damage. In this study, we developed an optimal HP protocol to enhance the survival and proangiogenic capacity of CV-MSCs for improving clinical outcomes in fetal applications. Total cell number, DNA quantification, nuclear area test, and cell viability test showed HP significantly protected CV-MSCs from ischemic damage. Flow cytometry analysis confirmed HP did not alter the immunophenotype of CV-MSCs. Caspase-3, MTS, and Western blot analysis showed HP significantly reduced the apoptosis of CV-MSCs under ischemic stimulus via the activation of the AKT signaling pathway that was related to cell survival. ELISA results showed HP significantly enhanced the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CV-MSCs under an ischemic stimulus. We also found that the environmental nutrition level was critical for the release of brain-derived neurotrophic factor (BDNF). The angiogenesis assay results showed HP-primed CV-MSCs could significantly enhance endothelial cell (EC) proliferation, migration, and tube formation. Consequently, HP is a promising strategy to increase the tolerance of CV-MSCs to ischemia and improve their therapeutic efficacy in fetal clinical applications.

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.

In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.

Effect of 2-octylcyanoacrylate on placenta derived mesenchymal stromal cells on extracellular matrix.

PURPOSE: Determine the effect of 2-octylcyanoacrylate on placenta derived mesenchymal stromal cells (PMSCs) seeded onto extracellular matrix (ECM) in order to assess its biocompatibility as a potential adhesive for in-vivo fetal cell delivery. METHODS: PMSCs isolated from chorionic villus tissue were seeded onto ECM. A MTS proliferation assay assessed cellular metabolic activity at various time points in PMSC-ECM with direct, indirect, and no glue contact. Conditioned media collected prior to and 24 hours after glue exposure was analyzed for secretion of human brain-derived neurotrophic factor, hepatocyte growth factor, and vascular endothelial growth factor. RESULTS: Direct and indirect contact with 2-octylcyanoacrylate results in progressively decreased cellular metabolic activity over 24 hours compared to no glue controls. Cells with direct contact are less metabolically active than cells with indirect contact. 24 hours of glue exposure resulted in suppression of growth factor secretion that is near complete with direct contact. DISCUSSION: Exposure to 2-octylcyanoacrylate results in decreased metabolic activity and decreased measurable secretion of growth factors by PMSCs seeded onto ECM. Thus, the application of 2-octylcyanoacrylate glue should be limited when working with cell-engineered scaffolds as its inhibitory effects on cell growth and secretory function can limit the therapeutic potential of cell-based interventions.

Fetal surgical repair with placenta-derived mesenchymal stromal cell engineered patch in a rodent model of myelomeningocele.

PURPOSE: The purpose of this study is to determine the feasibility of fetal surgical repair of myelomeningocele (MMC) in a rodent model using human placental mesenchymal stromal cells (PMSCs) seeded onto extracellular matrix (ECM) and to characterize the resulting changes in spinal cord tissue. METHODS: Fetal rodents with retinoic acid (RA) induced MMC underwent surgical repair of the MMC defect using an ECM patch on embryonic age (EA) 19 and were collected via caesarean section on EA 21. Various seeding densities of PMSC-ECM and ECM only controls were evaluated. Cross-sectional compression (width/height) and apoptotic cell density of the lumbosacral spinal cord were analyzed. RESULTS: 67 dams treated with 40mg/kg of RA resulted in 352 pups with MMC defects. 121 pups underwent MMC repair, and 105 (86.8%) survived to term. Unrepaired MMC pups had significantly greater cord compression and apoptotic cell density compared to normal non-MMC pups. Pups treated with PMSC-ECM had significantly less cord compression and demonstrated a trend towards decreased apoptotic cell density compared to pups treated with ECM only. CONCLUSION: Surgical repair of MMC with a PMSC-seeded ECM disc is feasible with a postoperative survival rate of 86.8%. Fetal rodents repaired with PMSC-ECM have significantly less cord deformity and decreased histological evidence of apoptosis compared to ECM only controls.

Isolation of myogenic progenitor cell population from human placenta: A pilot study.

PURPOSE: The purpose of this study was to demonstrate a method of isolating myogenic progenitor cells from human placenta chorionic villi and to confirm the myogenic characteristics of the isolated cells. METHODS: Cells were isolated from chorionic villi of a second trimester male placenta via a combined enzymatic digestion and explant culture. A morphologically distinct subpopulation of elongated and multinucleated cells was identified. This subpopulation was manually passaged from the explant culture, expanded, and analyzed by fluorescence in situ hybridization (FISH) assay, immunocytochemistry, and flow cytometry. Myogenic characteristics including alignment and fusion were tested by growing these cells on aligned polylactic acid microfibrous scaffold in a fusion media composed of 2% horse serum in Dulbecco's modified Eagle medium/high glucose. RESULTS: The expanded subpopulation was uniformly positive for integrin α-7. Presence of Y-chromosome by FISH analysis confirmed chorionic villus origin rather than maternal cell contamination. Isolated cells grew, aligned, and fused on the microfibrous scaffold, and they expressed myogenin, desmin, and MHC confirming their myogenic identity. CONCLUSION: Myogenic progenitor cells can be isolated from human chorionic villi. This opens the possibility for translational and clinical applications using autologous myogenic cells for possible engraftment in treatment of chest and abdominal wall defects.

Placental mesenchymal stromal cells seeded on clinical grade extracellular matrix improve ambulation in ovine myelomeningocele.

PURPOSE: The purpose of this study was to investigate the effects of placental mesenchymal stromal cells (PMSCs) seeded on a clinical grade porcine small intestinal submucosa (SIS)-derived extracellular matrix (ECM) on hindlimb motor function in an ovine fetal repair model of myelomeningocele (MMC). METHODS: MMC defects were surgically created in 21 fetuses at median gestational age 78 (range 76-83) days. Fetuses were randomly assigned to repair 25days later with ECM only or PMSC-ECM. Surviving fetuses were delivered at term. Motor function was evaluated using the Sheep Locomotor Rating (SLR) scale (0-15). Histologic analysis of the spinal cord (SC) was completed. RESULTS: Fetal viability was 71%. 5 of 8 (63%) lambs repaired with PMSC-ECM ambulated independently versus only 1 of 6 (17%) repaired with ECM only (p=0.04, χ2 test). SLR scores and large neuron densities were higher in the PMSC-ECM group. The cross-sectional areas of the SC and the gray matter were equally preserved. CONCLUSIONS: Fetal repair of MMC with PMSCs seeded on SIS-ECM improves hindlimb motor function in lambs. Using ECM helps to preserve the architecture of the SC, but adding PMSCs improves the lamb's ability to walk and increases large neuron density. Clinical studies are needed to show benefits in humans. LEVELS OF EVIDENCE/TYPE OF STUDY: Basic Science.

Early gestation chorionic villi-derived stromal cells for fetal tissue engineering.

AIM: To investigate the potential for early gestation placenta-derived mesenchymal stromal cells (PMSCs) for fetal tissue engineering. METHODS: PMSCs were isolated from early gestation chorionic villus tissue by explant culture. Chorionic villus sampling (CVS)-size tissue samples (mean = 35.93 mg) were used to test the feasibility of obtaining large cell numbers from CVS within a clinically relevant timeframe. We characterized PMSCs isolated from 6 donor placentas by flow cytometry immunophenotyping, multipotency assays, and through immunofluorescent staining. Protein secretion from PMSCs was examined using two cytokine array assays capable of probing for over 70 factors in total. Delivery vehicle compatibility of PMSCs was determined using three common scaffold systems: fibrin glue, collagen hydrogel, and biodegradable nanofibrous scaffolds made from a combination of polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA). Viral transduction of PMSCs was performed using a Luciferase-GFP-containing lentiviral vector and efficiency of transduction was tested by fluorescent microscopy and flow cytometry analysis. RESULTS: We determined that an average of 2.09 × 10(6) (SD ± 8.59 × 10(5)) PMSCs could be obtained from CVS-size tissue samples within 30 d (mean = 27 d, SD ± 2.28), indicating that therapeutic numbers of cells can be rapidly expanded from very limited masses of tissue. Immunophenotyping by flow cytometry demonstrated that PMSCs were positive for MSC markers CD105, CD90, CD73, CD44, and CD29, and were negative for hematopoietic and endothelial markers CD45, CD34, and CD31. PMSCs displayed trilineage differentiation capability, and were found to express developmental transcription factors Sox10 and Sox17 as well as neural-related structural proteins NFM, Nestin, and S100ß. Cytokine arrays revealed a robust and extensive profile of PMSC-secreted cytokines and growth factors, and detected 34 factors with spot density values exceeding 10(3). Detected factors had widely diverse functions that include modulation of angiogenesis and immune response, cell chemotaxis, cell proliferation, blood vessel maturation and homeostasis, modulation of insulin-like growth factor activity, neuroprotection, extracellular matrix degradation and even blood coagulation. Importantly, PMSCs were also determined to be compatible with both biological and synthetic material-based delivery vehicles such as collagen and fibrin hydrogels, and biodegradable nanofiber scaffolds made from a combination of PLA and PLGA. Finally, we demonstrated that PMSCs can be efficiently transduced (> 95%) with a Luciferase-GFP-containing lentiviral vector for future in vivo cell tracking after transplantation. CONCLUSION: Our findings indicate that PMSCs represent a unique source of cells that can be effectively utilized for in utero cell therapy and tissue engineering.

Placental mesenchymal stromal cells rescue ambulation in ovine myelomeningocele.

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.