Intra-amniotic mesenchymal stem cell therapy improves the amniotic fluid microenvironment in rat spina bifida aperta fetuses.

OBJECTIVES: Spina bifida aperta (SBA) is one of the most common neural tube defects. Neural injury in SBA occurs in two stages involving failed neural tube closure and progressive degeneration through contact with the amniotic fluid. We previously suggested that intra-amniotic bone marrow-derived mesenchymal stem cell (BMSC) therapy for fetal rat SBA could achieve beneficial functional recovery through lesion-specific differentiation. The aim of this study is to examine whether the amniotic fluid microenvironment can be improved by intra-amniotic BMSC transplantation. METHODS: The intra-amniotic BMSC injection was performed using in vivo rat fetal SBA models. The various cytokine expressions in rat amniotic fluid were screened by protein microassays. Intervention experiments were used to study the function of differentially expressed cytokines. RESULTS: A total of 32 cytokines showed significant upregulated expression in the BMSC-injected amniotic fluid. We focused on Activin A, NGF, BDNF, CNTF, and CXCR4. Intervention experiments showed that the upregulated Activin A, NGF, BDNF, and CNTF could inhibit apoptosis and promote synaptic development in fetal spinal cords. Inhibiting the activity of these factors weakened the anti-apoptotic and pro-differentiation effects of transplanted BMSCs. Inhibition of CXCR4 activity reduced the engraftment rate of BMSCs in SBA fetuses. CONCLUSION: BMSC transplantation can improve the amniotic fluid environment, and this is beneficial for SBA repair.

Intra-amniotic transplantation of brain-derived neurotrophic factor-modified mesenchymal stem cells treatment for rat fetuses with spina bifida aperta.

BACKGROUND: Spina bifida aperta (SBA) is a relatively common clinical type of neural tube defect. Although prenatal fetal surgery has been proven to be an effective treatment for SBA, the recovery of neurological function remains unsatisfactory due to neuron deficiencies. Our previous results demonstrated that intra-amniotic transplanted bone marrow mesenchymal stem cells (BMSCs) could preserve neural function through lesion-specific engraftment and regeneration. To further optimize the role of BMSCs and improve the environment of defective spinal cords so as to make it more conducive to nerve repair, the intra-amniotic transplanted BMSCs were modified with brain-derived neurotrophic factor (BDNF-BMSCs), and the therapeutic potential of BDNF-BMSCs was verified in this study. METHODS: BMSCs were modified by adenovirus encoding a green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) in vitro and then transplanted into the amniotic cavity of rat fetuses with spina bifida aperta which were induced by all-trans-retinoic acid on embryonic day 15. Immunofluorescence, western blot and real-time quantitative PCR were used to detect the expression of different neuron markers and apoptosis-related genes in the defective spinal cords. Lesion areas of the rat fetuses with spina bifida aperta were measured on embryonic day 20. The microenvironment changes after intra-amniotic BDNF-BMSCs transplantation were investigated by a protein array with 90 cytokines. RESULTS: We found that BDNF-BMSCs sustained the characteristic of directional migration, engrafted at the SBA lesion area, increased the expression of BDNF in the defective spinal cords, alleviated the apoptosis of spinal cord cells, differentiated into neurons and skin-like cells, reduced the area of skin lesions, and improved the amniotic fluid microenvironment. Moreover, the BDNF-modified BMSCs showed a better effect than pure BMSCs on the inhibition of apoptosis and promotion of neural differentiation. CONCLUSION: These findings collectively indicate that intra-amniotic transplanted BDNF-BMSCs have an advantage of promoting the recovery of defective neural tissue of SBA fetuses.

Transfection of STAT3 overexpression plasmid mediated through recombinant lentivirus promotes differentiation of bone marrow mesenchymal stem cells into neural cells in fetal rats with spina bifida aperta.

We investigated the influence of signal transducer and activator of transcription-3 (STAT3) on the spinal cord tissue grafts of rat fetuses with spina bifida aperta. In particular, we hoped to identify whether transfection of the STAT3 overexpression plasmid increases the survival of spinal cord transplantation in order to improve therapeutic efficacy. The fetal rat model of spina bifida aperta was established using retinoic acid and treated with a microsurgical injection of bone marrow mesenchymal stem cells (BMSCs). The animals were divided into either the blank control group, negative control group or the experimental group. The optical density (OD) value of BMSCs viability was determined using the Cell Counting Kit-8 (CCK-8). The expression of STAT3, phosphorylated STAT3 (pSTAT3), neural markers and apoptosis-related factors were evaluated using real-time PCR and Western blot. The OD value in the experimental group was highest at eight hours after transplantation using CCK-8. The expression of pSTAT3, glial fibrillary acidic protein, neuron-specific enolase, neurofilament and nestin in the experimental group was significantly higher compared to the blank control group and negative control group (P<0.05). However, STAT3 expression in the experimental group was statistically significantly decreased (P<0.05). The relative expression of caspase-8 and bcl-2 in the experimental group were significantly lower compared to the blank control group and negative control group (P<0.05). Transfection of the recombinant lentivirus-mediated STAT3 overexpression plasmid with BMSCs can help improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.

Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model.

Spina bifida aperta is a type of neural tube defect (NTD). Although prenatal fetal surgery has been an available and effective treatment for it, the neurological functional recovery is still need to be enhanced. Our previous results revealed that deficiencies of sensory, motor, and parasympathetic neurons were primary anomalies that occurred with the spinal malformation. Therefore, we emphasized that nerve regeneration is critical for NTD therapy. We delivered an adenoviral construct containing genes inserted for green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) into the amniotic fluid to investigate its prenatal therapeutic potential for rat fetuses with spina bifida aperta. Using immunofluorescence, TdT-mediated dUTP nick-end labeling staining, and real-time polymerase chain reaction analysis, we assessed cell apoptosis in the defective spinal cord and Brn3a positive neuron survival in the dorsal root ganglion (DRG); a protein array was used to investigate the microenvironmental changes of the amniotic fluid. We found that most of the overexpressed BDNF was present on the lesions of the spina bifida fetuses, the number of apoptosis cells in Ad-GFP-BDNF-transfected spinal cords were reduced, mRNA levels of Bcl2/Bax were upregulated and Casp3 were downregulated compared with the controls, the proportion of Brn3a positive neurons in DRG were increased by activating the BDNF/TrkB/Akt signaling pathway, and most of the significant changes in cytokines in the amniotic fluid were related to the biological processes of regulation of apoptotic process and generation of neurons. These results suggest that intra-amniotic Ad-GFP-BDNF gene delivery might have potential as a supplementary approach to treat congenital malformations of neural tubes.

Fetal myelomeningocele diagnosed in the antenatal period: Maternal-fetal characteristics and their relationship with pregnancy decision-making.

BACKGROUND: Spina bifida is the most common fetal anomaly of the central nervous system, which affects approximately 1:1000 live births in the United States. Myelomeningocele (MMC) is the most common presentation of spina bifida, representing half of these cases. Given the deformation to the spinal cord and the nerve roots, this defect may result in significant morbidity to infants and major life-long disabilities. In this study we aimed to identify maternal and fetal characteristics associated with expectant management or termination of pregnancy in the setting of antenatally diagnosed MMC. We hypothesized that the level of the defect would correlate with patient's decision to continue the pregnancy. METHODS: A retrospective cohort analysis was performed with patients who had presented to the Cleveland Clinic Fetal Care Center between 2005-2017. RESULTS: Our data showed 36% of patients with antenatal diagnosis of MMC elected for second trimester terminations versus 64% who chose to continue their pregnancy and deliver either by cesarean section or vaginal delivery. Based on ultrasound findings, there were no significant differences between these two groups. Maternal body mass index was significantly higher in those who continued pregnancies (p = 0.036). In addition, the fetal diagnostic methods chosen by patients were significantly different. Those who elected to terminate were more likely to pursue amniocentesis (p = 0.03) and less likely to opt for MRI characterization of the fetus (p = 0.007). CONCLUSION: We conclude, in the setting of fetal MMC diagnosed during pregnancy, patients often rely less on the associated ultrasonographic findings. Personal decisions likely influence the choice of other fetal diagnostic modalities. Other than BMI, we did not see an association between maternal factors and decisions regarding second trimester pregnancy termination.

[To act or not to act? Developments in prenatal and postnatal care for children with spina bifida aperta]. / Handelen of niet handelen?

To act or not to act? Developments in prenatal and postnatal care for children with spina bifida aperta Until the middle of the twentieth century, newborns with spina bifida aperta had low chances of survival. Advances in the treatment of hydrocephalus, among other conditions, led to increased chances of survival during the 1960s. This also revealed the downsides of the treatment of spina bifida patients since some considered the quality of life of a number of these patients to be unacceptable. But withholding treatment also had negative consequences, leading to an ethical deadlock. Over the past thirty years - besides postnatal closure of the neural tube defect - more emphasis has been put on selective pregnancy termination and sporadic active termination of life in newborns with very severe forms of spina bifida. At the same time, new treatment strategies, such as foetal surgery, are being developed. With this historical overview, we illustrate the way in which technological developments and ethical dilemmas are constantly affecting each other.

Contemporary management of prenatally diagnosed spina bifida aperta - an update.

Spina bifida aperta is a relatively common congenital defect that occurs in the general population. Once the disorder has been diagnosed, a discussion, that can be emotionally-charged, ensues about whether to treat it prenatally or to only offer surgery postnatally. Given that there are good arguments for and against both options, it is of paramount importance to gain a good understanding of the major advantages and disadvantages of the various surgical approaches. The aim of our paper is to summarize current knowledge about spina bifida and the potential benefits of prenatal surgery.

Reverse Thermal Gel for In Utero Coverage of Spina Bifida Defects: An Innovative Bioengineering Alternative to Open Fetal Repair.

Current state-of-the-art management of open spina bifida defects entails an open fetal surgery approach associated with significant morbidities. In an attempt to reduce these risks and provide for an earlier minimally invasive repair, it is aimed to develop and characterize an innovative alternative using a unique reverse thermal gel. This study focuses on characterization of the physical and biological properties of the polymer and its in vivo applicability. Based on the knowledge and benchmarking, the "ideal" biomaterial should have the following characteristics: stability in amniotic fluid, limited permeability, biocompatibility, biologically functional, nontoxic, ability to support cellular functions, and in vivo applicability. The results demonstrate that the polymer possesses a unique ultrastructure, is stable in amniotic fluid, possesses limited yet predictable permeability, biocompatible with cells exposed in neural tube defects, is nontoxic, and can support cellular migration. These characteristics make it a potential novel alternative to open fetal repairs.

Application potential of bone marrow mesenchymal stem cell (BMSCs) based tissue-engineering for spinal cord defect repair in rat fetuses with spina bifida aperta.

Spina bifida aperta are complex congenital malformations resulting from failure of fusion in the spinal neural tube during embryogenesis. Despite surgical repair of the defect, most patients who survive with spina bifida aperta have a multiple system handicap due to neuron deficiency of the defective spinal cord. Tissue engineering has emerged as a novel treatment for replacement of lost tissue. This study evaluated the prenatal surgical approach of transplanting a chitosan-gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in the healing the defective spinal cord of rat fetuses with retinoic acid induced spina bifida aperta. Scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMSCs. After transplantation of the scaffold combined with BMSCs, the defective region of spinal cord in rat fetuses with spina bifida aperta at E20 decreased obviously under stereomicroscopy, and the skin defect almost closed in many fetuses. The transplanted BMSCs in chitosan-gelatin scaffold survived, grew and expressed markers of neural stem cells and neurons in the defective spinal cord. In addition, the biomaterial presented high biocompatibility and slow biodegradation in vivo. In conclusion, prenatal transplantation of the scaffold combined with BMSCs could treat spinal cord defect in fetuses with spina bifida aperta by the regeneration of neurons and repairmen of defective region.

Current Management Strategies of Hydrocephalus in the Child With Open Spina Bifida.

Symptomatic hydrocephalus is a common condition associated with myelomeningocele (open spina bifida). Traditionally, hydrocephalus was treated with insertion of a ventriculo-peritoneal (VP) shunt. This has been the standard of treatment since the introduction of the Holter shunt valve for the VP shunt in the late 1950s. Now there are other treatments that offer alternatives to VP shunt diversion for hydrocephalus. This article is a review of hydrocephalus associated with myelomeningocele and its treatment options. Treatment in the form of a VP shunt, endoscopic third ventriculostomy (ETV), and conservative management are discussed.

Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida.

Myelomeningocele (MMC) is a devastating spinal cord birth defect, which results in significant life-long disabilities, impaired quality of life, and difficult medical management. The pathological progression of MMC involves failure in neural tube and vertebral arch closure at early gestational ages, followed by subsequent impairment in spinal cord and vertebral growth during fetal development. MMC is irreversible at term. Thus, prenatal therapeutic strategies that interrupt progressive pathological processes offer an appealing approach for treatment of MMC. However, a thorough understanding of pathological progression of MMC is mandatory for appropriate treatment to be rendered. This article is part of a Special Issue entitled SI: Spinal cord injury.

Different expression patterns of growth factors in rat fetuses with spina bifida aperta after in utero mesenchymal stromal cell transplantation.

BACKGROUND AIMS: In a previous study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stromal cells (MSCs) to fetal rat spinal column to treat retinoic acid-induced neural tube defects in rat. Our results show that MSCs survived, migrated and differentiated into neural lineage cells. We intended to study various growth factor expressions in rat fetal spinal cords with spina bifida aperta after in utero MSC transplantation and the effect of in vivo growth factor introduction for prenatal spina bifida treatment. METHODS: Pregnant rats were treated with retinoic acid on embryonic day 10 and then received fetal surgery for MSC transplantation and/or lentiviral epidermal growth factor (EGF) injection on embryonic day 16; various growth factor expression in spinal cords from embryonic day 20 fetuses were analyzed by means of quantitative reverse transcriptase-polymerase chain reaction. Terminal deoxynucleotidyl transferase dUTP nick end labeling analysis was performed to observe spinal tissue apoptosis. RESULTS: Growth factor expression was dysregulated in spinal cords with spina bifida. After MSC transplantation, we observed significantly increased expression of EGF, fibroblast growth factor (FGF)-8, FGF-2 and FGF-20 in the MSC transplantation group compared with blank injection; Furthermore, EGF expression positively correlated with surviving MSC amounts. Expression of other growth factors was not significantly different. In vivo EGF introduction reduced spinal tissue apoptosis. CONCLUSIONS: Our results suggest that intrinsic EGF and FGF-2, FGF-8 and FGF-20 might affect the in vivo fate of transplanted MSCs in a fetal rat spina bifida model. In vivo EGF introduction together with MSC transplantation might serve as a new strategy for prenatal spina bifida treatment.

Fetal surgery for spina bifida: past, present, future.

Open spina bifida or myelomeningocele (MMC) is a common birth defect that is associated with significant lifelong morbidity. Little progress has been made in the postnatal surgical management of the child with spina bifida. Postnatal surgery is aimed at covering the exposed spinal cord, preventing infection, and treating hydrocephalus with a ventricular shunt. Experimental and clinical evidence suggest that the primary cause of the neurologic defects associated with MMC is not simply incomplete neurulation, but rather chronic, mechanical and amniotic-fluid induced chemical trauma that progressively damages the exposed neural tissue during gestation. The cerebrospinal fluid leak through the MMC leads to hindbrain herniation and hydrocephalus. In utero repair of open spina bifida is now performed in selected patients and presents an additional therapeutic alternative for expectant mothers carrying a fetus with MMC. In the past, studies in animal models and clinical case series laid the groundwork for a clinical trial to test the safety and efficacy of fetal MMC repair. In the present, a prospective, randomized study (the MOMS trial) has shown that fetal surgery for MMC before 26 weeks' gestation may preserve neurologic function, reverse the hindbrain herniation of the Chiari II malformation, and obviate the need for postnatal placement of a ventriculoperitoneal shunt. However, this study also demonstrates that fetal surgery is associated with significant risks related to the uterine scar and premature birth. In the future, research will expand our understanding of the pathophysiology of MMC, evaluate the long-term impact of in-utero intervention, and to refine timing and technique of fetal MMC surgery using tissue engineering technology.

Therapeutic potential of in utero mesenchymal stem cell (MSCs) transplantation in rat foetuses with spina bifida aperta.

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.

Neoskin development in the fetus with the use of a three-layer graft: an animal model for in utero closure of large skin defects.

OBJECTIVE: To assess the ability of a three-layer graft in the closuse of large fetal skin defects. METHODS: Ovine fetuses underwent a large (4 × 3 cm) full-thickness skin defect over the lumbar region at 105 days' gestation (term = 140 days). A bilaminar artificial skin was placed over a cellulose interface to cover the defect (3-layer graft). The skin was partially reapproximated with a continuous nylon suture. Pregnancy was allowed to continue and the surgical site was submitted to histopathological analysis at different post-operative intervals. RESULTS: Seven fetuses underwent surgery. One maternal/fetal death occurred, and the remaining 6 fetuses were analyzed. Artificial skin adherence to the wound edges was observed in cases that remained in utero for at least 15 days. Neoskin was present beneath the silicone layer of the bilaminar artificial skin. CONCLUSIONS: Our study shows that neoskin can develop in the fetus using a 3-layer graft, including epidermal growth beneath the silicone layer of the bilaminar skin graft. These findings suggest that the fetus is able to reepithelialise even large skin defects. Further experience is necessary to assess the quality of this repair.

Neonatal euthanasia.

Despite advances in the care of infants, there remain many newborns whose medical conditions are incompatible with sustained life. At times, healthcare providers and parents may agree that prolonging life is not an appropriate goal of care, and they may redirect treatment to alleviate suffering. While pediatric palliative treatment protocols are gaining greater acceptance, there remain some children whose suffering is unrelenting despite maximal efforts. Due to the realization that some infants suffer unbearably (ie, the burdens of suffering outweigh the benefits of life), the Dutch have developed a protocol for euthanizing these newborns. In this review, I examine the ethical aspects of 6 forms of end of life care, explain the ethical arguments in support of euthanasia, review the history and verbiage of the United States regulations governing limiting and withdrawing life-prolonging interventions in infants, describe the 3 categories of neonates for whom the Dutch provide euthanasia, review the published analyses of the Dutch protocol, and finally present some practical considerations should some form of euthanasia ever be deemed appropriate.