| ObjectiveNeural tube defects (NTDs) are complex congenital malformations resulting from failure of complete neurulation during the fourth week of embryogenesis. Spina bifida and Anencephaly are the most common and severe forms of NTD, with a prevalence of about 1 in 1,000 births, although this varies throughout the world. Individuals with spina bifida have substantially enhanced survival rate thanks to recent improvements in medical and surgical management. However, these patients continue to be at increased risk for morbidity and mortality throughout their life, even though various prenatal or postnatal treatment strategies have been applied in clinic.Prenatal repair is an acceptable fetal surgery but with unproven benefits, updated reports suggest a reduced incidence of shunt-dependent hydrocephalus, as well as an improvement in hindbrain herniation. However, the expectations for improved neurological outcome have not been fulfilled and not all patients benefit from fetal surgery in the same way. Individuals with lumbosacral spina bifida continue to experience varying degrees of motor and sensory dysfunction of lower limbs and failure of anal and urethral sphincters after birth.Fetal cellular therapy is another option for the treatment of a variety of birth defects. There are several perceived advantages of in utero transplantation (IUT):(1) The rapid growth of the fetus provides opportunity for engraftment and expansion of donor cells, (2) the undeveloped immune system of the fetus cannot reject foreign tissues, and (3) early treatment of disease is beneficial or critical for effective treatment. Although transplantation of healthy cells into a fetus is suggested to offer the potential to treat a large number of birth defects (have therapeutic potential for a large number of genetic disorders), up to now, it is limited to the treatment of congenital hematologic disorders and immunodeficiency disease Only.Our previous studies have demonstrated the defective development of sensory and motor neurons in fetal rat spinal cord with spina bifida aperta, and the intrinsic neuron deficiency is a primary anomaly that coexists with the spinal malformation during fetal development, which likely to contribute to poor postoperative neural function (urea and rectal incontinience and leg paralysis) despite surgical repair. Thus we supposed that treatment based on neuron regeneration or replacement should be an alternative choice to achieve better functional outcome in spina bifida patients. Concerning regeneration medicine, mesenchymal stem cell transplantation has been widely used due to their convenient isolation, their lack of significant immunogenicity permitting allogenic transplantation without immunosuppressive drugs, their lack of ethical controversy, and their potential to differentiate into tissue-specific cell types with trophic activity. Based on all these, in this study we for the first time treated spina bifida aperta with in utero bone marrow derived mesenchymal stem cell (MSCs) transplantation using rat model.Method1. Experimental animal(1) MSCs primary culture Forty Wister rats aging 4 weeks, weighting 90-100g were involved. All rats were provided by laboratory of Affiliated Shengjing Hospital,China Medical University.(2) Animal model Outbred wister rats of 10-12 weeks of age (250-300gm,100 rats)purchased from animal center of china medical university were used in this study.2. Method(1) Primary culture and Transfection In sterile condition, rat marrow was flushed and cells were seeded in culture flask. Passage 0 to Passage 14 of marrow MSCs were transfected by adenovirus-EGFP(MOI value 300-500).(2) Rat congenital spina bifida model The appearance of vaginal plugs in the female rat the morning after mating was timed as the embryonic day 0(E0). Spinal bifida aperta were induced with a single intragastric 140g/kg retinoic acid administration on E10 morning.(3) Cell transplantation By fetus surgery EGFP transfected MSCs were injected to spinal cord through fine glass needle. Repair pregnant rats'uterus and feed them until day 20 gestation.(4) Cryosections and Counting cells Until day 20 gestation, pregnant rat underwent operation again. Spinal cord of fetus rats were removed following fixation, dehydration. Count cells with green fluorescence in frozen section.(5) Differentiation of transplanted cells Expression of Nestin, NF, GFAP and MyoD in transplanted MSCs were tested by immunofluorescence.Result1. Flowcytometry analysis showed the purity of our culture MSC was increased with longer culture.2. Total 86 pregnant rats received fetal surgery and microinjection on E16,E17 or E18,and 5 died before E20,81 rats were sacrificed on E20 for spine sample collection. The overall survival rate of rat fetus after fetal surgery was 94%. On average,2 to 3 fetuses could bi injected in one dam. Most of the operated fetus (> 75%) could survive after transplantation,and totally 24% of transplanted MSCs survived on fetal spinal vertebrate region.3. There is a tendency of higher survival rate with more cells injected, it is not statistically significant (P) 0,05). The passages of injected MSCs, only P0 cells showed lower survival rate,cells in P3-6 or in P10-14 presented similar surviving rate. it is not statistically significant (P) 0,05). The third factor is the stages of fetal development. Less eGFP positive MSCs survived on E16 fetal spinal vertebrate than on E17 and E18. MSCs migrated into different region of spinal vertebrate away from the injection site.4. During the 2-4 days of development transplanted MSCs defferented into various cell types of spinal vertebra,as eGFP positive MSCs expressed the specific markers of neural stem cell (Nestin), neuron (neurofilament), neurogliocyte (GFAP) and myoblast (MyoD) after transplantation. Concerning the effect of cell passages on MSCs showed higher neuronal differentiation rate in P10-14 cell group compared with in P3-6 cell group (P<0.01). Development stages also affect neual differentiation of MSCs, MSCs survived on E16 spinal vertebrate have significantly higher neuronal lineage marker expression compared with E17 and E18 group (P<0.01).Conclusion 1. It is an acceptable technique that we treated spina bidida aperta with in utero bone marrow derived mesenchymal stem (MSCs) transplantation using rat model by fetus surgery.2. Transplanted MSCs differentiate into neural stem cell, neuron, neurogliocyte and myoD in fetus spinal cord.3. The better neural differentiation obtained in this study with longer culture MSCs transplanted onto E16 rat fetus, which is the best condition in fetus transplantation. |
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