Feasibility of retroviral vector-mediated in utero gene transfer to the fetal rabbit

OBJECTIVES: Successful treatment or prevention of severe hereditary diseases could conceivably be achieved by genetic intervention early in development. Viral vector-mediated fetal gene transfer is proving a valuable tool to test the above concept in relevant animal models. Although the pregnant rabbit is a well-recognized model for fetal therapy, few preclinical assays have used it to validate fetal gene transfer approaches. In this preliminary study we assessed for the first time the feasibility of retroviral vector-mediated in utero gene transfer in the fetal rabbit. METHODS: Different amounts of the vesicular stomatitis virus G pseudotyped MFG(nls)LacZ retroviral vector, expressing a nuclear-localized beta-galactosidase reporter protein were injected intraperitoneally and -hepatically into 20- to 22-day-old fetuses. At 8-9 days post-treatment, the pups were sacrificed and the tissues harvested for analysis. Evidence of gene transfer was obtained by PCR amplification of proviral sequences within genomic DNA isolated from the treated samples. Transgenic beta-galactosidase expression was assessed by X-gal histochemical staining. RESULTS: By intraperitoneal injection 43% of the viable fetuses treated (3/7) showed evidence of successful LacZ gene transfer and low-level beta-galactosidase expression into liver and heart, whereas by intrahepatic injection roughly 38% (3/8) of the livers were positive for LacZ gene transfer and expression. The success rate for the viable fetuses rose to 67% positive livers (4/6) when a near double amount of recombinant virus was injected using a 10-fold concentrated virus stock. In terms of short-term safety, fetal and maternal survival rates approached 80% of treated fetuses, and 100% of treated does. CONCLUSIONS: The pregnant rabbit is a useful and reliable model allowing the design of further studies to optimize the conditions for effective, safer, and persistent retroviral vector-mediated fetal gene transfer.     

Ethical questions raised by in utero therapeutics with stem cells and gene therapy

The plasticity of living beings is limitless and our guidelines are constantly upset. Several ethical questions arise with this new in utero therapeutic approach: (1) The possibility of a mother giving truly informed consent when she is divided between a proposal to terminate her pregnancy and resignation to the birth of an abnormal child. How can she choose between three therapeutic proposals, when two of them are unbearable and only one carries some hope? (2) If a dead fetus is used as a donor of hepatic or hematologic stem cells, should this be based on the mother's consent? If so, what status is given to this fetus? Is it possible to take into consideration at the same time the growing demand to recognize the fetus as a person and his gift? (3) It is important to separate the services that receive dead fetuses from those that perform the injection of fetal stem cells so that the fetus does not become a mere therapeutic tool. (4) Even if the technique is not very difficult, can ex vivo transduction of mesenchymatous cells be performed as a kind of gene therapy without causing great anxiety over the embryo's germinal future? The main question is that even though surgery and medical therapeutics for the fetus have made great progress and become commonplace, the use of stem cell transplantations and eventually gene therapy privileges experimental medicine and research that are more and more difficult to conceive on an ethical level. If indeed fetal therapeutics seems to hold out promise for the future, the very speed with which we pass from conception to its experimental realization with a child must necessarily challenge our thinking.     

Ultrastructural evolution of nucleoli of female rat germ cells.

The nucleoli of oogonia and oocytes of rat ovaries during fetal and neonatal life were studied by light and electron microscopy. An evolution from atypical to typical nucleoli was shown. Nucleoli undergo a gradual increase in size and change from dense fibrillar bodies to complete nucleoli with pars fibrosa, pars granulosa, pars amorpha, and associated chromatin.     

Ultrasonographically guided direct gene transfer in utero: successful induction of beta-galactosidase in a rabbit model.

OBJECTIVE: We sought to determine whether the transfer of enzyme-encoding genes in utero can be detected after birth.Study Design: An adenoviral vector carrying the gene for beta-galactosidase was injected under ultrasonographic guidance into the livers of 4 rabbit fetuses per litter (3 litters total) at 27 days' gestation. On delivery of the pups 2 to 3 days later, the livers were analyzed for beta-galactosidase activity by using 5-bromo-4-chloro-3-indolyl-beta-D -galactopyranoside (X-gal) staining. Polymerase chain reaction was also performed on liver extracts as an additional independent measure of successful vector delivery. RESULTS: Successful targeting of the livers of fetal rabbits was demonstrated by beta-galactosidase activity in the nuclei of liver serosal cells, parenchymal hepatocytes, or columnar cells of the gallbladder in 7 (58%) of 12 injected pups and by polymerase chain reaction in liver extracts from 10 (83%) of 12 injected pups. CONCLUSIONS: These results suggest that vectors that carry genes for specific enzymes can be delivered to fetal organs in utero and that expression of the enzyme can be detected after delivery.     

Inefficient transduction of sheep in utero after intra-amniotic injection of retroviral producer cells

OBJECTIVE: Our purpose was to test the hypothesis that the intra-amniotic injection of a retroviral vector producer cell line into pregnant sheep will result in retrovirus-mediated transduction and stable gene transfer to the ovine fetus. STUDY DESIGN: Thirteen pregnant ewes at various gestational ages underwent amniocentesis and injection of cells producing the retrovirus vector LIRESgeo, which is derived from Maloney murine leukemia virus and encodes Escherichia coli LacZ (beta-galactosidase) as a marker gene. Pregnant ewes and fetuses were killed, and amniotic fluid, placenta, and fetal tissues were collected and assayed for transgene expression 7 to 77 days after intraamniotic injection. In addition, serum was collected and analyzed for evidence of specific immune responses against the producer cells, and amniotic fluid was collected and analyzed for deleterious effects on producer cell viability, vector production, and vector transduction. RESULTS: Only 1 of 10 fetuses exposed to the retroviral producer cells demonstrated beta-galactosidase activity that correlated with positive immunohistochemistry for LacZ in lung, trachea, pancreas, and small intestine. However, the presence of the LacZ gene could not be confirmed by polymerase chain reaction. Thus, we could not confirm transduction after any of the injections. The retroviral producer cells survived well in amniotic fluid and continued to produce high levels of retroviral vector after intra-amniotic injection, although amniotic fluid inhibited the transducing activity of the vector in a manner dependent on gestational age. CONCLUSIONS: Intra-amniotic retroviral gene transfer with the use of these amphotropic producer cells does not result in reproducible gene transfer in the ovine fetus although amniotic fluid sustains producer cell viability and vector production. Possible reasons for the inefficient transduction are discussed.     

High-efficiency retroviral transduction of fetal liver CD38-CD34++ cells: implications for in utero and ex utero gene therapy

OBJECTIVES: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. METHODS: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38-CD34++lineage- (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (Delta NGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 microg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10-21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. RESULTS: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). CONCLUSIONS: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.     

Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses

OBJECTIVE: Mesenchymal stem cells (MSC) have multiorgan differentiation capacity, providing the potential for prenatal treatment of genetic disorders. We address the question if in utero transplantation of MSC results in short-term organ-specific engraftment in the fetal sheep. STUDY DESIGN: Sheep fetal liver-derived MSC selected by adherence culture (passage 1) were transplantated into the fetal peritoneal cavity with ultrasound-guidance (mean gestational age, 59 days). After 14 days recipient fetuses were analyzed by fluorescence-activated cell sorting (FACS), real-time polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: Fetuses (n = 11) were transplanted with 7.7 x 10(6) MSCs (mean). All surviving fetuses (n = 5) showed engraftment with mean levels of 3.2% (lung), 0.8% (spleen), 0.6% (liver, brain), 0.4% (bone marrow), 0.1% (blood, thymus), and <0.1% (kidneys) by flow cytometry. Immunohistochemistry showed organ-specific distribution. CONCLUSION: In utero transplantation of allogeneic MSC results in low level, multiorgan engraftment at 14 days post transplant. This supports the potential of in utero MSC transplantation for the treatment of nonhematopoietic genetic disorders of the fetus.     

The sheep model of in utero gene therapy

Once its full clinical potential has been realized, hematopoietic stem cell based gene therapy (GT) promises to cure a wide array of both inborn and acquired diseases. For many genetic disorders, early onset and irreparable tissue and organ damage necessitate innovative methods that allow therapeutic intervention early in development, if a full cure is to be realized. Performing GT in utero would allow early correction prior to disease onset and is thus one of the few therapeutic modalities that could promise the birth of a healthy infant. Several features of the developing fetus may circumvent obstacles that have thus far been observed in GT trials. For example, the immune na?veté of the early gestational fetus may evade immune reactions to the vector and transgene product. Furthermore, fetal exposure to foreign antigens can result in sustained tolerance, suggesting that induction of tolerance to the vector/transgene product could allow postnatal treatment to be performed successfully. In addition to these immunologic advantages, the fetal hematopoietic system promises to be more amenable to retrovirus-mediated gene transfer than either the neonate or adult as a result of both proliferation and expansion of the stem/progenitor cell pool that take place during fetal development. To investigate whether these characteristics of the developing fetus could be used to advantage to efficiently transduce hematopoietic stem cells, we developed an approach to in utero GT, in which retroviral vectors were directly injected into the peritoneal cavity of preimmune fetal sheep. This approach resulted in the transfer and long-term (>5 years) expression of exogenous genes within the hematopoietic system of primary and secondary recipients, albeit at relatively low levels that would not likely be therapeutic in most diseases. These studies also demonstrated that the direct injection of retroviral vectors into preimmune fetal sheep results not only in the successful transduction of long-term engrafting hematopoietic stem cells, but also in the widespread distribution of vector to all other tissues examined, including the reproductive organs. In an effort to increase the hematopoietic cell transduction to clinically relevant levels, we repeated our initial studies with 1,000-fold higher titer vectors. This led to only a modest (two- to fourfold) increase in the transduction levels, suggesting that factors other than absolute vector dosage were responsible for the low levels of gene transfer. For this reason, we have more recently begun evaluating the effect of recipient gestational age on the efficiency of gene transfer to both hematopoietic and nonhematopoietic tissues. Thus far, we have observed an inverse relation between the gestational age at the time of vector administration and the level of transduction and expression of the transgene within the hematopoietic system, such that fetuses injected earlier in gestation have higher levels of hematopoietic cell transduction. These elevated levels have persisted for at least 1 year after injection, suggesting that the enhancement is at the level of primitive stem/progenitor cells. When analyzing the liver sections from animals that had received the vector at different gestational ages, we also observed an inverse correlation between recipient age and efficiency of gene transfer to the hepatocytes, such that a high efficiency of gene transfer occurred at early ages, while very little occurred at later stages of gestation. In contrast to the findings in the hematopoietic system and in the liver, analysis of the lungs of these same animals revealed that the efficiency of transduction of nonhematopoietic lung tissue increased with increasing gestational age. These results demonstrate that both hematopoietic cells and nonhematopoietic cells within liver and lung are transduced following direct injection of murine retroviral vector supernatants into the peritoneal cavity of preimmune fetal sheep and suggest that the developmental stage of each organ at the time of injection may determine its etermine its susceptibility to in utero gene transfer.     

Engraftment of human cord blood-derived stem cells in preimmune ovine fetuses after ultrasound-guided in utero transplantation

OBJECTIVE: The fetal sheep in utero transplantation model has developed into an important tool to study the efficacy of human in utero stem cell transplantation and gene therapy because of similarities in both the scale and development of immunocompetence relative to gestational age. The aim of this study was to determine whether human stem cells can be successfully transplanted to the first-trimester ovine fetus by use of a newly developed minimally invasive technique. STUDY DESIGN: Human cord blood-derived, CD34(+)-enriched stem cells were injected into the peritoneal cavity of 45- to 60-day-old ovine fetuses by using ultrasound-guided transabdominal percutaneous needle puncture. Engraftment was determined 1 to 3 months after birth by flow cytometry with use of human-specific anti-CD45 antibodies. RESULTS: In contrast to previous studies that used surgical techniques, we observed a fetal loss rate of 24%, significantly below previous values and only marginally higher than natural loss. Successful human cell engraftment was achieved in 18% of lambs available for analysis. Engraftment levels of human cells in bone marrow of the recipient were up to 0.8% of total nucleated cells. CONCLUSION: Ultrasound-guided percutaneous transplantation of stem cells to fetal sheep in the first trimester is feasible. Although we were unable to observe a significant improvement in the level of engraftment of human cells in sheep, the decreased fetal loss rate associated with this technique allows greater use for further studies that use this model of in utero transplantation.     

Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia

OBJECTIVE: To evaluate the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after repair of testicular torsion in rats. DESIGN: Controlled laboratory study. SETTING: Department of Urology, Yamagata University School of Medicine, Yamagata, Japan. ANIMAL(S): Fifty-one male Sprague-Dawley rats. INTERVENTION(S): Dexamethasone, 10 mg/kg of body weight. MAIN OUTCOME MEASURE(S): Testicular germ-cell apoptosis (percentages of apoptotic tubules and apoptotic cells) and vascular neutrophil adhesion were assessed by using DNA nick-end labeling and the endothelial-neutrophil adhesion score, respectively. RESULT(S): Intravenous administration of dexamethasone at repair of 90-minute testicular torsion significantly inhibited testicular germ-cell apoptosis and vascular neutrophil adhesion. This inhibition was suppressed by intravenous injection of mifepristone, a glucocorticoid-receptor antagonist. CONCLUSION(S): Glucocorticoids can be administered for torsion in addition to conventional torsion repair.     

Prolonged neonatal complications after in utero exposure to fluoxetine

This article presents the case of a female newborn with irritability, increased tonus, jitteriness, and eating difficulties who was referred to our institution. Her mother had been taking fluoxetine (20 mg daily) during the second and third trimesters of her pregnancy. The infant's signs began on the first day after birth and persisted for 6 weeks. Extensive investigations excluded infective, genetic, and metabolic causes, and a provisional diagnosis of fluoxetine withdrawal was made. There have been few reports of neonatal complications following maternal fluoxetine hydrochloride treatment. According to these, signs occurred within a few days after birth and lasted up to 4 weeks. To our knowledge, our patient demonstrated the longest duration of signs reported to date. We recommend that physicians be aware of these complications in newborns after fetal exposure in utero to selective serotonin reuptake inhibitors. These neonates should be followed-up closely for adverse signs during the first days of life so that signs can be recognized and treated if necessary.     

Cervical-vaginal adenosis after in utero exposure to synthetic estrogens

A study to determine the incidence of adenosis and/or adenocarcinoma of the vagina in 528 females between 13 and 25 years who were exposed to diethlstilbesterol in utero and to define the clinical and microscopic changes in their vaginas was undertaken, using primary physicians records as a source for patient identification. Lugol's staining was evaluated and was found to be a simple technique for identifying the lesion of adenosis. Of the young women with documented exposure to diethylstilbesterol or dienestrol, over 90% had adenosis of the vagina. In almost all cases the drug treatment began before the twelfth gestational week. In some patients, the appearance of the cervix was unusual. Of the 188 patients who underwent excisional biopsy, 2 showed a small focus of clear cell adenocarcinoma which was not detected clinically. A causal relationship between in utero exposure to diethylstilbesterol or dienestrol is suggested. It is also suggested that the lesion is developmental since it has been found in prepubertal girls.     

Pregnancy after gamete intrafallopian transfer in a woman with primary infertility and in utero exposure to diethylstilbestrol. A case report

In utero exposure to diethylstilbestrol (DES) has an adverse effect on reproductive performance and may be associated with infertility. Gamete intrafallopian transfer (GIFT) is a new reproductive technique that has been advocated as an alternative to in vitro fertilization in women with at least one normally functioning fallopian tube. The process involves the translaparoscopic placement of oocytes and sperm into the fallopian tube. The technique has been successful in treating infertility due to endometriosis, male factors and immunologic factors as well as unexplained infertility. We accomplished the first successful GIFT procedure in a woman with significant uterine effects from prenatal DES exposure. This technique may prove to be an effective treatment for infertile women with DES exposure who have no adequate explanation for their infertility.     

原始生殖细胞和胚胎生殖细胞发育多能性

精卵结合是动物藉以繁衍后代维持种族延续的基本方式,受精卵经过不断的分裂、分化产生内中外三个胚层以及支持胚胎发育的胚外组织,最终形成完整的个体。个体所有组成细胞均来源于受精卵且具有相同的基因型,因而受精卵作为整个发育程序（ｐｒｏｇｒａｍ）的起点其发育潜能（ｐｏｔｅｎｃｙ）为全能性（ｔｏｔｉｐｏｔｅｎｃｙ）。随着发育程序的推进,基因差次表达积累到一定程度导致分化细胞形成,在此过程中,发育潜能由全能、多能而至限能,最后发育全能性或多能性仅局限于少数细胞群,即不同类型的干细胞。有趣的是,具发育全能性的受…