Human muscular fetal cells: a potential cell source for muscular therapies.

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Whole-cell bioprocessing of human fetal cells for tissue engineering of skin.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity (simple culture conditions), maintenance of differentiated phenotype for primary human cell culture and transmission of communicable diseases. Cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. Master and working cell banks from one fetal organ donation (skin) can be developed in short periods of time and safety tests can be performed at all stages of cell banking. For therapeutic use, fetal cells can be used up to two thirds of their life-span in an out-scaling process and consistency for several biological properties includes protein concentration, gene expression and biological activity. As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in out-scaling for the preparation of whole-cell tissue-engineering products.

Heterogeneity of the induction of HLA-DR expression by human immune interferon on glioma cell lines and their clones.

The modulation of HLA-DR and HLA-A, -B, and -C by human recombinant immune interferon (IFN-gamma) was studied on 10 malignant glioma cell lines established in our laboratory, on 8 clones or subclones derived from these lines, and on a fetal astrocyte cell line. Comparative studies were performed with recombinant leukocyte interferon (IFN-alpha). The results not only confirmed the selective activity of IFN-gamma on the modulation of HLA-DR expression, as opposed to that of IFN-alpha, but also demonstrated a marked heterogeneity in the response of glioma cell lines and their clones to the two types of IFN tested. For example, all 3 clones of an inducible cell line could be modulated to express HLA-DR, whereas only 2 of 5 clones derived from a noninducible line were modulated. This heterogeneity did not seem to be due to the absence of the receptor for IFN-gamma on the surface of these cells, since almost all of the cell lines or clones tested (17 of 19) responded to IFN-gamma by the induction or enhancement of the expression for either HLA-DR or HLA-A, -B, and -C (or both). The heterogeneity of induction was also demonstrated between clones derived from a glioma line that did not express HLA-DR after IFN-gamma treatment. The production of HLA-DR by one of the clones was abundant enough to be confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Dexamethasone stimulates the biochemical differentiation of fetal forebrain cells in reaggregating cultures.

The influence of dexamethasone on the development of neurons and oligodendrocytes was studied in serum-free, aggregating rat brain cell cultures. Synaptogenesis and myelination occur in this culture system. The concentration of myelin basic protein and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase were used as oligodendroglia and myelin markers. Choline acetyltransferase and acetylcholinesterase served as neuronal markers, glutamine synthetase reflected astrocyte differentiation, while ornithine decarboxylase served as a general marker for cell growth and maturation. This study showed that dexamethasone stimulated the differentiation of cholinergic neurons and astrocytes. The effect of dexamethasone on oligodendroglial differentiation and myelination depended on the stage of development: during the early phase of myelination dexamethasone had a stimulatory effect, whereas at a later stage it showed a significant inhibition.

Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein.

INTRODUCTION: Intrauterine growth restriction (IUGR) affects ∼8% of all pregnancies and is associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiovascular diseases in adulthood. Despite identification of several risk factors, the mechanisms implicated in the development of IUGR remain poorly understood. In case of placental insufficiency, reduced delivery of oxygen and/or nutrients to the fetus could be associated with alterations in the umbilical circulation, contributing further to the impairment of maternal-fetal exchanges. We compared the structural and functional properties of umbilical cords from growth-restricted and appropriate for gestational age (AGA) term newborns, with particular attention to the umbilical vein (UV). METHODS: Human umbilical cords were collected at delivery. Morphological changes were investigated by histomorphometry, and UV's reactivity by pharmacological studies. RESULTS: Growth-restricted newborns displayed significantly lower growth parameters, placental weight and umbilical cord diameter than AGA controls. Total cross-section and smooth muscle areas were significantly smaller in UV of growth-restricted neonates than in controls. Maximal vasoconstriction achieved in isolated UV was lower in growth-restricted boys than in controls, whereas nitric oxide-induced relaxation was significantly reduced in UV of growth-restricted girls compared to controls. CONCLUSION: IUGR is associated with structural alterations of the UV in both genders, and with a decreased nitric oxide-induced relaxation in UV of newborn girls, whereas boys display impaired vasoconstriction. Further investigations will allow to better understand the regulation of umbilical circulation in growth-restricted neonates, which could contribute to devise potential novel therapeutic strategies to prevent or limit the development of IUGR.

Congenital diaphragmatic hernia: current status and review of the literature.

Treatment of congenital diaphragmatic hernia (CDH) challenges obstetricians, pediatric surgeons, and neonatologists. Persistent pulmonary hypertension (PPHT) associated with lung hypoplasia in CDH leads to a high mortality rate at birth. PPHT is principally due to an increased muscularization of the arterioles. Management of CDH has been greatly improved by the introduction of prenatal surgical intervention with tracheal obstruction (TO) and by more appropriate postnatal care. TO appears to accelerate fetal lung growth and to increase the number of capillary vessels and alveoli. Improvement of postnatal care over the last years is mainly due to the avoidance of lung injury by applying low peak inflation pressure during ventilation. The benefits of other drugs or technical improvements such as the use of inhaled nitric oxide or extracorporeal membrane oxygenation (ECMO) are still being debated and no single strategy is accepted worldwide. Despite intensive clinical and experimental research, the treatment of newborn with CDH remains difficult.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Neuronal nitric oxide synthase does not contribute to the modulation of pulmonary vascular tone in fetal lambs with congenital diaphragmatic hernia (nNOS in CDH lambs).

AIM: The aim of this study was to determine the presence of the neuronal nitric oxide synthase (nNOS) in near full-term lambs with congenital diaphragmatic hernia (CDH) and its role in the modulation of pulmonary vascular basal tone. METHODS: We surgically created diaphragmatic hernia on the 85th day of gestation. On the 135th, catheters were used to measure pulmonary pressure and blood flow. We tested the effects of 7-nitroindazole (7-NINA), a specific nNOS antagonist and of N-nitro-L-arginine (L-NNA), a nonspecific nitric oxide synthase antagonist. In vitro, we tested the effects of the same drugs on isolated pulmonary vessels. The presence of nNOS protein in the lungs was detected by Western blot analysis. RESULTS: Neither 7-NINA nor L-NNA modified pulmonary vascular basal tone in vivo. After L-NNA injection, acetylcholine (ACh) did not decrease significantly pulmonary vascular resistance (PVR). In vitro, L-NNA increased the cholinergic contractile-response elicited by electric field stimulation (EFS) of vascular rings from lambs with diaphragmatic hernia. CONCLUSION: We conclude that nNOS protein is present in the lungs and pulmonary artery of near full-term lamb fetuses with diaphragmatic hernia, but that it does not contribute to the reduction of pulmonary vascular tone at birth

A post-processing pipeline to reconstruct the developing fetal brain using low-resolution MRI

Introduction. Development of the fetal brain surfacewith concomitant gyrification is one of the majormaturational processes of the human brain. Firstdelineated by postmortem studies or by ultrasound, MRIhas recently become a powerful tool for studying in vivothe structural correlates of brain maturation. However,the quantitative measurement of fetal brain developmentis a major challenge because of the movement of the fetusinside the amniotic cavity, the poor spatial resolution,the partial volume effect and the changing appearance ofthe developing brain. Today extensive efforts are made todeal with the âeurooepost-acquisitionâeuro reconstruction ofhigh-resolution 3D fetal volumes based on severalacquisitions with lower resolution (Rousseau, F., 2006;Jiang, S., 2007). We here propose a framework devoted tothe segmentation of the basal ganglia, the gray-whitetissue segmentation, and in turn the 3D corticalreconstruction of the fetal brain. Method. Prenatal MRimaging was performed with a 1-T system (GE MedicalSystems, Milwaukee) using single shot fast spin echo(ssFSE) sequences in fetuses aged from 29 to 32gestational weeks (slice thickness 5.4mm, in planespatial resolution 1.09mm). For each fetus, 6 axialvolumes shifted by 1 mm were acquired (about 1 min pervolume). First, each volume is manually segmented toextract fetal brain from surrounding fetal and maternaltissues. Inhomogeneity intensity correction and linearintensity normalization are then performed. A highspatial resolution image of isotropic voxel size of 1.09mm is created for each fetus as previously published byothers (Rousseau, F., 2006). B-splines are used for thescattered data interpolation (Lee, 1997). Then, basalganglia segmentation is performed on this superreconstructed volume using active contour framework witha Level Set implementation (Bach Cuadra, M., 2010). Oncebasal ganglia are removed from the image, brain tissuesegmentation is performed (Bach Cuadra, M., 2009). Theresulting white matter image is then binarized andfurther given as an input in the Freesurfer software(http://surfer.nmr.mgh.harvard.edu/) to provide accuratethree-dimensional reconstructions of the fetal brain.Results. High-resolution images of the cerebral fetalbrain, as obtained from the low-resolution acquired MRI,are presented for 4 subjects of age ranging from 29 to 32GA. An example is depicted in Figure 1. Accuracy in theautomated basal ganglia segmentation is compared withmanual segmentation using measurement of Dice similarity(DSI), with values above 0.7 considering to be a verygood agreement. In our sample we observed DSI valuesbetween 0.785 and 0.856. We further show the results ofgray-white matter segmentation overlaid on thehigh-resolution gray-scale images. The results arevisually checked for accuracy using the same principlesas commonly accepted in adult neuroimaging. Preliminary3D cortical reconstructions of the fetal brain are shownin Figure 2. Conclusion. We hereby present a completepipeline for the automated extraction of accuratethree-dimensional cortical surface of the fetal brain.These results are preliminary but promising, with theultimate goal to provide âeurooemovieâeuro of the normal gyraldevelopment. In turn, a precise knowledge of the normalfetal brain development will allow the quantification ofsubtle and early but clinically relevant deviations.Moreover, a precise understanding of the gyraldevelopment process may help to build hypotheses tounderstand the pathogenesis of several neurodevelopmentalconditions in which gyrification have been shown to bealtered (e.g. schizophrenia, autismâeuro¦). References.Rousseau, F. (2006), 'Registration-Based Approach forReconstruction of High-Resolution In Utero Fetal MR Brainimages', IEEE Transactions on Medical Imaging, vol. 13,no. 9, pp. 1072-1081. Jiang, S. (2007), 'MRI of MovingSubjects Using Multislice Snapshot Images With VolumeReconstruction (SVR): Application to Fetal, Neonatal, andAdult Brain Studies', IEEE Transactions on MedicalImaging, vol. 26, no. 7, pp. 967-980. Lee, S. (1997),'Scattered data interpolation with multilevel B-splines',IEEE Transactions on Visualization and Computer Graphics,vol. 3, no. 3, pp. 228-244. Bach Cuadra, M. (2010),'Central and Cortical Gray Mater Segmentation of MagneticResonance Images of the Fetal Brain', ISMRM Conference.Bach Cuadra, M. (2009), 'Brain tissue segmentation offetal MR images', MICCAI.

Biochemical characterization of a myelin fraction isolated from rat brain aggregating cell cultures.

Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.

High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Determinants of maternal sex steroids during the first half of pregnancy.

OBJECTIVE: To examine the associations of maternal and child characteristics with early pregnancy maternal concentrations of testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, and estradiol (E2). METHODS: We analyzed these hormones among 1,343 women with singleton pregnancies who donated serum samples to the Finnish Maternity Cohort from 1986 to 2006 during the first half of pregnancy (median 11 weeks). The associations of maternal and child characteristics with hormone concentrations were investigated by correlation and multivariable regression. RESULTS: Women older than age 30 years had lower androgen and E2 but higher progesterone concentrations than women younger than that age. Multiparous women had 14% lower testosterone, 11% lower androstenedione and 17-hydroxyprogesterone, 9% lower progesterone, and 16% lower E2 concentrations compared with nulliparous women (all P<.05). Smoking mothers had 11%, 18%, and 8% higher testosterone, androstenedione, and 17-hydroxyprogesterone levels, respectively, but 10% lower progesterone compared with nonsmoking women (all P<.05). E2 concentrations were 9% higher (P<.05) among women with a female fetus compared with those with a male fetus. CONCLUSION: Parity, smoking, and, to a lesser extent, maternal age and child sex are associated with sex steroid levels during the first half of a singleton pregnancy. The effects of smoking on the maternal hormonal environment and the possible long-term deleterious consequences on the fetus deserve further evaluation. LEVEL OF EVIDENCE: II.