The enigmatic primitive streak: prevailing notions and challenges concerning the body axis of mammals

The primitive streak establishes the antero‐posterior body axis in all amniote species. It is thought to be the conduit through which mesoderm and endoderm progenitors ingress and migrate to their ultimate destinations. Despite its importance, the streak remains poorly defined and one of the most enigmatic structures of the animal kingdom. In particular, the posterior end of the primitive streak has not been satisfactorily identified in any species. Unexpectedly, and contrary to prevailing notions, recent evidence suggests that the murine posterior primitive streak extends beyond the embryo proper. In its extraembryonic site, the streak creates a node‐like cell reservoir from which the allantois, a universal caudal appendage of all amniotes and the future umbilical cord of placental mammals, emerges. This new insight into the fetal/umbilical relationship may explain the etiology of a large number of umbilical‐associated birth defects, many of which are correlated with abnormalities of the embryonic midline.     

A correlative study of the allantois in pig and rabbit highlighting the diversity of extraembryonic tissues in four mammalian species,including mouse and man

Despite its conserved role in placenta and umbilical cord formation, the mammalian allantois shows remarkable diversity in size and form as well as in the timing of its appearance and attachment to the chorion. In the mouse, the common allantoic diverticulum is lacking; instead, the allantoic core domain is defined as a progenitor center for allantoic development. In this study, the allantoises of the pig and the rabbit as two nonrodent mammals of increasing significance in biomedical research are compared (1) morphologically using high resolution light and electron microscopy and (2) molecularly using brachyury mRNA expression as a mesodermal marker. Multiple small allantoic diverticula in the rabbit contrast with a single large cavity filling the entire allantois of the pig, but neither pig nor rabbit allantois expresses brachyury . The mesothelium on the allantois surface shows regional variability of cell contacts and microvilli, while blood vessels appear randomly around the allantoic diverticula in a mesodermal layer of variable thickness. Primordial germ cell‐like cells are found in the allantois of the pig but not of the rabbit. To understand further the relevance of this developmental and morphological diversity, we compare the allantois development of pig and rabbit with early developmental landmarks of mouse and man. Our findings suggest that (1) tissue interaction between endoderm and mesoderm is important for allantoic development and vascular differentiation in species with a rudimentary allantoic diverticulum, (2) allantoic mesothelium plays a specific role in chorioallantoic attachment, allantoic differentiation and vascularization, and (3) there is a pronounced diversity in the extraembryonic migratory pathways of primordial germ cells among mammals. Finally, the phylogenetically basal characteristics of the pig allantois are suggestive of a functional similarity in mammals with a large allantois before placentation and in (aplacental) sauropsids with a chorioallantoic membrane well‐adjusted to material exchange function.     

Collagen of umbilical cord vein and its alterations in pre-eclampsia

The state of the vascular system of the mother and of placenta is known to exert a great influence on intrauterinal development of the fetus. Pre-eclampsia is the most common pathological syndrome connected with pregnancy. Since collagen is one of the main constituents of the vessel wall a comparison was made with collagen content and its molecular polymorphism in umbilical cord veins of newborns from healthy and pre-eclamptic mothers. It was found that umbilical cord veins of newborns from mothers with pre-eclampsia contained 18% less collagen than those of the newborns from normal pregnancies. This decrease was accompanied by a slight decrease of collagen solubility, but all its types (I, II, IV, V and VI) were present. However, the umbilical vein wall of newborns from mothers with pre-eclampsia contained relatively less of type I and more of type III collagen than the normal umbilical cord. These differences may be connected with a disturbance of blood flow in fetus of a woman with pre-eclampsia.     

Current Perspectives on Umbilical Cord Abnormalities Including Blood Flow Parameters Based on Ultrasound Observations

The umbilical cord is a vital structure between the fetus and placenta for the growth and well-being of the fetus. Although the umbilical cord may be the only organ that dies at the beginning of life, it is one of the most important parts of the feta-placental unit and plays a role in determining how life begins. In general, the prenatal examination of the umbilical cord is limited to the observation of the number of vessels and the evaluation of umbilical artery blood flow parameters. Pathologists have done more research on the morphological characteristics of the umbilical cord and linked them to perinatal outcomes. The introduction of advanced imaging technology makes it possible to study the characteristics of fetal umbilical cord from early to late gestation. Many studies have shown that the changes of umbilical cord structure may be related to pathological conditions, such as preeclampsia, fetal growth restriction. Prenatal morphometric umbilical cord characteristics and arterial blood flow parameters in normal and pathologic conditions are discussed in this review.     

Ceramides, sphinganine, sphingosine and acid sphingomyelinases in the human umbilical cord blood.

Ceramides, sphingosine, sphinganine, as well as Zn (++)-dependent and Zn (++)-independent acid sphingomyelinase are present in the plasma of adults. The aim of the present study was to examine the concentrations of these compounds and activities of both enzymes in the umbilical cord blood in humans. Twenty-two women with uncomplicated term pregnancy volunteered for the study. Blood was taken from the umbilical cord artery and from the antecubital vein of the mother immediately after delivery. Free ceramides were isolated by thin layer chromatography, and their fatty acids were identified and quantified by gas-liquid chromatography. Free sphingosine and sphinganine concentrations were determined using high-performance liquid chromatography. Acid Zn (++)-dependent and Zn (++)-independent sphingomyelinase activity was measured using sphingomyelin [choline-methyl-14C] as a substrate. We found that the compounds examined are present in the umbilical cord blood. The total fatty acid-containing ceramide concentrations in fetal blood were lower than in mother's blood. The mean sphingosine and sphinganine concentrations in the fetal and maternal serum were similar. The examined enzymes were present in the fetal serum, and their mean activity did not differ from that in the mother. In conclusion, we have shown the presence of ceramides, sphingosine and sphinganine and both isoforms of acid sphingomyelinase in the human fetal cord blood. They are most likely the product of the fetus itself.     

Mesothelium of the murine allantois exhibits distinct regional properties

The rodent allantois is thought to be unique amongst mammals in not having an endodermal component. Here, we have investigated the mesothelium, or outer surface, of murine umbilical precursor tissue, the allantois (～7.25–8.5 days postcoitum, dpc) to discover whether it exhibits the properties of an epithelium. A combination of morphology, challenge with biotinylated dextran amines (BDAs), and immunohistochemistry revealed that the mesothelium of the mouse allantois exhibits distinct regional properties. By headfold stages (～7.75–8.0 dpc), distal mesothelium was generally squamous in shape, and highly permeable to BDA challenge, whereas ventral proximal mesothelium, referred to as “ventral cuboidal mesothelium” (VCM) for the characteristic cuboidal shape of its cells, was relatively impermeable. Although “dorsal cuboidal mesothelium” (DCM) resembled the VCM in cell shape, its permeability to BDA was intermediate between the other two regions. Results of immunostaining for Zonula Occludens‐1 (ZO‐1) and Epithelial‐cadherin (E‐cadherin), together with transmission electron microscopy (TEM), suggested that impermeability in the VCM may be due to greater cellular contact area between cells and close packing rather than to maturity of tight junctions, the latter of which, by comparison with the visceral yolk sac, appeared to be rare or absent from the allantoic surface. Both VCM and DCM exhibited an ultrastructure more favorable for protein synthesis than did the distal squamous mesothelium; however, at most stages, VCM exhibited robust afadin (AF‐6), whereas the DCM uniquely contained alpha‐4‐integrin. These observations demonstrate that the allantoic mesothelium is not a conventional epithelium but possesses regional ultrastructural, functional and molecular differences that may play important roles in the correct deployment of the umbilical cord and its associated vascular, hematopoietic, and other cell types. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Hindlimb glucose and lactate metabolism during umbilical cord compression and acute hypoxemia in the late-gestation ovine fetus

The metabolic adaptation of the hindlimb in the fetus to a reversible period of adverse intrauterine conditions and, subsequently, to a further episode of acute hypoxemia has been examined. Sixteen sheep fetuses were chronically instrumented with vascular catheters and transit-time flow probes. In nine of these fetuses, umbilical blood flow was reversibly reduced by 30% from baseline for 3 days (umbilical cord compression), while the remaining fetuses acted as sham-operated, age-matched controls. Acute hypoxemia was subsequently induced in all fetuses by reducing maternal fractional inspired oxygen concentration for 1 h. Paired hindlimb arteriovenous blood samples were taken at appropriate intervals during cord compression and acute hypoxemia, and by using femoral blood flow and the Fick principle, substrate delivery, uptake, and output were calculated. Umbilical cord compression reduced blood oxygen content and delivery to the hindlimb and increased hindlimb oxygen extraction and blood glucose and lactate concentration in the fetus. However, hindlimb glucose and oxygen consumption were unaltered during umbilical cord compression. In contrast, hindlimb oxygen delivery and uptake were significantly reduced in all fetuses during subsequent acute hypoxemia, but glucose extraction, oxygen extraction, and hindlimb lactate output significantly increased in sham-operated control fetuses only. Preexposure of the fetus to a temporary period of adverse intrauterine conditions alters the metabolic response of the fetal hindlimb to subsequent acute stress. Additional data suggest that circulating blood lactate may be derived from sources other than the fetal hindlimb under these circumstances. The lack of hindlimb lactate output during acute hypoxemia in umbilical cord-compressed fetuses, despite a significant fall in oxygen delivery to and uptake by the hindlimb, suggests that the fetal hindlimb may not respire anaerobically after exposure to adverse intrauterine conditions. hypoxia     

Measuring fetal and maternal temperature differentials: a probe for clinical use during labour

The healthy fetus maintains a higher temperature than that of its mother during gestation and labour. This results from the thermal balance between the heat generated by the fetus and the heat loss to its maternal surroundings. The heat loss can be by heat exchange via blood flowing in the umbilical cord and placenta, and via conduction through the fetal skin and amniotic fluid to the maternal wall. The temperature difference between the fetal and maternal tissue may reflect the metabolic state of the fetus and the magnitude and changing patterns of placental blood flow during labour. Physiological changes, such as those induced by epidural analgesia, and fetal infection have been shown to exhibit an increase in the absolute temperature. An intrauterine probe, previously used for non-invasive ECG detection, has been equipped with temperature sensors that measure fetal and maternal skin temperature in utero. Laboratory tests to characterize the performance of the probe reveal that absolute and differential temperatures can be resolved to around 0.01° C with a thermal time constant of approximately 9 s. Ideally the probe body should have infinite thermal insulation or thermal shunting across the probe will occur reducing the measured temperature difference. In this initial probe design, a high thermal isolation between sensors has been achieved but is not perfect, resulting in around 85% of the actual temperature difference across the probe being registered. Average feto-maternal differences of 0.2° C have been measured in a clinical investigation.     

Immunohistochemical localization of bovine placental retinol-binding protein.

An immunogold staining method was used in combination with epipolarization microscopic detection to demonstrate the presence of bovine placental retinol-binding protein in bovine extraembryonic membranes. Amnion, chorion and allantois were fixed in Bouin fixation fluid and embedded in polyethylene glycol 1500. Sections (5 mm) were cut and transferred onto Digene silanated slides and immunostained using rabbit antiserum raised against bovine placental retinol-binding protein followed by goat anti-rabbit IgG labeled with 1 nm gold. Gold particles after silver enhancement were viewed and photographed under epipolarization microscopy. Epithelial cells of all three membranes (i.e. amniotic ectoderm, chorionic trophectoderm, and allantoic endoderm) were immunoreactive, while mesodermal cells, collagen, and blood cells were not. These data, together with our previous observation that these three placental membranes synthesize and secrete retinol-binding protein, indicate that epithelial cells lining the amnion, chorion and allantois are the major sources of this protein. The presence of retinol-binding protein in placental membranes and their fluids may be indicative of an important role for retinol in placental differentiation and development.     

Acute cord occlusion increases blood ionized magnesium concentration in preterm fetal sheep during maternal magnesium sulfate exposure

This study tested the hypothesis that a pathophysiologic insult to the fetus that decreases pH (umbilical cord occlusion) produces an increase in physiologically active (i.e., ionized) magnesium concentration. Preterm pregnant sheep (n = 7) were instrumented with maternal and fetal catheters and an inflatable vascular occluder was placed around the umbilical cord. After a 2-day recovery period, each ewe received a 4-g loading dose, followed by continuous intravenous infusion of 1 g magnesium sulfate/h. After 48 h, an episode of acute fetal distress was produced by inflation of the umbilical occluder for 10 min. Maternal and fetal arterial blood samples were collected at regular intervals to quantitate ionized magnesium concentration and monitor physiologic status. Magnesium sulfate infusion increased maternal and fetal blood ionized magnesium concentration. In vitro blood analysis demonstrated that there was a linear inverse correlation (r2 = 0.99) between fetal sheep blood pH and ionized magnesium concentration. In vivo, 10 min of umbilical cord occlusion produced an increase in fetal blood ionized magnesium concentration in all animals (P = 0.02) that was temporally related to the decrease in fetal blood pH. Whether this increase in physiologically active magnesium concentration is beneficial (via neuroprotection) or deleterious (via suppression of stress response) to the distressed fetus remains to be determined.     

The carnivore pregnancy: the development of the embryo and fetal membranes

The aim of this research was to compare the morphological aspects during the development of pregnancy in dogs and cats, distinguishing features of the fetal membranes, such as yolk sac evolution and differentiation of hemangioblasts, and the degree of elaboration of the amnion and allantois. Canine and feline placentae from 20, 24, 35, 45 and 55 d of pregnancy were perfusion-fixed for histological investigation and vascular corrosion casts were produced. The casts were prepared for scanning electron microscopy (SEM) and the embryo and fetal membrane development was analyzed. The growth patterns of the conceptuses were compared with the organization of the placentation process, and changes of the morphology during pregnancy were recorded. In feline placentae, an incomplete zonary shape was present in 62.5% out of 60 studied cases. This was located distal to the insertion of the umbilical cord. In the lamellar zone, the interhemal membrane or placental barrier resembled endotheliochorial conditions, and the maternal-fetal microvascular blood flow interrelationship was of simple crosscurrent type. Dogs have a zonary placenta, completely surrounding the fetus, and complex lamellar organization of maternal and fetal tissues. At the border, two marginal hematomes with green colouration delimited the central placental girdle. The yolk sac consisted of one large sacculation with an inverted "T" shape and an enormous number of blood vessels; it had hemangioblast cells in contact with the epithelium. The amnion was avascular in early stages, but became vascularized by blood vessels of the internal allantoic membrane in later stages of pregnancy by intrinsic relation.     

Histology of the extraembryonic membranes of an oviparous snake: towards a reconstruction of basal squamate patterns

An understanding of the evolutionary morphology of extraembryonic membranes in reptiles requires information about oviparous as well as viviparous species. We are studying histology and ultrastructure of the extraembryonic membranes of snakes to clarify the evolutionary history of reptilian fetal membranes, including determination of basal (ancestral) ophidian and squamate patterns. Microscopic anatomy of the membranes of oviparous corn snakes (Elaphe guttata) was examined using light and electron microscopy. At mid-development the inner surface of the eggshell is lined by two extraembryonic membranes, the chorioallantois and the omphalallantoic membrane. The chorioallantois consists of a bilayered cuboidal epithelium that overlies the allantoic blood vessels. During development, allantoic capillaries become more abundant, and the chorionic epithelium thins, decreasing the diffusion distance for respiratory gas exchange. The abembryonic pole of the egg is delimited by a bilaminar omphalopleure and isolated yolk mass, the latter of which is lined on its inner face by the allantois. The isolated yolk mass regresses developmentally, and patches of yolk droplets become isolated and surrounded by allantoic blood vessels. By late development, the abembryonic hemisphere has been fully vascularized by allantoic vessels, forming a "secondary chorioallantois." With regard to its extraembryonic membranes, Elaphe gutatta is similar to viviparous snakes. However, this species exhibits features that have not previously been reported among squamates, perhaps reflecting its oviparous reproductive habits. Morphological evidence for the uptake of eggshell material by epithelia of the chorion and omphalopleure suggests that the potential for absorption by extraembryonic membranes predates the origin of viviparity.     

Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis

The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.     

Relationship between the appearance of preantral follicles in the fetal ovary of Antarctic minke whales (Balaenoptera bonaerensis) and hormone concentrations in the fetal heart, umbilical cord and maternal blood

The present study aimed to determine the relationship among changes in the number of preantral follicles and concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), androstenedione (A) and estradiol-17beta (E2) in the fetal heart, umbilical cord and maternal blood. Primordial follicles had already appeared in a 20 cm fetus and primary follicles were observed in a 50 cm fetus. In a 70 cm fetus, the number of primordial and primary follicles increased rapidly and secondary follicles were present. The concentrations of LH and FSH did not change between 20 cm and 160 cm in fetal length. When the fetal length became > 70 cm, serum levels in the fetus, umbilical cord and mothers, and E2 levels in umbilical cord increased synchronously (p < 0.05). These results showed increases in the number of preantral follicles in the Antarctic minke whale fetal ovary along with fetal growth during the early gestation period. These findings suggest that the change in preantral follicles was associated with changes in the concentration of steroids in early gestation periods. The changes in steroid concentrations in the fetal and umbilical cord blood and the increased number of preantral follicles were coincident at around 70 cm in fetal length, whereas the growth and differentiation of primordial and primary follicles appeared to be independent of FSH and LH.     

Fetal blood sampling in baboons (Papio spp.): Important procedural aspects and literature review

Background  The baboons ( Papio cynocephalus ) have similarities with human placentation and fetal development. Fetal blood sampling allows investigators to assess fetal condition at a specific point in gestation as well as transplacental transfer of medications. Unfortunately, assessing fetal status during gestation has been difficult and fetal instrumentation associated with high rate of pregnancy loss. Our objectives are to describe the technique of ultrasound guided cordocentesis (UGC) in baboons, report post-procedural outcomes, and review existing publications.
Methods  This is a procedural paper describing the technique of UGC in baboons. After confirming pregnancy and gestational age via ultrasound, animals participating in approved research protocols that required fetal assessment underwent UGC.
Results  We successfully performed UGC in four animals (five samples) using this technique. Animals were sampled in the second and third trimesters with fetal blood sampling achieved by sampling a free cord loop, placental cord insertion site or the intrahepatic umbilical vein. All procedures were without complication and these animals delivered at term.
Conclusions  Ultrasound guided fetal umbilical cord venipuncture is a useful and safe technique to sample the fetal circulation with minimal risk to the fetus or mother. We believe this technique could be used for repeated fetal venous blood sampling in the baboons.     

Disposition of methadone in the ovine maternal-fetal unit

The distribution of methadone between mother and fetus after a single dose and at steady state was determined using the chronic pregnant ewe preparation. Chronic indwelling catheters were placed in the maternal aorta and vena cava, umbilical vein and fetal aorta. Following a single i.v. dose (0.5 mg/kg) to the mother, methadone was rapidly distributed to the fetus, with peak concentration in the umbilical vein occurring within two min. An umbilical venous-arterial gradient existed for 10–15 min after drug administration, indicating uptake of methadone by fetal tissues. Methadone concentration in the fetus was 2–5 times lower than those in the mother even in the post-distribution phase. The terminal half-life of methadone in 4 animals was 57±7.6 (S.E.) min in the mother, and 58.5±10.0 (S.E.) min in the fetus. When methadone was infused at a constant rate to the mother (0.01 mg/kg/min), steady state was achieved in both mother and fetus by 4–5 hrs. In 5 animals, maternal steady state was found to be 203±18.8 (S.E.) ng/ml, and fetal steady state was found to be 29.7±2.9 (S.E.) ng/ml. These studies show that methadone is rapidly distributed to the fetus, but fetal concentration remain lower than maternal concentration at all times.     

The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.

The chorio-allantoic placenta forms through the fusion of the allantois (progenitor tissue of the umbilical cord), with the chorionic plate. The murine placenta contains high levels of hematopoietic stem cells, and is therefore a stem cell niche. However, it is not known whether the placenta is a site of hematopoietic cell emergence, or whether hematopoietic cells originate from other sites in the conceptus and then colonize the placenta. Here, we show that the allantois and chorion, isolated prior to the establishment of circulation, have the potential to give rise to myeloid and definitive erythroid cells following explant culture. We further show that the hematopoietic potential of the allantois and chorion does not require their union, indicating that it is an intrinsic property of these tissues. These results suggest that the placenta is not only a niche for, but also a source of, hematopoietic cells.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.