Microscopic aspects of the yolk sac hematopoiesis from ... · Microscopic aspects of the yolk sac hematopoiesis from ovine embryos ... that hematopoietic stem cells ... stem cells,

Microscopic aspects of the yolk sac hematopoiesis from ovine embryos

A. G. T. Pessolato*, 1, D. S. Martins 2, A. Galdos-Riveros 1, A. M. Fontes 3, C. E. Ambrósio 4, R. E. Grassi Rici1, D. A. R. Magalhães3, A. Castilho-Fernandes3, D. T. Covas3 and M. A. Miglino1

1Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo/São Paulo, 05508-270 - São Paulo, Brazil. *Corresponding author: [email protected]

2Department of Animal Welfare, Faculty of Animal Sciences and Food Engineering, University of São Paulo/Pirassununga, 13635-900 - São Paulo, Brazil.

3Center of Cell Therapy and Regional Blood Center of Ribeirão Preto, Faculty of Medicine of Ribeirão Preto, University of São Paulo/Ribeirão Preto, 14051-140 - São Paulo, Brazil.

4Faculty of Animal Sciences and Food Engineering, Department of Basic Sciences, University of São Paulo/Pirassununga, 13635-900 - São Paulo, Brazil.

The yolk sac (YS) is the single embryo attachment present in all species of vertebrate embryos, reptiles, birds and mammals. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called "blood islands" that represent the progenitors of vascular and hematopoietic systems. The initial development of the YS hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from its sow other intraembryos sites. We analysed 40 samples of YS from ovine embryos with estimated age assessed by Crown Rump measures from 15 to 29 days by optical microscopy, confocal, scanning and transmission eletron microscopy. It was observed in the microscopic analysis that there is indeed a relationship between the two cell lineages. Our study provides important microscopic findings related to ovine placentation and especially to embryonic hematopoiesis. Thus, this study contribute not only to a better understanding of the relation YS – placenta, nor only with all the initial embryonic hematopoietic system, but also enables studies of cell therapy for various diseases of blood origin.

Keywords yolk sac; hematopoiesis; stem cells; ovine; embryos.

1. Introduction

The beginning of placental development in sheep occurs before the "implantation" final of the fertilized egg and placental morphogenesis during early pregnancy is closely related to extra-embryonic membranes, differentiated in chorion, allantois, amnion and yolk sac [1, 2]. Given the importance of placental membranes, the foccus of our attention in the present work is the yolk sac, which is the only membrane attached to the embryo present in all species of both viviparous and oviparous vertebrates [3, 4]. The yolk sac, despite its variety of shapes and relations with the embryonic and fetal membranes, it has the task of forming primitive multipotent stem cells that are capable of forming blood cells and tissues of the veins, arteries and capillaries [5] through a precursor of both hematopoietic and endothelial cell lines that is named hemangioblast [6-11]. Currently, other theories state that the hemangioblast can generate hematopoietic cells through a hemogenic endothelium stage [12] or that the hematopoietic stem cells originate de novo from mesenchymal cells [13]. Despite disagreements about the hemangioblast, the possible existence of this precursor cell common to both lines, in part enables the understanding of the occurrence of hematopoiesis, a fundamental biological process for proper embryonic development and that has its origin in this primitive site that is the yolk sac. The initial embryonic hematopoiesis is another point of discussion. In early embryonic development, a hematogen tissue is formed in the extra-embryonic esplancnopleura of the yolk sac. This tissue is represented initially by niches of mesenchymal cells called hemangioblasts, which together are called of islets of Wolff. The central hemangioblast of each islet of Wolff give rise to stem cells responsible for the formation of all blood cells and the immune system and are also referred to as stem cells, supporting the theory monophyletic origin of the blood. A percentage of the population of stem cells differentiate into erythrocytes very large, which enter in the embryo through the vessel also in formation [14, 15]. These blood cells are short lived and are constantly renewed by the proliferation of mitotic cells in the hematopoietic organs [16]. Thus, this study sought to demonstrate microscopic evidence of the emergence of this precursor cell type in the vitelline membrane at different stages of sheep development, in order to ground possible evidences of origin, emergence and maintenance of the initial embryonic hematopoiesis.

2. Material and methods

2.1 Ovine embryos dissection

In this study 40 ovine embryos with estimated gestational age varying between 15 and 29 days from slaughterhouse were used. Initially the uteri were collect and kept in PBS solution supplemented with antibiotics and antifungals, which

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

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were transferred to the laboratory of Histology and Embryology, Faculty of Veterinary Medicine and Animal Science at USP – São Paulo, where they were dissected and subjected to microscopic technique. Our studies were conducted according to ethical guidelines approved by the Regional Scientific-Ethical Committee on the Use of Animals at the School of Veterinary Medicine of São Paulo, University of São Paulo, Brazil (protocol number #1713/2009).

2.2 Optical microscopy

The vitelline membranes were fixed in Metacarn for 7 hours and kept in 70% alcohol until processing. The material was dehydrated in ascending ethanol series (70-100%), diaphanized in xylene and embedded in Paraplast® [17]. The sections were obtained with a thickness of 5 µm Polyartmicrótomo microtome (Leica) and then routinely stained with HE (hematoxylin eosin) [17] for further photomicroscopic analysis.

2.3 Scanning electron microscopy

After 48 hours of fixation in Karnowisky, the material was washed three times in 0.1 M phosphate buffer (pH 7.4) for 10 minutes, totaling 30 minutes of immersion. Subsequently the materials were post-fixed in aqueous 4% osmium tetroxide (PolyScience family, Inc. USA) for 2 hours and again washed 3 times in 0.1 M phosphate buffer (pH 7.4) for 10 minutes. Dehydration was imposed by battery of 15 minutes with increasing concentrations of alcohol (50%, 70%, 80%, 90% and 95%) then moving in absolute ethanol for 15 minutes four times. The critical point was obtained through the apparatus (CPD 020 - BALZERS UNION), where all the alcohol was removed from the material leaving it completely dry to metallization in gold. As a final processing step, the samples were fixed in "stubs" of aluminum with carbon glue, left in an oven for 12 hours for drying and subjected to gold sputter bath (EMITECH - 550 K). Analyzes were performed on electron micrographs and scanning electron microscope (LEO - 435 VP).

2.4 Transmission electron microscopy

The yolk sacs fixed in Karnowisky were washed in phosphate buffer and post-fixed with aqueous 4% osmium tetroxide (PolyScience family, Inc. USA), washed in saline and embedded in uranyl acetate (PolyScience family, Inc. USA). The vitelline membranes were washed again in phosphate buffer and dehydrated in increasing concentrations of acetone (30-100%). Then continued for 1 hour under stirring in a 3:1 mixture of acetone and resin (Araldite resin, grade 502, PolyScience family, Inc. USA + DDSA, PolyScience family, Inc. USA + DMP-30, PolyScience family, Inc. USA) for 1 hour in a 1:1 mixture of acetone and resin, and for 1 hour in pure resin. Subsequently, the vitelline membranes were placed in a mold of silicone containing pure resin and in an oven at 60°C for 72 hours, so that polymerization of the resin occurred. We obtained the first semi-fine sections of 1μm thick for observation under light microscope. The ultrathin sections, about 60nm in thickness were harvested copper screens and contrasted by uranyl acetate 2% (PolyScience family, Inc. USA) in distilled water (5 min) and 0.5% lead citrate (PolyScience family, Inc. USA) in distilled water (10 minutes). The observations and photomicrographs were performed subcellular Morgagni electron microscope (model 268d, Philips).

2.5 Confocal Microscopy

The vitelline membrane of embryos at different developmental periods previously fixed in Metacarn were embedded in Tissue-Tek ® (Embedding Medium for Frozen Tissue Specimens to Ensure Optimal Cutting Temperature - Sakura ®). Sections were stained with DAPI (100ng/ml, dihydrochloride of 4,6-diamino-2-phenylindole) (Abbott Molecular Inc.) for identifying the core and labeled primary antibodies made in mice: CD31 endothelial cells (Novus Biologicals) , CD41/CD61 of primitive hematopoietic cells and progenitors (ABD Serotec) and CD48 lymphocytes and macrophages (New Ab) and the secondary antibody with Alexa Fluor 488 (Invitrogen) made in goat. Cuts were obtained with the vitelline membrane into 5μm thick cryostat. All steps necessary for the marking of cuts were performed at room temperature. The sections were immersed in PBS overnight. To reduce autofluorescence, the sections were incubated in a solution of glycine (One Plus) 0.1 M in PBS for 30 minutes and washed repeatedly in PBS. Then, they were permeabilized with Triton X-100 solution (Sigma) 0.3% in PBS for 1 hour, washed consecutive times with PBS and incubated for 1 hour in blocking solution Bovine Serum Albumin (BSA) 1% (Sigma) PBS. During the incubation the primary and secondary antibodies were diluted in 1% BSA solution, centrifuged at 10000rpm for 40 minutes at 4°C. The supernatant of the primary antibodies (10μg/mL) was added to the cut and incubated for 1 hour in a moist chamber. It was followed by successive washing and subsequent incubation with the secondary antibody with the DAPI (100ng/ml) for 1 hour in a moist chamber and away from light.



Sections were then incubated consecutive times in PBS, immersed in distilled water prior to assembly of the blade. It was added to mounting medium Fluoromount G (Electron Microscopy Sciences) for cuts and cover with coverslip. The analyzes were made on cell confocal microscope LSM 710 software and Zein 2008 (Carl Zeiss).

3. Results of our microscopic study in ovine yolk sacs

3.1 Optical microscopy

The Figure 1A shows that the yolk sacs obtained presented as a trilaminar structure, with each of its distinct and well defined layers. A coating layer of yolk sac cavity, called the endodermis, it had a single layer of endodermal cells; mesenchymal vascular an intermediate layer, called mesenchymal or mesodermal and the other intermediate to the single layer mesothelial exoceloma called mesothelium. Figure 1A is a sample from the embryo of 15 days which there was lots of blood islands with small amount of primitive blood cells - erythrocytes probably primary - inside, surrounded by endothelial cells. Figure 1B shows samples of embryos with an estimated age of 22 days presenting major blood islands located in vitelline mesenchyme, possessing large amounts of primary erythrocytes inside of them. During this embryo period the vitelline membranes were similar to the previous period, however, the vascular islands presented more developed and greater amount of primitive nucleated blood cells. The yolk sacs of the animals with an estimated age of 23 days were similar to those described for the previous period. Most of the islands had developed and was filled with large amounts of blood cells. However, throughout the vitelline membrane was observed in Figure 1C some blood islands small quantities of blood cells inside. In Figure 1D the vitelline membrane of embryos with an estimated age of about 25 days, the blood islands had merged and formed initial endothelial tubes.

Fig. 1 Photomicrograph A shows the three layers that compose the yolk sac, i.e. endodermal (End), mesenchymal (Msc), mesotelial (Mst), blood islands (arrows) newly formed and the small amount of blood cells inside, at 15 days. Photomicrograph B shows an increase in blood islands (arrows) and larger amount of primitive blood cells within them at 22 days. Photomicrograph C shows some small amount of blood islands (arrows) with blood cells inside at 23 days. Scale bar of 100µm. Photomicrograph D shows the fusion of blood islands (arrows), giving rise to the first vitelline blood vessels at 25 days. Scale bar of 200µm. H&E staining.



3.2 Scanning electron microscopy

In the Figure 2A the analysis of scanning electron microscopy showed that in the age of 25 days, the vitelline membranes showed blood vessels with cells being released into the lumen of the vessels and vitelline duct and in Figure 2B cavities of small size inside the membrane. In embryonic age estimated 27 days was observed blood cells being released into the outer membrane in Figure 2C, and in Figure 2D empty vesicles, probably possessed cells previously released. At 29 days showed in the Figure 2E the vitelline membrane had released niche blood cells within the membrane, and the Figure 2F shows a large amount of particles and cells released through the vesicles from the superficial vessels.

Fig. 2 A shows the blood vessel releasing blood cells (arrows) from the lumen of the vessel into the embryo. B highlights the presence of cavities (full arrow) inside of the membrane at 25 days. C shows cells (arrow) being released to the outside of the membrane and D shows a large flaccid empty bladder (full arrow) in a sample of 27 days. E and F reveal the liberation of niches of the blood cells (arrow) within the membrane and of vesicles (full arrow) from the surface vessels (v) at 29 days.

3.3 Transmission electron microscopy

The ultrastructural analysis of the vitelline membrane was performed by transmission electron microscopy. The Figure 3A shows at 23 days-old embryonic estimated that were observed extravasation of cytoplasm from endothelial cells to the extracellular region in contact with primitive blood cell and in Figure 3B blood vessel localized in the mesenchymal region with primitive blood cells inside. At 25 days old embryo estimated the Figure 3C and 3D shows the analysis of transmission electron microscopy of the vitelline membrane presenting blood islands in the mesenchymal region of the



yolk sac with a large amount of blood cells and also blood cells located in the endodermal region with absence of endothelium.

Fig. 3 A shows communication between the endothelial (ec) and blood cytoplasm (full arrows). B indicates blood vessel (v) in the mesenchymal region with primitive blood cells (full arrow) at 23 days. C and D reveal blood islands (arrows) present in the mesenchyme region of the membrane and also blood cells located in the endodermal region with absence of endothelium (full arrows).

3.4 Confocal Microscopy

Figure 4A shows the immunofluorescence of the vitelline membrane, analyzed by confocal microscopy at 15 days, presenting a strong labeling of CD31 (specific of endothelial cells) in endothelial cells in the mesenchymal region of the yolk sac, and also, interestingly, labeling on endodermal cells.

Fig. 4 A shows labeling of CD31 on endothelial (arrows) and endodermal cells (full arrows). Scale bar is 10µm.



4. Discussion and Conclusion

During ontogeny of vertebrates, hematopoietic sites were modified as they emerged new populations of stem cells. Rohen and Drecoll [18] reported that the embryonic hematopoiesis may occur in stages, beginning with the vascular system in extra-embryonic mesenchyme that is megaloblastic erythropoiesis in the yolk sac blood islands and in the corium, which emerges large nucleated megaloblasts. The second stage of the period called hepatosplenic hematopoiesis arises in the liver and from the 12th week in the spleen. Subsequently the liver and spleen function as organ hemocitopoietic temporary, however, the second month of intrauterine life human clavicle begins to ossify, and begins the formation of hematogenous bone marrow inside. As ossification progresses in the rest of the body, the bone marrow becomes increasingly important as the hematopoietic organ [16, 18-19]. Coordinated activity of different hematopoietic sites ensure rapid production of differentiated blood cells to the immediate needs of the embryo, and maintain a pool of undifferentiated hematopoietic cells. The hypothesis that early embryonic hematopoiesis occurring in the region of the lumen of the yolk sac blood islands and is surrounded by endothelial cells [20-21] is thoroughly discussed. Jaffredo et al. [11] stated that the yolk sac produces a supply of hematopoietic cells, where part of these cells differentiate in situ, i.e. in the same tissue, while others colonize another organs. However, the ability of hematopoietic cells is probably influenced by the microenvironment of the yolk sac, and may induce myeloid cells, erythroid and hematopoietic. Pereda and Niimi [22] reported that human, the initial generation of blood cells occurs in the extra-embryonic yolk sac during the third week post-conception. Therefore, the yolk sac is considered the primary source of hematopoietic progenitor cells, circulating through the bloodstream of the embryo and transferring the first blood cells. According to Sadler [23]; Zago and Covas [24], the central hemangioblasts form the first hematopoietic stem cells, whereas the peripheral hemangioblasts differentiate into angioblasts, endothelial precursors. Our observations made in optical microscope of the yolk sac showed that at 15 days of gestation estimated there is in the mesenchymal region, a large quantity of blood islands across the length of the membrane, but with a small amount of primitive blood cells within. The blood islands observed at ages 21 to 23 days, unlike the one found in the previous period, were filled, with lots of primitive blood cells surrounded by endothelial cells. In the analyzes carried out in embryo at 25 days the blood islands presented a merger between itself and a large quantity of blood cells. Corroborating our findings, this intimate association of development of hematopoietic and endothelial lineages in the region of blood islands confirm the probable hypothesis that both lines originate from this common precursor called hemangioblast [8, 9, 25, 26] . The vitelline membrane analyzed by SEM presented data similar to those found in optical microscopy. The blood islands present as globular elevations on the membrane surface when analyzed by this type of microscopy. These elevations are visible from 25 to 29 days. However, over the course of embryonic development of these blood islands cluster and merge into each other, forming endothelial tubes [23]. The membrane surface has cavities and blood vessels and cells released into the vessel lumen, blood cells released into the membrane, and a large amount of particles and cells released from the entire outer surface of the membrane, as observed by Pereda in human yolk sac in his various works [22, 27-31], which proposes that before the movement yolk sac-embryo is established primitive blood cells carrying to the embryo through the vitelline vessels, a transfer of material and mature blood cells are produced to the embryo through the vitelline duct. Furthermore, Pereda, Monge and Niimi [31] also reported that these cavities seen across the surface of the vitelline membrane are a continuation of the luminal opening of endodermal vesicles, which supply the demand blood-cell embryos from the establishment of the corporeal circulation. In transmission electron microscopy, in general, the vitelline membrane has vascular mesenchymal region [32, 33], with presence of blood islands formed by endothelial cells surrounding blood nucleated cells inside. Below the mesenchymal region is the region mesothelial formed by a single layer of endothelium, which has on its surface microvilli, as described by Pereda, Monge and Niimi [31], vesicles and secretion in the cytoplasmic region, suggesting high secretory activity of these cells [34]. McGrath and Palis [35] re-cultured cells obtained from explants of the yolk sac to understand the process of development of blood cells and endothelial cells of the extraembryonic mesoderm. The authors suggested so that the presence of the endoderm, yolk sac, or its extracellular matrix is necessary for the coalescence of endothelial cells in the development of capillary networks. In connection with this hypothesis, we observed in confocal microscopy both endothelial and endodermal cells labeling by antibody CD31 (marker for endothelial progenitor cells)in the yolk sac. This fact further reinforces the theory of emergence can also occur in blood a layer other than the mesodermal [22, 31]. These results confirm the existence of a differential hematopoiesis capacity of extra-embryonic living tissues, indicating that these cells are capable of producing long-term adult hematopoiesis. Thus, this study contribute not only to a better understanding of the relation YS – placenta, nor only with all the initial embryonic hematopoietic system, but also enables studies of cell therapy for various diseases of blood origin [36].

Acknowledgements: The support by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and by INCTC (Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular) is gratefully acknowledged.



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Microscopic aspects of the yolk sac hematopoiesis from ... · Microscopic aspects of the yolk sac hematopoiesis from ovine embryos ... that hematopoietic stem cells ... stem cells,