Cristiane Valverde Wenceslau

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results.

PURPOSE Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

During early development, human dental pulp is originated from neural crest, which is a transient embryonic structure (Fig. 1). According to current knowledge, neural crest stem cells (NCSCs) have the capacity to self-renewal and display a developmental potential almost the same as embryonic stem (ES) cells (Kerkis and Caplan, 2012). These postmigratory NCSCs generate all craniofacial bones, the majority of the peripheral nervous system cells and tissues, as well as several non-neural cell types, such as smooth muscle cells of the cardiovascular system, pigment cells in the skin, cartilage, connective tissue, corneal epithelium and dental pulp among them. Although postmigratory, postnatal NCSCs are of restricted developmental potential they maintain functional characteristics resembling their embryonic counterparts and an ability to differentiate into a broad spectrum of cell types (Le Douarin et al., 2004, 2007, 2008; Dupin et al., 2007; Le Douarin & Dupin, 2003, 2012).

Allogeneic Mesenchymal Stem Cell Transplantation in Dogs with Keratoconjunctivitis Sicca

Keratoconjunctivitis sicca (KCS) is a dysfunction in tear production associated with clinical signs, which include conjunctival hyperemia, ocular discharge, discomfort, pain, and, eventually, corneal vascularization and pigmentation. Immunosuppressive drugs are routinely administrated for long periods to treat KCS but with side effects and limited results. Evaluation of the clinical benefits of intralacrimal transplantation of allogeneic mesenchymal stem cells (MSCs) in dogs with mild-moderate and severe KCS was done. A total of 24 eyes with KCS from 15 dogs of different breeds were enrolled in the present study. A single transplantation of MSCs (1 × 106) directly into lacrimal glands (dorsal and third eyelid) was performed. The Schirmer tear tests (STTs) and ocular surface improvements were used to assess short- and long-term effects of these cells. The STTs were carried out on day 0 (before MSCs transplantation) and on days 7, 14, 21, and 28, as well as 6 and 12 months after MSC transplantation. Our data demonstrate that allogeneic MSC transplantation in KCS dogs is safe since no adverse effects were observed immediately after transplantation and in short- and long-term follow-ups. A statistically significant increase in the STT and ocular surface improvements was found in all eyes studied. In all the eyes with mild-moderate KCS, STT values reverted to those of healthy eyes, while in eyes with severe KCS, although complete reversion was not found, there was improvement in tear production and in other clinical signs. Our study shows that a single dose of a low number of MSCs can be used to treat KCS in dogs. In contrast to immunosuppressive drug use, MSC transplantation has an effect over a long period (up to 12 months), even after a single administration, and does not require daily drug administration.

Mesenchymal Stem Cell Benefits Observed in Bone Marrow Failure and Acquired Aplastic Anemia

Acquired aplastic anemia (AA) is a type of bone marrow failure (BMF) syndrome characterized by partial or total bone marrow (BM) destruction resulting in peripheral blood (PB) pancytopenia, which is the reduction in the number of red blood cells (RBC) and white blood cells (WBC), as well as platelets (PLT). The first-line treatment option of AA is given by hematopoietic stem cell (HSCs) transplant and/or immunosuppressive (IS) drug administration. Some patients did not respond to the treatment and remain pancytopenic following IS drugs. The studies are in progress to test the efficacy of adoptive cellular therapies as mesenchymal stem cells (MSCs), which confer low immunogenicity and are reliable allogeneic transplants in refractory severe aplastic anemia (SAA) cases. Moreover, bone marrow stromal cells (BMSC) constitute an essential component of the hematopoietic niche, responsible for stimulating and enhancing the proliferation of HSCs by secreting regulatory molecules and cytokines, providing stimulus to natural BM microenvironment for hematopoiesis. This review summarizes scientific evidences of the hematopoiesis improvements after MSC transplant, observed in acquired AA/BMF animal models as well as in patients with acquired AA. Additionally, we discuss the direct and indirect contribution of MSCs to the pathogenesis of acquired AA.

Comportamento dos nervos glossofaríngeo e vago, na região retrofaríngea de ovinos: origem aparente no crânio, trajeto, ramificação e distribuição

In 60 hemiheads of sheep of the Santa Ines breed the apparent origin in the skull of itinerary, ramification and distribution of the glossopharingeal nerve (Gf) and the vagus nerve (Vg) in the retropharyngeal region (Rr) were studied. By fixation and dissection of the specimens it was seen that the glossopharyngeal nerve and the vagus nerve arise from the jugular foramen in 100% of the cases. The right and the left glossopharingeal nerve (Glde) are frequently (86.6%) located more medially to the tympanic bulla, pass caudally to the stylohyoid bone, bypass the margin of the caudal stylopharyngeal muscle, the tonsilla, of the pharyngeal and the lingual mucous membrane. These branches are distributed, respectively, in the carotid sinus, pharyngeal musculature, soft palate, stylopharyngeal muscle, palatine tonsil, pharyngeal mucosa and the caudal third of the tongue (100%). The right and the left vagus nerve run caudally in association with the accessory nerves (Ac) up to the atlas (70%) and go then medially to the occipital artery, dorsally to the common carotid and the sympathetic trunk in the cervical region (80%). The vague nerves have pharyngeal (86.6%) and cranial laryngeal (100%) branches.

Pluripotent Stem Cells to Model and Treat Huntington’s Disease

Stem cell therapies hold considerable promise for the treatment of neurodegenerative diseases. Pluripotent stem cells (PSCs) have been of particular clinical interest because of their ability to generate neuronal cells and to be used in animal models of neurodegenerative disease as well as for testing new drugs. Several PSCs isolated from humans and animals that carry the genotype of Huntington’s disease (HD) have been used in aforementioned studies. HD-PSCs obtained can produce in vitro neural progenitor cells (NPCs). These NPCs applied in HD models show several advantages: they engraft into the brain in animal models and differentiate into neuronal cells, thus promoting behavioral recovery and motor impairment. Although progress has been made using PSCs, additional tests should be done to overcome several limitations as, for example, tumorigenicity, before their clinical application. We focus this chapter on current knowledge regarding HD-PSC lines and their helpfulness as an in vitro model for basic research. Next, we discuss the advances of disease-free PSCs in preclinical HD models aiming to their potential application in patients. Additionally, we discuss their potential use as a test system for anti-HD drug screening by the pharmaceutical industry, especially considering HD patients’ welfare.

Induced Pluripotent Stem Cells Derived from Dental Stem Cells: A New Tool for Cellular Therapy

Induced pluripotent stem cells (iPSCs) are a type of experimentally produced pluripotent stem cell (PSC), which share similar features with embryonic stem cells (ES) isolated directly from early embryos. Shinya Yamanaka’s lab in Kyoto, Japan was the first to develop iPSCs in 2006 by the introduction of four genes that encode transcription factors of PSC into mouse embryonic fibroblasts—a process known as “reprogramming”. Later on, different animal and human fetal or adult somatic cell types have been converted into iPSCs using this technology, demonstrating similarities and slight differences between iPSCs lines, which are known to depend on the origin of the cells used in reprogramming. The present chapter will provide an overview of iPSCs derived from dental stem cells (DSCs), such as stem cells isolated from apical papilla (SCAPs), stem cells from exfoliated deciduous teeth (SHEDS), from pulp of third molars and adult permanent teeth (DPSCs). We will discuss the origin of the cells used for reprogramming, factors which may favor or hinter the reprogramming process, methods and efficiency of cell reprogramming; the differentiation ability of iPSCs derived from DSCs; their safety, tolerance by the host and regenerative potential in preclinical models, as well as the use of these cells in toxicological studies, disease modeling and drug discovery. The possible use of iPSCs obtained from DSCs as a new tool for regenerative therapy will also be shortly discussed.

Dental Stem Cells: Risk and Responsibilities

In early embryo development, the tooth tissues are originated from the neural crest anatomical site. Neural crest stem cells are considered embryonic-like stem cells, which are maintained under the control of Hox genes. Moreover, they are clonogenic cells and are able to differentiate into various cell types, such as odontoblasts, osteoblasts, chondrocytes, neurons, melanocytes, and the muscles. Following the migration of neural crest stem cells during fetal development, the oral epithelium- and cranial crest-derived mesenchymal cells arise, which next form the dental follicle and dental pulp.