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.

Identificação do nicho de progenitores mesenquimais no fígado de embriões e fetos caninos: uma fonte de células-tronco para terapia celular / Identification of mesenchymal progenitor niches from liver of embryo and fetuses of canines: a source of stem cells for cell therapy

As células-tronco (CT) derivadas dos tecidos fetais (TF) foram as mais recentes descobertas entre as CT, e ultimamente tem demonstrado amplo potencial terapêutico, dentre os TF o fígado fetal (FF) apresenta grande potencial terapêutico. Este órgão durante o período fetal em mamíferos é um nicho hematopoético transitório, sendo o principal órgão responsável pela hematopoese no feto, além de contribuir com a formação do nicho definitivo na medula óssea adulta, portanto pode ser considerado um nicho de células-tronco mesenquimais (CTM) e progenitores. No entanto, pouco se sabe sobre a localização destas células no FF, desta forma o presente estudo visa identificar o nicho de CTM e progenitores em FF de cães, a fim de contribuir com as técnicas de isolamento e extração celular. Em conjunto foi realizada a verificação da expressão do fator de transcrição Oct-3/4 e da proteína delta polimerase do DNA (PCNA). Para a análise foram utilizados cinco embriões e 11 fetos caninos com idades gestacionais variando de 25-60 dias. Os resultados elucidaram a partir de 25 dias de gestação o FF apresentou-se volumoso e composto por todas as estruturas típicas, dentre elas a tríade portal, ductos biliares e ramos das artérias hepáticas. Com 30 dias de gestação foram identificados os primeiros sitos de progenitores mesenquimais (PM) enquanto que aos 60 dias os nichos estavam completamente formados com localização semelhante ao fígado adulto (FA). No entanto, células imunopositivas para Oct-3/4 não foram identificadas; sendo assim, destacamos que o FF é uma fonte de PM, apresentando-se como uma alternativa para a utilização terapêutica, bem como para os estudos da biologia do desenvolvimento das CTM e progenitores.(AU)

Stem cells (SC) derived from fetal tissues (FT) were the most recent discoveries among the SC, and lately had demonstrated broad therapeutic potential; the FT from the fetal liver (FL) presents great therapeutic potential. This organ during the fetal period in mammals acts as a transient hematopoietic niche, being the main organ responsible for hematopoiesis in the fetus, and contribute to the formation of permanent niche in the adult bone marrow, thus can be considered a niche for mesenchymal stem cells (MSC) and parents. However, little is known about the location of these cells in FF, so the present study aims to identify the niche of mesenchymal progenitors in FF and dogs in order to contribute to the techniques of cell isolation and extraction. Together was performed to verify the expression of the transcription factor Oct-3/4 of the protein and DNA polymerase delta (PCNA). For the analysis of five embryos were used and 11 canine fetuses with gestational ages ranging from 25-60 days. The results elucidated from 25 days of gestation showed the FF is bulky and composed of all the typical structures, among them the portal triad, bile ducts and hepatic artery branches. With 30 days of gestation were identified the first requirements of mesenchymal progenitors (MP) at 60 days while the niches were completely formed with location similar to adult liver (AL). However, cells immunoreactives for Oct-3/4 were not identified, therefore, point out that the FL is a source of PM, presenting as an alternative for therapeutic purposes as well as for studying the developmental biology of MSC and parents.(AU)

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord.

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.

Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells.

Induced pluripotent stem cells (iPSCs) can be created by forcing expression of certain genes in fibroblasts or other somatic cell types, reversing them to a pluripotent state similar to that of embryonic stem cells (ESC). Here, we used human immature dental pulp stem cells (hIDPSCs) as an alternative source for creating iPSC. hIDPSCs can be easily isolated from accessible tissue of young and adult patients. hIDPSCs possess a fibroblast-like morphology, retaining characteristics of adult multipotent stem cells. Reprogramming of hIDPSCs was fast, producing primary hIDPSC-iPSC colonies even under feeder-free conditions. hIDPSCs acquired ESC-like morphology, expressed pluripotent markers, possessed stable, normal karyotypes, and demonstrated the ability to differentiated in vitro and in vivo. Our data demonstrate that hIDPSCs-iPSCs offer an advantageous cell system for future cell therapy and basic studies, particularly as a model for pediatric developmental disorders.

Morfofisiologia da inervação do diafragma de ovinos / Morphophysiology of diaphragm innervation in sheep

Thirty diaphragms of sheep of Santa Inês breed were studied regarding their origin, division and arrangement of the right and left phrenic nerves (Fde), and the participation of other nerves in the innervation of the diaphragm. By fixing and dissecting pieces, it was found that phrenic nerves (F) frequently come from the ventral branches of the 5th (C5) and 6th (C6) cervical spinal nerves (Ec), at right (46.67 percent) and at left (43.33 percent). The F often form a lumbocostal trunk, sternal branches at right (40.00 percent) and lumbar, costal and esternal branches at left (36.68 percent). The lumbar branches of F innervate frequently at left (96.67 percent) the homolateral pillar of the diaphragma, and at right (50.00 percent) they give fillets to Vena cava caudalis. The costal branches of the F innervate at left (90.00 percent) and at right (76.66 percent) the dorsal and ventral regions of the pars costalis. The sternal branches of the F innervate at right (100.00 percent) and at left (83.33 percent) the pars sternalis and the ventral region of the pars costalis at the same side. The intercostal nerves (VII to XII pairs, 63.33 percent) contribute to innervate the diaphragm of Santa Inês sheep.

Foram estudados em 30 diafragmas de ovinos da raça Santa Inês, a origem, a divisão e a distribuição dos nervos frênicos direito e esquerdo (Fde) e a participação de outros nervos na inervação do diafragma. Mediante fixação e dissecação das peças foi observado que os nervos frênicos (F) originam-se a partir dos ramos ventrais do 5º (C5) e 6º (C6) nervos espinhais cervicais (Ec) tanto à direita (46,67 por cento) como à esquerda (43,33 por cento). Os F finalizam em tronco lombocostal e ramo esternal à direita (40,00 por cento) e em ramo lombar, costal e esternal à esquerda (36,68 por cento). Os ramos lombares dos F inervam à esquerda (96,67 por cento) o pilar homolateral do diafragma e, à direita (50,00 por cento) fornecem filetes à veia caudal. Os ramos costais dos F ramificam à esquerda (90,00 por cento) e à direita (76,67 por cento) as regiões dorsal e ventral da pars costalis. Os ramos esternais dos F inervam à direita (100,00 por cento) e à esquerda (83,33 por cento) a pars sternalis e a região ventral da pars costalis do mesmo lado. Os nervos intercostais (VIII ao XII pares, 63,33 por cento) contribuem na inervação do diafragma de ovinos da raça Santa Inês.

Comportamento dos nervos glossofaríngeo e vago, na região retrofaríngea de ovinos: origem aparente no crânio, trajeto, ramificação e distribuição / Behavior of the glossopharyngeal and vagus nerves in the retropharyngeal region of sheep: apparent origin in cranium, course, branching and distribution

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 percent of the cases. The right and the left glossopharingeal nerve (Glde) are frequently (86.6 percent) 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 percent). The right and the left vagus nerve run caudally in association with the accessory nerves (Ac) up to the atlas (70 percent) and go then medially to the occipital artery, dorsally to the common carotid and the sympathetic trunk in the cervical region (80 percent). The vague nerves have pharyngeal (86.6 percent) and cranial laryngeal (100 percent) branches.

Em 60 hemicabeças de ovinos da raça Santa Inês foram estudadas a origem aparente no crânio, trajeto, ramificação e distribuição do nervo glossofaríngeo (Gf) e do nervo vago (Vg), na região retrofaríngea (Rr). Mediante fixação e dissecação das peças, foi observado que os nervos glossofaríngeos e vagos originam-se no forame jugular em 100 por cento dos casos. Os nervos glossofaríngeos direito e esquerdo (Gfde) são localizados, com maior freqüência (86,6 por cento), medialmente à bula timpânica, passam caudalmente ao osso estiloióide, contornam a margem caudal do músculo estilofaríngeo caudal, tonsilar, da mucosa faríngea e lingual. Estes ramos distribuemse, respectivamente, no seio carotídeo, musculatura faríngea, palato mole, músculo estilofaríngeo caudal, tonsila palatina, mucosa faríngea e terço caudal da língua (100 por cento). Os nervos vagos direito e esquerdo (Vgde) correm caudalmente em associação com os nervos acessórios (Ac) até o atlas (70 por cento), após o que passam medialmente à artéria occipital, dorsalmente à carótida comum e ao tronco simpático, na região cervical (80 por cento). Os ramos dos nervos vagos são os faríngeos (86,66 por cento) e os laríngeos craniais (100 por cento).