derivation-differentiation-canine-ovarian-mesenchymal-stem

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

University of São Paulo

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are  the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences  between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that  improve knowledge of the pathology of stroke in both human and veterinary medicine.

The use of animal models for stroke research: a review.

Pteridium aquilinum, one of the most important poisonous plants in the world, is known to be carcinogenic to animals and humans. Moreover, our previous studies showed that the immunosuppressive effects of ptaquiloside, its main toxic agent, were prevented by selenium in mouse natural killer (NK) cells. We also verified that this immunosuppression facilitated development of cancer. Here, we performed gene expression microarray analysis in splenic NK cells from mice treated for 14 days with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg) to identify gene transcripts altered by ptaquiloside that could be linked to the immunosuppression and that would be prevented by selenium. Transcriptome analysis of ptaquiloside samples revealed that 872 transcripts were expressed differentially (fold change>2 and p<0.05), including 77 up-regulated and 795 down-regulated transcripts. Gene ontology analysis mapped these up-regulated transcripts to three main biological processes (cellular ion homeostasis, negative regulation of apoptosis and regulation of transcription). Considering the immunosuppressive effect of ptaquiloside, we hypothesized that two genes involved in cellular ion homeostasis, metallothionein 1 (Mt1) and metallothionein 2 (Mt2), could be implicated because Mt1 and Mt2 are responsible for zinc homeostasis, and a reduction of free intracellular zinc impairs NK functions. We confirm these hypotheses and show increased expression of metallothionein in splenic NK cells and reduction in free intracellular zinc following treatment with ptaquiloside that were completely prevented by selenium co-treatment. These findings could help avoid the higher susceptibility to cancer that is induced by P. aquilinum-mediated immunosuppressive effects.

Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.

Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

Amnion-derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases. 

Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation.

Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine. 

Parthenogenesis and Human Assisted Reproduction.

Skin is an extensive and easily accessible organ possessing various cell types that are constantly renewed. Previous studies have suggested the presence of a stem cell niche at the bulge region of the hair follicle, which contains cells positive for CD200 and CD34. Thus, this study sought to identify these cell populations in canine skin cells using the following methods 1- collecting samples of adult and fetal skin and isolating and culturing these cells using a method of simple enzymatic digestion and 2- testing the cell cultures for CD200 and CD34 in vitro and comparing them with skin tissue samples (in situ). Immunofluorescence results were negative for both CD200 and CD34 in frozen and paraffin embedded tissue, whereas the analysis showed that cultured cells positive for CD34, CD200 and double positive cells could be visualized in different percentages. Additionally, the pluripotency marker OCT4 was positive in the isolated cells. Analysis of CD34, CD200 and OCT4 by RT-qPCR showed that there is expression in fetal and adult cells, although no difference was observed between groups. Our results suggest that bulge stem cells from both fetuses and adult dogs were reported with the use of CD34 and CD200 markers in this study, and further techniques for cell isolation and in vitro cultivation are needed in order to obtain enriched populations of skin stem cells in dogs.

In vitro identification of a stem cell population from canine hair follicle bulge region.

Primordial germ cells (PGCs) are precursors of gametes that can generate new individuals throughout life in both males and females. Additionally, PGCs have been shown to differentiate into embryonic germ cells (EGCs) after in vitro culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (Canis lupus familiaris). Here, we elucidated the dynamics of the expression of pluripotent (POU5F1 and NANOG), germline (DDX4, DAZL and DPPA3), and epigenetic (5mC, 5hmC, H3K27me3 and H3K9me2) markers that are important for the development of male canine germ cells during the early (22-30 days post-fertilization (dpf)), middle (35-40 dpf) and late (45-50 dpf) gestational periods. We performed sex genotype characterization, immunofluorescence, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. Furthermore, in a preliminary study, we evaluated the capacity of canine embryo PGCs (30 dpf) to differentiate into EGCs. To confirm the canine EGCs phenotype, we performed alkaline phosphatase detection, immunohistochemistry, electron and transmission scanning microscopy and RT-qPCR analyses. The PGCs were positive for POU5F1 and H3K27me3 during all assessed developmental periods, including all periods between the gonadal tissue stage and foetal testes development. The number of NANOG, DDX4, DAZL, DPPA3 and 5mC-positive cells increased along with the developing cords from 35-50 dpf. Moreover, our results demonstrate the feasibility of inducing canine PGCs into putative EGCs that present pluripotent markers, such as POU5F1 and the NANOG gene, and exhibit reduced expression of germinative genes and increased expression of H3K27me3. This study provides new insight into male germ cell development mechanisms in dogs.

Dynamics of male canine germ cell development.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

Article	Year
Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle. Cellular reprogramming\| PubMed ID: 21241190	2011
The use of animal models for stroke research: a review. Comparative medicine\| PubMed ID: 22330245	2011
Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium. Toxicology\| PubMed ID: 23274088	2013
Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer. In vitro cellular & developmental biology. Animal\| PubMed ID: 23519559	2013
Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation. Stem cells and cloning : advances and applications\| PubMed ID: 25249758	2014
Parthenogenesis and Human Assisted Reproduction. Stem cells international\| PubMed ID: 26635881	2016
In vitro identification of a stem cell population from canine hair follicle bulge region. Tissue & cell\| PubMed ID: 29429517	2018
Dynamics of male canine germ cell development. PloS one\| PubMed ID: 29489867	2018
ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells. Veterinary and comparative oncology\| PubMed ID: 30047225	2018

Fabiana F. Bressan

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