Jaromír Vašíček

Effect of the MACS technique on rabbit sperm motility

Magnetic-activated cell sorting (MACS) separates apoptotic spermatozoa by the use of annexin V-conjugated nanoparticles which bind to phosphatidylserine that is externalized on the outer leaflet of the sperm plasma membrane. This technique yields two fractions: annexin V-negative (AnV−) and annexin V-positive (AnV+). The aim of the study was to evaluate the effect of MACS application on the motility parameters of rabbit spermatozoa. Rabbit semen samples collected separately from 4 bucks (I, II, III, and IV) were filtered and separated in a MACS system. The semen samples from a control (untreated) group, AnV− and AnV+ fraction were evaluated using CASA system. The experiment was replicated 4 times for each buck. The AnV+ sperm had significantly lower concentration than the AnV− fractions and the control samples (P<0.05 for bucks I, II, III, but not IV). We observed that the proportion of apoptotic spermatozoa in the semen of NZW bucks is about 20%. There was no significant difference in the percentage of motile and progressively motile spermatozoa between the AnVfractions and control samples. In conclusion, the MACS technique has no harmful effect on the rabbit sperm concentration and motility.

Cryopreservation of chicken blastodermal cells and their quality assessment by flow cytometry and transmission electron microscopy

The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to −80°C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live‐Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P < 0.05) between control (8.37 ± 1.04%) slow freezing (83.73 ± 2.72%) and vitrification group (84.39 ± 1.77%) in the percentage of Trypan blue positive (necrotic) BCs. Moreover, differences (P < 0.05) between control and slow freezing (5.08 ± 1.94%, 73.31 ± 3.90%) and control and vitrification group (2.97 ± 0.30%, 79.02 ± 1.56%) in results on portion of necrotic cells (DRAQ5+/LD+) analyzed by flow cytometry were also observed. The large percentage of necrotic BCs was found in all freezing methods. However, based on ultrastructural analysis, our study showed, that BCs contain lipid granules which prevent successful freezing even though different methods of cryopreservation were used. Thus, freezing of BCs probably required subsequent culture to eliminate lipid droples and yolk granules in the cells, which could possibly improve the success.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti‐CD34 and anti‐CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti‐human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti‐human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic‐activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti‐rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs.

Phenotype and ultrastructure of stem cells derived from amniotic fluid of Nitra rabbit

The isolation of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) has been already shown in human and several other species including rabbit. However, prior to the preclinical research on various animal models it is desirable to define AF-MSCs by a panel of surface protein markers. Therefore, the aim of this preliminary study was to detect the expression of several protein markers on the surface of AF-MSCs isolated from local breed of Nitra rabbit. Amniotic fluid was collected from humanely sacrificed rabbits (n = 3) and AF-MSCs were cultured to a third passage. Flow cytometry was used to detect surface protein marker expression and for viability testing. Rabbit AF-MSCs (rAF-MSCs) were also analyzed by transmission electron microscopy to define the ultrastructure. rAF-MSCs showed both sufficient viability (more than 80%) and low apoptosis rates at third passage and highly expressed CD29 (88.17 ± 7.17%) and CD44 (80.00 ± 2.28%). However, a dim expression of CD90 (17.24 ± 1.31%) and negative expression of CD73 (1.21 ± 0.56% and 4.41 ± 1.46%), CD105 (1.67 ± 0.37%) and CD166 (0.96 ± 2.26%) was observed. Additionally, ultrastructure analysis revealed eccentrically located nucleoli, an abundance of thin pseudopodia on cells’ surfaces and proved the presence of typical mesenchymal stem cell features. In conclusion, this set of data contributes to more detailed information on rAF-MSCs, which were previously proposed feasible for preclinical stem cell research and as a suitable source for the cryopreservation of animal genetic resources in gene bank.

Different RNA and protein expression of surface markers in rabbit amniotic fluid-derived mesenchymal stem cells

Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species‐specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid‐derived mesenchymal stem cells (rAF‐MSCs) and to reflect upon the current identification of AF‐MSCs by providing a summary of detected surface markers in different species. The expression of rAF‐MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF‐MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT‐PCR (reverse transcription‐polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF‐MSCs, required for the identification of these cells in preclinical investigations.