SUMMARY OCT4A transcription factor is one of the main regulators of pluripotency. It is expressed in embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells) and primordial germ cells. In recent years, the expression of OCT4 has also been detected in various tumors, normal adult tissues (bone marrow, umbilical cord and peripheral blood), and even in certain adult cell lines. The OCT4 gene has three alternative transcripts: OCT4A, OCT4B and OCT4B1, which are translated into four different protein isoforms: OCT4A, OCT4B-265, OCT4B-190, and OCT4B-164. There are also at least six known OCT4 pseudogenes scattered through the genome (PG1 to PG6). We showed that pseudogenes OCT4-pg1, OCT4-pg3 and OCT4-pg4 are expressed in human umbilical cord blood, adult human bone marrow and peripheral blood. We were not able to confirm the expression of embryonic OCT4A in any of tested somatic cell types. Undifferentiated embryonic stem cells and EC cells (NTERA and NCCIT) did not express any of the OCT4 pseudogenes. During differentiation, the expression of pluripotency markers OCT4A, SOX2, NANOG and CRIPTO-1 was down regulated and expression of pseudogenes was turned on and they were exclusively expressed in differentiated cells. During the dediferentiaion of fibroblasts pseudogenes were turned down and cells started to express embryonic OCT4A. Several reports argue that the detection of OCT4A in somatic cells is false-positive result due to the detection of all OCT4 isoforms, pseudogene transcripts and genomic DNA contamination. To avoid falsely positive results the use of specific primers for PCR, followed by cloning and sequencing, is mandatory. Immunocytochemistry imaging is often used as method to confirm protein expression and localization of stem cell-specific transcription factors to cell nuclei. During our immunocytochemistry studies of stem cells we came accross an interesting phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. We tested multiple DNA dyes, mounting media and three different fixatives. Our data provides a comprehensive insight in the various factors that affect DAPI photoconversion and shows how the effect can be avoided. Our work is the first report about the expression of OCT4A pseudogenes 1, 3 and 4 during ES cells differentiation. Our results show that during differentiation of stem cells there is a developmentally regulated balance between the expression of embryonic genes and pseudogenes. The switch between one transcription pattern to the other depends on differentiation status of the cells. Since pseudogenes were expressed only in mature somatic cells we propose that the expression of pseudogenes can be used as a novel marker of cell differentiation status. Our data also indicates that it is necessary to critically evaluate existing published conclusions on the expression of OCT4A in different cell types, because pseudogene expression can lead to false positive results of molecular analysis. We examined the specificity of published primers for PCR and our data show that most of them are not specific and in addition to embryonic form also amplify several pseudogenes and isoform B.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 266048512The term describes the treatment of advanced cell therapy , tissue engineering and gene therapy. Cell therapy is the treatment of autologous or allogeneic cells . The transplanted cells can replace missing or diseased cells or their components , or with others to achieve therapeutic effects (eg changes in the immune system attack cancer cells ) . The most prevalent form of the stem cell transplant , which treated many , mainly hematological diseases . As a source of hematopoietic stem cells in the peripheral blood largely replaced bone marrow . Establishment of a voluntary register of donors has greatly increased the availability of donors. Suitable source of allogeneic hematopoietic stem cell is the cord blood , which is widely available , the donor does not bring risks and causes less graft versus host disease . Therapy with specific populations of leukocytes to allow for both a selective anti-tumor effects as well as the inhibition of graft vs. host disease and autoimmunity . The therapy with stem cell transplant in the future will probably also be used to treat diseases such as Parkinson's disease , diabetes , arthritis , multiple sclerosis and cardiovascular diseases . Similar to cellular therapies , even in tissue engineering are the main therapeutic ' active ' cells . The paper describes the modalities of advanced therapies that are already used routinely.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 30934489Cord blood (CB) is a rich source of stem cells. It contains both hematopoietic and mesenchymal stem cells, which have become of interest for use in regenerative medicine. Public banks of umbilical cord blood are storing more than 570,000 units of the CB, which are associated to international registries of CB. It is estimated that an even greater number is stored as autologous units of CB in private banks. Majority of professional associations do not support the storage of autologous CB based on existing indications for UCB transplantation. Autologous CB has potential importance in developing regenerative medicine. In Slovenia there is one public cord blood bank at the Blood Transfusion Center Ljubljana, where we store 859 units. Only the units that meet stringent quality requirements are used, however.
F.22 Improvement to existing health/diagnostic methods/procedures
COBISS.SI-ID: 30611417