Telomerase reverse transcriptase (TERT) vectors were transfected into bone marrow mesen- chymal stem cells (BMSCs) which were then cultured and selected to establish TERT-BMSC cell lines whilst sequencing BMSCs and TERT-BMSCs via transcriptome in this study to explore their regulatory mechanism and effect on osteogenic differentiation after TERT ectopic expres- sion in sheep BMSCs. After sequencing and analysing differential genes, PI3K/Akt signalling pathway related to osteogenic differentiation was investigated. Western blot was used before and after applying the PI3K/Akt signalling pathway inhibitor LY294002 to detect protein expression levels of AKT and p-AKT. On the twenty-first day of osteogenic differentiation, RT-qPCR and Western blot were used to detect mRNA and protein expression levels of RUNX2 and OPN and alizarin red staining was utilised to analyse calcium salt deposition. Results showed that pro- tein expression levels of AKT and p-AKT were significantly up-regulated, mRNA and protein expression levels of RUNX2 and OPN increased and calcium salt deposition increased after ectopic expression of TERT. After applying LY294002, the protein expression of AKT and p-AKT was down-regulated, mRNA and protein expression levels of RUNX2 and OPN were reduced and calcium salt deposition was reduced. These results confirmed the stable integration and expression of the exogenous TERT gene in BMSCs to promote the differentiation of BMSC osteoblasts, which may be mediated by the PI3K/Akt signalling pathway.

Emerging researches in humans, pigs and mice, highlighted that estrogen plays a pivotal role in self-renewal and differentiation of bone marrow mesenchymal stem cells (BMSCs). The present study aimed at evaluating effects of 17 beta-estradiol (E2) on proliferation and apoptosis of canine-derived bone marrow mesenchymal stem cells (cBMSCs) in vitro. The results showed that E2 supplementation at the concentration of 10-11 M promoted the proliferation of cBMSCs by CCK-8 assay and RT-qPCR analysis for the proliferation-related genes, with proliferating cell nuclear antigen (PCNA), cyclin-D1 (CCND1) being up-regulated and cyclin-dependent kinase inhibitor 1B (CDKN1B) being down-regulated. Contrarily, analysis of fluorescence-activated cell sorting (FACS) and RT-qPCR demonstrated that E2 supplementation above 10-11 M had inhibitory effects on the proliferation of cBMSCs and induced apoptosis. Intriguingly, cBMSCs still possessed the capability to differentiate into osteoblasts and adipocytes with 10-11 M E2 addition. Taken together, this study determined the optimal culture condition of cBMSCs in vitro, and has important implications for further understanding the regulatory effect of E2 on the self-renewal of cBMSCs, which are helpful for the clinical application of BMSCs.

Cellular therapy, as a part of regenerative medicine, implies to the treatment of human disorders with cells as a medical product, so called – “living drugs”. Usually such therapy is applied when other alternative efficient pharmacological therapies are not available. Stem cells of different origin: 1) tissue specific e.g. hematopethic, epithelial, neuronal, limbal; 2) mesenchymal stem cells (MSC) harvested from variety of tissues; 3) pluripotent stem cells: embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) – serve as a source of cells for regenerative medicine application, depending upon disease and application requirements. Currently MSC are the type of stem cells that are most frequently used in registered regenerative medicine clinical trials. In this paper we provide the information on the application of cell therapy in orthopedics, hematology, ophthalmology, dermatology, gastrology and neurology. The influence of origin of MSCs and iPSCs on their mode of action as therapeutic, regenerative agents are discussed. Advantages and disadvantages of application of different cell types for cell therapy are underlined. Last, but not least current low regulations in Poland and requirements of European regulatory bodies for cell therapy are pointed out and discussed.