Influence of Glucose Concentration on Colony-Forming Efficiency and Biological Performance of Primary Human Tissue–Derived Progenitor Cells

Objective Glucose concentrations used in current cell culture methods are a significant departure from physiological glucose levels. The study focuses on comparing the effects of glucose concentrations on primary human progenitors (connective tissue progenitors [CTPs]) used for cartilage repair. Design Cartilage- (Outerbridge grade 1, 2, 3; superficial and deep zone cartilage), infrapatellar fatpad-, synovium-, and periosteum-derived cells were obtained from 63 patients undergoing total knee arthroplasty and cultured simultaneously in fresh chondrogenic media containing 25 mM glucose (HGL) or 5 mM glucose (NGL) for pairwise comparison. Automated ASTM-based quantitative image analysis was used to determine colony-forming efficiency (CFE), effective proliferation rates (EPR), and sulfated-proteoglycan (GAG-ECM) staining of the CTPs across tissue sources. Results HGL resulted in increased cell cultures with CFE = 0 compared with NGL in all tissue sources ( P = 0.049). The CFE in NGL was higher than HGL for superficial cartilage ( P < 0.001), and contrary for synovium-derived CTPs ( P = 0.046) when CFE > 0. EPR of the CTPs did not differ between the media in the 6-day assay time period ( P = 0.082). The GAG-ECM area of the CTPs and their progeny was increased in presence of HGL ( P = 0.027). Conclusion Glucose concentration is critical to progenitor’s physiology and should be taken into account in the setting of protocols for clinical or in vitro cell expansion strategies.

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE

Objective. Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. Materials and methods. The colony forming efficiency (CFE) was assayed in stage I–II plasma cell myeloma (PCM) patients (n = 37) aged 42–73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons not exposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM, 9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stage I PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in the control group. Results. Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposed and not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs. (11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients. Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosis was lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission up to (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients during diagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in the stage I and stage II PCM patients. Conclusion. Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39, p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients was significantly longer vs. cases of CFE < 20. Key words: plasma cell myeloma, bone marrow, granulocyte-macrophage colony-forming unit, fibroblast colony-forming unit, cluster.

ENDOMETRIAL STROMAL CELLS: ISOLATION, EXPANSION, MORPHOLOGICAL AND FUNCTIONAL PROPERTIES

Aim: We aimed to study biological properties of human endometrial stromal cells in vitro. Materials and Methods: The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incubator at 5% CO2 and 5% O2. At P3 the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit. Results: Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90+CD105+CD73+CD34-CD45-HLA-DR-. The cells also expressed CD140a, CD140b, CD146, and CD166 antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3 h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48 h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8 and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10 and MCP-1. Conclusion: Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use.

284 CHARACTERIZATION OF GOAT MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW AND ADIPOSE TISSUE

Gene modification of cells in vitro followed by somatic cell nuclear transfer (SCNT) currently offers the best route for creating genetically modified livestock species. However, low cloning efficiencies in differentiated somatic cells have been attributed to the possibility of improper nuclear reprogramming. Adult stem cells may have greater developmental potential and better nuclear reprogramming potential following cloning. There is considerable interest in using goats as models for genetically engineering dairy animals and for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSC) are adult stem cells that that have been isolated and characterised from various species, but are poorly characterised in goats. Three MSC lines were isolated from bone marrow (9004 BM-MSC, 9003 BM-MSC) and adipose tissue (9003 A-MSC) of neonatal goats. In this study, these MSC lines were characterised to verify MSC-specific characteristics and assess their amenability to genetic modification in vitro. Passage 5 cells were evaluated for capacity to differentiate into osteogenic, adipogenic, and chondrogenic lineages, as well as for colony-forming efficiency after 10 days of culture from low-density plating. Expression of MSC-specific positive cell surface markers CD90, CD73, and CD105, as well as pluripotency markers Nanog, Oct-4, and Sox-2, was examined by RT-PCR. Oct-4 protein localization was examined by immunofluorescence. The MSC were also assessed for their potential for gene modification by nucleofection with circular and linearized plasmids expressing green fluorescent protein (GFP) and neomycin resistance. Differences between cell lines were statistically analysed using ANOVA. The 9003 BM-MSC cells were also utilised for SCNT. All 3 MSC lines showed a normal karyotype. The MSC lines were capable of undergoing osteogenic, adipogenic, and chondrogenic differentiation, with observed differences in capacities between the BM-MSC and A-MSC lines, as shown by staining with Alizarin Red S, Oil Red O, and Alcian Blue. Expression of CD90, CD73, CD105, Nanog, Oct-4, and Sox-2 was detected, and Oct-4 was localised in the cytoplasm. There were significant differences in clonability between the cell lines, with 9004 BM-MSC showing the highest colony-forming efficiency (61% ± 5.4; P < 0.05). There were no significant differences in the percentage of GFP-positive cells from transfections done with the circular plasmid, but 9003 BM-MSC yielded a significantly lower number of integrant colonies per 500 000 cells transfected with the linear plasmid and G418 selection (12.75 ± 3.24; P < 0.05). Somatic cell nuclear transfer was able to reprogram 9003 BM-MSC and produce pregnancies. One hundred forty-four embryos were reconstructed, 101 embryos were transferred into 8 recipients, and the resulting pregnancy rate was 73%. Our findings provide characterisation information on goat MSC, and show that significant differences can exist between MSC isolated from different tissues and from within the same tissue.