Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn's disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.

This study investigated conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells (S-MSC). Forty primary skin-derived precursor cell (SKP) cultures were established from both male and female donors (age 29-65 years) and eight of them were randomly selected for in-depth characterization. Effects of basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), leukemia inhibiting factor (LIF) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on S-MSC proliferation were investigated. Primary SKP cultures were >95% homogenous for CD90, CD73, and CD105 marker expression enabling to classify these cells as S-MSC. FGF-2 dose-dependent stimulation was observed in low serum medium only, whereas EGF neither stimulated S-MSC proliferation nor potentates the effect of FGF-2. Pronounced donor to donor differences among S-MSC cultures were observed in 3-day proliferation assay. This study demonstrates that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age. Optimal cell culture conditions for in vitro propagation of S-MSC are B27 supplemented or low serum media with FGF-2 (4 ng/ml). EGF and LIF as well as db-cAMP are dispensable for S-MSC proliferation.

The epigenetic status of integrated adenovirus type 12 (Ad12) DNA in hamster cells cultivated for about 4 decades has been investigated. Cell line TR12, a fibroblastic revertant of the Ad12-transformed epitheloid hamster cell line T637 with 15 copies of integrated Ad12 DNA, carries one Ad12 DNA copy plus a 3.9-kbp fragment from a second copy. The cellular insertion site for the Ad12 integrate, identical in both cell lines, is a >5.2-kbp inverted DNA repeat. The Ad12 transgenome is packaged around nucleosomes. The cellular junction is more sensitive to micrococcal nuclease at Ad12-occupied sites than at unoccupied sites. Bisulfite sequencing reveals complete de novo methylation in most of the 1,634 CpGs of the integrated viral DNA, except for its termini. Isolated unmethylated CpGs extend over the entire Ad12 integrate. The fully methylated transgenome segments are characterized by promoter silencing and histone H3 and H4 hypoacetylation. Nevertheless, there is minimal transcriptional activity of the late viral genes controlled by the fully methylated major late promoter of Ad12 DNA.A major part of mammalian genomes is made up of repetitive DNA, and viral retrotransposons comprise a substantial part of it. Little is known about the stability or numerical plasticity of the repetitive sequences. Repetitive DNA in mammalian genomes is heavily methylated (for recent reviews, see references 6, 29, and 47), possibly as a defense against the potential activity of foreign genes in an established genome (5, 50). The degree of methylation, particularly of the repetitive cellular DNA sequences, is subject to alterations in cells with integrated foreign DNA in the genome (13,32). Transgenomes are frequently generated and exploited in experimental and applied molecular biology. Their structure and the effects of their insertion on the stability and functionality of the recipient genome require more detailed studies.In a model system, we have investigated the stability as well as the methylation and transcriptional profiles of integrated adenoviral genomes in hamster cells. The transformed cell line T637 has been generated by infecting baby hamster kidney cell line BHK21 (40) with human adenovirus type 12 (Ad12) (41). The genomes of T637 cells carry about 15 copies of Ad12 DNA integrated at a single chromosomal site (18,35,39). Upon continuous cultivation of cell line T637, morphological revertants, from epitheloid to more fibroblastic, arose spontaneously (10). In one of these revertants, TR12, only one copy and a fragment of a second copy of Ad12 DNA persist stably (7).The genomes of human adenoviruses are always chromosomally integrated and hypermethylated in Ad12-transformed cells and in Ad12-induced hamster tumor cells (26,42,43). Virion DNA or free intracellular adenoviral DNA is not methylated (11,17). The integrated Ad12 genomes in the revertant cell line TR12 are more extensively methylated than those in the parental cell line, T637 (27). Hence, hypermethylation of transgenic DNA might be related to its stability in t...

Objectives To evaluate the main symptoms of knee osteoarthritis (OA) and tissue structure changes after a single dose bone marrow-derived mononuclear cell (BM MNC) intra articular injection. Case series study. Patients with knee OA Kellgren Lawrence (K-L) grade II and III received 1 injection of BM MNC. The clinical results were analyzed with the Knee injury and Osteoarthritis Outcome Score (KOOS) and Knee Society Score (KSS) before, 3, 6, and 12 months after injection. Radiological evaluation was performed with a calibrated x-ray and the magnetic resonance (MR) imaging before and 6 to 7 months postinjection. Results A total of 34 knees were treated with BM MNC injections. Mean (±SD) age of patient group was 53.96 ± 14.15 years; there were 16 males, 16 females, KL grade II, 16; KL grade III, 18. The average injected count of BM MNCs was 45.56 ± 34.94 × 10 cells. At the endpoint of 12 months 65% of patients still had minimal perceptible clinical improvement of the KOOS total score. The mean improvement of KOOS total score was +15.3 and of the KSS knee score was +21.45 and the function subscale +27.08 ( P < 0.05) points. The Whole Organ Magnetic Resonance Imaging Score (WORMS) improved from 44.31 to 42.93 points ( P < 0.05). No adverse effects after the BM-MNC injection were observed. Conclusions The single dose BM MNC partially reduces clinical signs of the knee osteoarthritis stage II/III and in some cases, decreases degenerative changes in the joint building tissue over 12-month period.