Guido Stadler

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, stem cells (SC)s derived from other adult tissue sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. The aim of our study was to compare the immunomodulatory potential of human amniotic mesenchymal and human amniotic epithelial cells with that of human adipose-derived SCs under identical experimental conditions. We have demonstrated a dose-dependent inhibition of peripheral blood mononuclear cell (PBMC) immune responses in mixed lymphocyte reactions (up to 66-93% inhibition) and phytohemagglutinin activation assays (up to 67-96% inhibition). The lowest SC-to-PBMC ratio able to inhibit PBMC proliferation significantly was 1:8. Subcultivation (passage 2-6) did not alter immunoinhibitory properties, whereas cryopreservation significantly reduced the immunomodulatory potential. Using transwell systems, we have demonstrated an inhibition mechanism that is dependent on cell contact. Additionally, in coculture with allogeneic PBMCs, SCs were well tolerated and at most provoked mild alloreactions in singular cases. This study demonstrates, for the first time, contact- and dose-dependent immunosuppression of mesenchymal and epithelial amniotic SC populations, as well as of adipose tissue-derived SCs. All three cell types may be considered as possible alternatives to BMSCs for allogeneic application in tissue engineering.

While global chromatin conformation studies are emerging, very little is known about the chromatin conformation of human telomeres. Most studies have focused on the role of telomeres as a tumor suppressor mechanism. Here we describe how telomere length regulates gene expression long before telomeres become short enough to produce a DNA damage response (senescence). We directly mapped the interactions adjacent to specific telomere ends using a Hi-C (chromosome capture followed by high-throughput sequencing) technique modified to enrich for specific genomic regions. We demonstrate that chromosome looping brings the telomere close to genes up to 10 Mb away from the telomere when telomeres are long and that the same loci become separated when telomeres are short. Furthermore, expression array analysis reveals that many loci, including noncoding RNAs, may be regulated by telomere length. We report three genes (ISG15 [interferon-stimulated gene 15 kd], DSP [Desmoplakin], and C1S [complement component 1s subcomplement]) located at three different subtelomeric ends (1p, 6p, and 12p) whose expressions are altered with telomere length. Additionally, we confirmed by in situ analysis (3D-FISH [three-dimensional fluorescence in situ hybridization]) that chromosomal looping occurs between the loci of those genes and their respective telomere ends. We term this process TPE-OLD for "telomere position effect over long distances." Our results suggest a potential novel mechanism for how telomere shortening could contribute to aging and disease initiation/progression in human cells long before the induction of a critical DNA damage response.

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.