npg Differentiation, Senescence and Stem Cell S54 Cell Research (2006) 16:S54-S72 npg © 2006 IBCB, SIBS, CAS All rights reserved 1001-0602/06 www.nature.com/cr APOCB2006-05-001 hTERT to immortalize a variety of human cell types includ- The role of telomeres and telomerase in aging and cancer ing skin keratinocytes, dermal fibroblasts, muscle satellite Jerry W Shay (stem) cells, endothelial cells, retinal-pigmented epithe- UT Southwestern Medical Center, Dallas, USA lial cells, breast epithelial cells, both corneal fibroblasts (keratocytes) and corneal epithelial cells and lastly human Cellular senescence is a general stress-response program bronchial epithelial and stromal cells. The epithelial and that restrains cellular proliferation. There are pathways stromal cells can be reconstituted in 3D to produce tissue- leading to an irreversible arrest of cell proliferation that like structures. The production of hTERT engineered cells are independent of telomere length (overexpression of offer the possibility of producing tissues to treat a variety oncogenes, reactive oxygen species). However, under opti- of diseases and aged-related medical conditions that are mal growth conditions, the onset of replicative senescence due to telomere-based replicative senescence. depends on telomere status. When telomeres are sufficiently Keywords: senescence; stem cells; immortalization; re- short (uncapped) there is a DNA damage response-in- generative medicine; telomeres; duced growth arrest. The senescence pathway involves Correspondence: Jerry W. Shay the formation of telomere dysfunction induced foci that E-mail: [email protected] contain DNA damage response factors. The DNA-damage response in senescent cells is not a transient phenomenon, but consists of a permanent activation of the DNA dam- APOCB2006-05-002 age checkpoint machinery. The long-term growth arrest Myogenesis potential of porcine adipose-derived mesen- at senescence may be thought of as an initial anti-tumor chymal stem cells protection mechanism. In situations where normal cell Changqing Qu1,2, Guohua Zhang1, Gongshe Yang1 cycle checkpoints are altered, cells can bypass the normal 1Laboratory of Animal Fat Deposition and Muscle Development, senescence signaling pathway and continue to grow until Northwest A&F University, Yangling, China; 2Department of they reach a second growth arrest state known as crisis. In biology, Fuyang normal college, Fuyang, China crisis, telomeres are terminally short and, in the presence of other genetic and epigenetic changes, can result in telomeric Recently, some studies suggested that human adipose tissue fusions, subsequent breakage-fusion-bridge cycles and in contains pluripotent stem cells similar to bone marrow- rare cells, up-regulation or reactivation of telomerase, the derived stem cells which have the capability to colonize cellular RNP that is able to add telomeric repeats to the different tissues, replicate, and differentiate into multilin- ends of chromosomes and thus prevent their shortening. eage cells. Animals such as farm animals provide both a In both senescence and crisis ectopic expression of the source of fat-derived stem cells and an immunocompetent, catalytic subunit of telomerase (hTERT) leads directly to autologous host animal in which to investigate the capacity an immortal state demonstrating that telomeres are im- of the fat-derived cells to differentiate and form tissues in portant in both replicative senescence and crisis. In most vivo. Our goal was the isolation and characterization of por- pre-neoplasias, telomeres are exceptionally short. Almost cine adipose tissue mesenchymal to study their myogenic all human malignant tumors express telomerase to maintain potential in vitro. Porcine adipose mesenchymal stem cells these short telomeres and this activity is probably required (pAMSCs) were selected from the dorsal neck, shoulder, for the long-term growth of most advanced malignancies. and back regions of Large-White pigs after extensive wash- Telomerase activity is absent or at lower levels in normal ing with PBS and treatment with collagenase. After 24h in tissues, making the inhibition of telomerase an attractive primary culture in growth medium (Dulbecco’s modified target for cancer therapeutics. Cancer stem cells (a subset Eagle’s medium +10% fetal bovine serum), pAMSCs ad- of cells within a tumor mass that is capable of regenerating hered to the plastic surface, presenting a small population of the tumor) are both telomerase expressing and contain short single cells. 7 to 10 days after initial plating, the cells looked telomeres. Telomerase inhibitors are thus likely to target like long spindle-shaped fibroblastic cells and began to form both the bulk of the more differentiated tumor cells as well colonies. When the cells approached densities over 80%, as the stem cells that are thought to be the source of che- the cell morphology changed to a more spindle-shaped, motherapy and radiotherapy resistance in most advanced fibroblastic morphology. After expansion to 20 passages, cancers. The expression of the catalytic subunit of human the fifth passage cells were selected and Flow cytometry telomerase (hTERT) reconstitutes telomerase activity and analysis of isolated pAMSCs demonstrated the following circumvents the induction of senescence. We have used phenotype, CD44 (pos), CD105 (pos) and CD14 (neg), Cell Research | www.cell-research.com npg S55 CD106 (neg), HLA-DR and S100(neg), similar to that de- examined passages. Although Thy1.2 was not expressed scribed for human AMSCs. To induce optimal myogenesis, in primary culture, it was observed on about half to one the fifth passage pAMSCs were plated in myogenic medium fifth of the cells in passage 3. CD31 antigen was never (Dulbecco’s modified Eagle’s medium +10% fetal bovine expressed on the cells. The expressional level of CD135, serum+5% horse serum+0.1 mM dexamethasone +50 mM CD45 and CD11b were gradually decreased and c-kit, Sca1 hydrocortisone) for 3 weeks. Myogenic differentiation was and CD34 expression were slightly increased from primary assessed by desmin and myoglobin staining, and expression culture on to passage 3. Two studied mice strains showed of myogenic specific genes were detected by RT-PCR.At some differences in expressing certain markers. Unlike first week, desmin was expressed in the treated cells. Three Balb/c cells, some cells of NMRI strain expressed VCAM weeks later, cells were stained with a monoclonal antibody antigen. Passage-3 cells from both strains were easily dif- specific to skeletal muscle myoglobin . Low levels of Myf5 ferentiated into bone, fat and cartilage when being cultured and MyoD were observed in induced pAMSCs at first week in appropriate culture condition. In conclusion: First, the and Myosin heavy chain was observed when induced for culture of mMSCs remains heterogonous in terms of their 3 weeks. In control groups, expression of these genes was surface antigens although it gradually becomes morpho- barely detectable. Thus, pAMSCs derived from adipose logically homogenous during cultivation period from the tissue were able to differentiate "ex vivo" into myoblast-like primary culture to passage 3. Second, more than 90% of the cells. These elements may be a suitable source for research cells were CD 44 positive throughout the culture period. on skeletal muscle development and studying relationship Third, the cells with hematopoietic specific antigens were between muscle and adipose. not eliminated even in the passage 3 in which the majority of the cells were typically fibroblastic cells. Fourth, the Keywords: porcine; adipose tissue; myogenic; cell culture MSCs from two mouse strains were somewhat different Correspondence: Gongshe Yang E-mail: [email protected] in terms either of morphology in culture or having certain surface antigens. Keywords: murine mesenchymal stem cells; surface anti- APOCB2006-05-003 gens; bone, adipocyte and cartilage differentiation; Balb/c Surface antigens of murine mesenchymal stem cells and NMRI strains 1 2 Mohamadreza B Eslaminejad , Samad Nadri , Correspondence: Mohamadreza B Eslaminejad Reza H hosseini2 E-mail: [email protected] 1Stem Cell Department, Royan Institute, Tehran, Iran; 2Biochem- istry Department, Payam Nour University, Tehran, Iran APOCB2006-05-004 The aim of this study is to issue the surface antigen deter- PPARα is involved in cardiomyocyte differentiation of murine minants of murine mesenchymal stem cells (mMSCs) from embryonic stem cells the bone marrow samples. For this purposes, 6-8 weeks old Ling Ding, Yijia Lou either NMRI or Balb/c mice were sacrificed, their bone mar- Institute of Pharmacology-Toxicology and Biochemical Pharma- row were harvested and plated in 75-cm2 flask at a density ceutics, College of Pharmaceutical Sciences, Zhejiang University, of 107 cells per ml. Two weeks after culture initiation, the HangZhou, China cells were tripsinized, the majority of them sub cultured in a new culture dishes in split ratio of 1:2 and 2 ×105 cells of Peroxisome Proliferator-activated Receptors α (PPARα) is primary culture were prepared for flow cytometry analysis a fatty acid-activated nuclear receptor which plays a key to detect some surface antigens including CD135, CD44, role in the transcriptional regulation of genes involved CD31, Thy1.2, CD11b, CD45, CD34, Vcam1, Sca-1and in cellular lipid and energy metabolism. It is abundantly c-Kit. Cultivation of the cells was continued for 3 passages expressed in tissues with high energy demand, including