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Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10 per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmml mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmml. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4IB-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 104 copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophila.The rRNA genes (rDNA) in the ciliated protozoan Tetrahymena thermophila provide an excellent opportunity for studying DNA replication in a eucaryotic system. In the somatic nuclei of T. thermophila, the rDNA is a relatively short (21-kilobase pair [kb] somally integrated rDNA copy in each haploid genome. During macronuclear development, the rDNA is excised from the chromosome and converted into a 21-kb palindromic molecule. The palindrome is made up of two inversely oriented copies of the micronuclear rDNA sequence. The extrachromosomal rDNA acquires telomeres (1) and is amplified to a copy number of about 104 molecules per macronucleus. These maturation events are completed prior to the onset of vegetative growth, approximately 18 h after conjugation is initiated. The macronuclear rDNA is maintained at a high copy number throughout vegetative cell divisions. Figure 1A diagrams one-half of an rDNA palindrome. The origin of replication is within the 5' nontranscribed spacer (5'NTS), upstream from the transcription initiation site (Fig. 1A) (4). Two nuclease-hypersensitive regions within the 5'NTS (15), domains 1 and 2, have very similar sequences and contain evolutionarily conserved repeated sequence elements (5, 11). By mutational analysis, one of these repeated elements (type I) was implicated in the activation of rDNA replication (8).We have previously shown that rDNA from a T. thermophila inbred strain C3 (C3 rDNA) has a replicative advantage over rDNA from inbred strain B (B rDNA), causing C3 rDNA to completely replace B rDNA in C3/B heterozygotes during vegetative growth (8)....

Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor b2 (TGF-b2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGFb2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-b. These data collectively indicate that TGF-b2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-b2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.