Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC-derived mesenchymal-like progenitor population. We found the direct plating of undifferentiated iPSCs into high-density micromass cultures in the presence of BMP-2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP-2 treatment of iPSC-derived MSC-like (iPSC-MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage-specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell-based approaches to repair joint cartilage damage.
The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with γ-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.
Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1L148A) and generated Runx1L148A/− mice in which >50% of Runx1 activity was abrogated. Runx1L148A/− mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9‐, Runx2‐, and Runx3‐positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1‐Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud–derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral‐expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage. © 2012 American Society for Bone and Mineral Research.
The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOP AP , a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic ␣-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3 region of the SLAP gene indicated that SLAP1 and TOP AP are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOP AP were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOP AP , and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOP AP transmembrane domains mutually exclusive. These data reveal that TOP AP and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins.
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