Insights into the present and future of cartilage regeneration and joint repair.

Knee osteoarthritis is the most common joint disease. It causes pain and suffering for affected patients and is the source of major economic costs for healthcare systems. Despite ongoing research, there is a lack of knowledge regarding disease mechanisms, biomarkers, and possible cures. Current treatments do not fulfill patients' long-term needs, and it often requires invasive surgical procedures with subsequent long periods of rehabilitation. Researchers and companies worldwide are working to find a suitable cell source to engineer or regenerate a functional and healthy articular cartilage tissue to implant in the damaged area. Potential cell sources to accomplish this goal include embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The differentiation of stem cells into different tissue types is complex, and a suitable concentration range of specific growth factors is vital. The cellular microenvironment during early embryonic development provides crucial information regarding concentrations of signaling molecules and morphogen gradients as these are essential inducers for tissue development. Thus, morphogen gradients implemented in developmental protocols aimed to engineer functional cartilage tissue can potentially generate cells comparable to those within native cartilage. In this review, we have summarized the problems with current treatments, potential cell sources for cell therapy, reviewed the progress of new treatments within the regenerative cartilage field, and highlighted the importance of cell quality, characterization assays, and chemically defined protocols.

GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient.

Organs and tissues, such as cartilage and limbs, are formed during development through an orchestration of growth factors that function as morphogens. Examples of growth factors include growth differentiation factor 5 (GDF5) and transforming growth factors beta 1 and 3 (TGFß-1 and TGFß-3) which can specify creation of more than one cell type after forming a concentration gradient in vivo. Here, we studied the impact of morphogen gradients during differentiation of induced pluripotent stem cells (iPSCs) into the chondrocyte lineage. Cell budding zones, consisting of condensed cell aggregates, were observed only in gradients of GDF5. T-box transcription factor 3 (TBX3) was detected specifically in the budding zones (ranging from 500-1,500 particles/µm2) of nuclei and cell vesicles. A homogenous density of GDF5 of 900 particles/µm2 on a surface induced budding and expression of TBX3 after five days in iPSCs. Therefore, we conclude that a gradient of GDF5, as well as the specific homogenous density of GDF5, support the induction of TBX3 in iPCSs. Moreover, differentiation of iPSCs first on GDF5 gradient or homogenous surfaces for five days and then in a three-dimensional structure for five weeks resulted in pellets that expressed TBX3.

Nuclear reprogramming by xenopus oocytes.

The aim of this contribution is to relate amphibian nuclear transplantation work to prospects for stem cell creation and hence to the long-term aim of cell replacement in humans. The methods used include the transplantation of single somatic cell nuclei to enudeated unfertilized eggs of Xenopus, and also the transfer of multiple somatic cell nuclei to the nucleus (germinal vesicle) of a growing ovarian oocyte. A key difference between these types of recipient cell is that eggs immediately induce DNA replication in transplanted nuclei, whereas an oocyte induces no DNA replication, but directly reprograms an injected nucleus to a new pattern of transcriptional activity. We summarize the extent and success of past and current nuclear reprogramming in experiments with enucleated frog eggs, and also those carried out with growing oocytes. We discuss possible mechanisms of nuclear reprogramming, and the possible contribution of such knowledge for stem cell creation and cell replacement in humans.

Nuclear reprogramming and stem cell creation.

The transplantation of a somatic cell nucleus to an enucleated egg results in a major reprogramming of gene expression and switch in cell fate. We review the efficiency of nuclear reprogramming by nuclear transfer. The serial transplantation of nuclei from defective first-transfer embryos and the grafting of cells from such embryos to normal host embryos greatly increases the proportion of nuclei that can be seen to have been reprogrammed. We discuss possible reasons for the early failure of most nuclear transfers from differentiated cells and describe the potential value of growing oocytes, rather than unfertilized eggs, as a source of nuclear reprogramming molecules and for the eventual identification of these molecules. Nuclear transfer provides a possible route for the creation of stem cells from adult somatic cells.

From intestine to muscle: nuclear reprogramming through defective cloned embryos.

Nuclear transplantation is one of the very few ways by which the genetic content and capacity for nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding Xenopus larvae. We have analyzed single nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 10(4) times, within a few hours of nuclear transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication.

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III-box I hairpin with a 3' single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III-box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III-box I hairpin in partially unwound DNA.

A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein.

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA.

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.

Identification and characterization of genomic nucleosome-positioning sequences.

Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.

The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding protein and the single strand DNA-binding protein, ICP-8.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.