Direct Conversion Of Fibroblasts To Chondrocyte-like Cells By C-Myc

Cell reprogramming is a progress in which the memory of a mature cell is erased and then the cell develops novel phenotype and function; ultimately, the fates of the cell change. Cell reprogramming usually occurs at gene expression levels in which no genomic DNA sequence change will be involved. By changing the program of the gene expression of cells in a spatiotemporal pattern, cell reprogramming alters the differentiation of cells and thus produces the required cells. In recent years, researchers around the world are strongly interested in cell reprogramming, promoting the rapid development of cell reprogramming technology.In2006and2007, the induced pluripotent stem cells (iPS cells)[with characteristics identical to those of embryonic stem cells (ES cells)] directly in vitro reprogrammed from mouse and human fibroblasts by some defined factors (i.e., Oct4, Sox2, c-Myc and Klf4or Oct4, Sox2, Nanog and Lin28) have captured great attentions in both scientific community and general public; somatic reprogramming, dedifferentiation and the resource of pluripotent stem cells become the research hot points of stem cells and developmental biology. In2012, Japanese stem cell scientist Shinya Yamanaka and John B. Gurdon of the U.K. have jointly won the Nobel Prize in Physiology or Medicine2012for the discovery that mature cells can be reprogrammed to become pluripotent.Another argument of direct reprogramming is lineage reprogramming; in other words, direct transdifferentiation of somatic cells becomes other types of functional cells or progenitor cells. Both reprogramming somatic cells into iPS cells and lineage reprogramming belong to cell reprogramming. Conversion of terminally differentiated somatic cells to iPS cells is a de-differentiation, while lineage reprogramming is involved in trans-differentiation. For security reasons, iPS cells are required to in vitro differentiate into a variety of different cells of types, and then iPS cell-derivatives are applied in regenerative medicine, while lineage reprogramming can directly convert a cell to another terminally differentiated cells. At present, fibroblasts are directly reprogrammed into the various functional cells, such as skeletal muscle cells, cardiac muscle cells, neuronal cells, chondrocytes, hepatocytes and etc, indicating the advantages (simple, fast and high-efficiency) of lineage reprogramming.c-Myc protein belongs to the transcription factors of Myc family, which also includes the N-Myc and L-Myc genes. Studies have shown that c-Myc gene regulates almost15%of all gene’s expression by modifying the expression of its target genes. c-Myc protein plays an important role in the regulation of normal cell proliferation, growth, differentiation, apoptosis, survival, self-renewal of stem cell and the multi-potential of establishment and maintenance.iPS cells directly in vitro reprogrammed from mouse and human fibroblasts by some defined factors (i.e., Oct4, Sox2, c-Myc and Klf4) have demonstrated the important roles of c-Myc. Therefore, c-Myc also becomes one of Yamanaka’s classic four-factors and has a great impact on cellular reprogramming process. In2011, Kunihiko Hiramatsu directly reprogrammed mouse fibroblasts into cartilage-like cells by c-Myc, klf4and sox9, which arouses our interest in c-Myc gene.Cartilage tissue is composed of chondrocytes and cartilage matrix; cartilage consists of cartilage tissue and the surrounding perichondrium. Cartilage is the main bracket ingredients in early embryo and is gradually replaced by bone as fetal developed. In adult body, it plays an important role in body, for example, articular cartilage has a role to support the weight and reduce the friction. Cartilage, with a simple structure, is composed of specialized cells called chondroblasts without vessels and nerve fiber. However, these traits of cartilage tissue limit the cartilage repair after injury. Clinical patients can hardly self-repair after cartilage defect. In the meantime, it has become a big problem in surgical treatment because there is no suitable cartilage transplantation. Therefore, it will be a big success if the cartilage tissue can be built artificially and also makes sense in theoretical innovation and brings a significant clinical application value. With the long-term development of several stages of cartilage tissue engineering, people become aware of that seed cells is one of the major barriers to bring the cartilage tissue engineering into clinical. Researchers use many kinds of cells to gain seed cells, such as embryonic stem cells, mesenchymal stem cells, mogeneity variant chondrocytes, and so on. But it brings the inevitable influences such as difficulty in separation and purification, the low efficiency, immunological rejection, etc. Therefore, finding out a fast and convenient access to the cartilage seed cells will undoubtedly promote the development of cartilage tissue engineering and large-scale clinical applications of cartilage tissue. The advantage of lineage reprogramming and the c-Myc gene in cartilage reprogramming worth further study.Our preliminary experiments show that the overexpression of c-Myc gene in porcine fibroblasts will lead to epithelial-like morphological changes in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)], followed by in vitro and in vivo assassing the identity of the c-Myc-expressing fibroblasts undergoing epithelial-like morphological changes and dissecting the related mechanisms.The results follow as:1. Generation of c-Myc transgene-expressing pig embryos and skin fibroblasts1.1Preparation of swine embryos and skin fib rob last cells (PEFs and PDFs)PEFs and PDFs were produced by sow of gestational age for35days and the newborn Tibet miniature pigs (1-3days after birth), the cells turn out to be fusiform, polygonal and flat star typical fibroblast morphology (Figure1-2).1.2Identification of the lentivirus plasmidThe results of enzyme and sequencing identification confirmed that pLenti-EFla-c-Myc-IRES-EGFP from other lab is right.1.3The preparation of lentivirus carrying c-Myc or vector controlpLenti-EF1-c-Myc-IRES-EGFP or pLenti-EFl-IRES-EGFP and the packaging plasmids (i.e., psPAX2and pMD2.G) co-transfected293T cells to produce two lentiviruses, such as LV-c-Myc (harboring EF1-c-Myc-IRES-EGFP) and LV-con (harboring EF1-IRES-EGFP). followed by determination of virus titers. The titers of LV-c-Myc and LV-con were6.2×106TU/ml and9.2×106TU/ml, respectively.1.4Generation of c-Myc transgene-expressing pig embryos and skin fibroblastsLV-c-Myc and LV-con were used to infect PEFs and PDFs, respectively.48h later, green fluorescence were observed in PEFs and PDFs. Western blotting and immunofluorescence confoirmed the normal expression of Myc transgene in PEF-c-Myc (referred to as P-C cells) and PDF-c-Myc.2. c-Myc reprograms pig embryo fibroblasts to chondrocyte-like cells2.1Preparation of Tibetan miniature primary chondrocytesThe primary chondrocytes of newborn Tibetan miniature pigs (1-3days) were prepared by standard method, followed by identifying the identity of the primary cells via toluidine blue staining, alcian blue staining and immunofluorescence. The prepared primary cells demonstrated positive toluidine blue staining and alcian blue staining, while immunofluorescence showed that the prepared primary cells indicated staining positive for Type II collagen and Aggrecan, but staining negative for Type I collagen, suggesting the successful preparation of pig primary chondrocytes.2.2The proliferation assay for P-C cells by CCK-8Compared with pig primary chondrocytes (referred to as Pr Ch) and cells carrying LV-con, P-C cells showed the stronger proliferative capacity.2.3Cell morphological changes in c-Myc-expressing PEFs (P-C cells)4days after infection, epithelial-like morphological changes were onserved in c-Myc-expressing PEFs (P-C cells). Additionally, P-C cells could still keep the epithelial-like morphological changes after P-C cells were trypsinized and passaged.2.4Identify the identity of P-C cells by special stainingAs cartilage cells secrete acidic glycosaminoglycan, toluidine blue staining and alcian blue staining are used to detect glycosaminoglycan to identify whether the cells have the characteristics of chondrocytes. We intend to preliminarily determine whether P-C cells have the characteristics of chondrocytes by detecting acidic glycosaminoglycans using toluidine blue staining and alcian blue staining. P-C cells demonstrated positive toluidine blue staining and alcian blue staining, while control cells were stained negative for toluidine blue and alcian blue.2.5Detection of cartilage-related gene expression in P-C cells by qRT-PCRqRT-PCR demonstrated the up-regulated expression of cartilage-related genes (i.e., Col2a1, Acan, Sox5and Sox6) and the down-regulated expression of Collal and Col1a2.2.6Detection of cartilage-related gene expression in P-C cells by immunofluorescenceImmunofluorescence analysis showed that P-C cells were stained positive for Type II collagen and Aggrecan, and negative for Type I collagen, which is consistent with the results of qRT-PCR.2.7The karyotype analysis of P-C cellsKaryotype analysis showed that there are19pairs of chromosomes, no deletions, chromosomal aberrations and ectopic and other abnormal changes in P-C cells.2.8Histological detection About4weesk after P-C cells were inoculated subcutaneously in nude mice, there has visible lump with hard texture at transplantation site, while control cells did not form lump at transplantation site. Next, we sacrificed a nude mouse, and found white cartilage-like tissue contained in stripped lump, followed by tissue fixation, paraffin embedding and sections. The results of HE staining and toluidine blue staining show a large number of cartilage cells stained positive for EGFP and toluidine blue.3. c-Myc reprograms pig skin fibroblasts to chondrocyte-like cells3.1Cell morphological changes in c-Myc-expressing PDFs (i.e., PDFs-c-Myc cells or D-C cells)4days after infection, epithelial-like morphological changes were onserved in c-Myc-expressing PDFs (D-C cells). Additionally, D-C cells could still keep the epithelial-like morphological changes after D-C cells were trypsinized and passaged.3.2The proliferation assay for D-C cells by CCK-8Compared with pig primary chondrocytes (referred to as Pr Ch) and PDFs carrying LV-con, D-C cells showed the stronger proliferative capacity.3.3Identify the identity of D-C cells by special stainingAs cartilage cells secrete acidic glycosaminoglycan, toluidine blue staining and alcian blue staining are used to detect glycosaminoglycan to identify whether the cells have the characteristics of chondrocytes. We intend to preliminarily determine whether D-C cells have the characteristics of chondrocytes by detecting acidic glycosaminoglycans using toluidine blue staining and alcian blue staining. D-C cells demonstrated positive toluidine blue staining and alcian blue staining, while control cells were stained negative for toluidine blue and alcian blue. The mentioned-above results demonstrates that D-C cells and chondrocytes share common features.3.4Detection of cartilage-related gene expression in P-C cells by immunofluorescenceImmunofluorescence analysis showed that D-C cells were stained positive for Type II collagen and Aggrecan, and negative for Type I collagen, which is consistent with the results of P-C cells.4. Mechanisms involved in c-Myc reprogramming of fibroblasts into cartilage-like cells4.1MET-like cellular marker alterations induced in c-Myc-expressing PEFs and PDFsDuring c-Myc transgene was reexpressed in PEFs and PDFs, we rapidly found that c-Myc-expressing PEFs and PDFs underwent epithelial-like morphological changes around day4after virus infection, indicating MET induction in c-Myc-expressing PEFs and PDFs because MET is a reversible biological process that involves the transition from motile, multipolar or spindle-shaped mesenchymal cells to planar arrays of polarized cells called epithelia.This further prompted us to characterize the existence of a MET at the molecular level. Firstly, at5or10days after virus infection, qRT-PCR analysis showed the significantly increased expression of epithelial markers, such as cell adhesion proteins (E-cadherin and Bves), tight junction protein occludin, cytokeratins (Krt8and Krt18), and the significantly reduced expression of mesenchymal markers [vimentin and fibronectin1(FN1), collagen family of proteins (COL1A1and COL5A2) and matrix metalloproteinase (MMP) family (MMP12and MMP14)] in c-Myc-expressing PEFs and PDFs. Immunofluorescence assay demonstrated that c-Myc overexpression in PEFs and PDFs activated epithelial genes including E-cadherin and a-catenin, while Western blot analysis validated the increase in a-catenin, Bves or Krt18, and showed a concomitant reduction of the mesenchymal-like marker vimentin. Furthermore, the more obvious upregulation of the epithelial genes including E-cadherin, occludin, Krt8and Krt18was observed when c-Myc-expressing PEFs were subjected to successive subculturing by standard trypsinization procedure every several day until the15th passage (P15).In summary, c-Myc activates an epithelial gene expression program in c-Myc-expressing PEFs and PDFs.4.2Decreased cell motility and increased cell adhesion demonstrated in c-Myc-expressing cells undergoing epithelial-like morphological conversionEMT is a process characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility, while as mentioned above, MET is the reverse process of EMT. Therefore, we further determined the changes in motile properties and adhesion ability of c-Myc-expressing PEFs and PDFs which have undergone MET by in vitro migration assay using transwell chamber and cell adhesion assay, respectively. c-Myc-expressing PEFs and PDFs with an epithelial-like phenotype exhibited significantly decreased mobility compared with vector control cells. In addition, c-Myc-expressing PEFs and PDFs with MET-like cellular marker alterations demonstrated the significantly enhanced cell adherent ability compared with control cells, which was consistent with the above-described increased expression of cell adhesion proteins (E-cadherin, α-catenin and Bves) in c-Myc-expressing PEFs and PDFs. Collectively, the enforced expression of c-Myc in PEFs and PDFs triggers MET accompanied by the reduced cell migration and increased cell adhesion.4.3Ectopic expression of c-Myc in PEFs and PDFs impaired cytoskeletal events and resulted in RhoA/Rock pathway inactivationIt has been illustrated that RhoA/ROCK-dependent pathway participates in regulating EMT, MET and cytoskeletal signalling events, and is crucial for cell motility; the cytoskeleton regulation is associated with EMT and MET; moreover, cytoskeletal reorganization exemplified by the formation of stress fibre bundling arrays is a prerequisite for processes like endocytosis, cell motility and cancer cell invasion. This prompted us to dissected the status of stress fibre formation and polymerized actin in c-Myc-expressing cells and vector-expressing cells. Using phalloidin staining, we observed that stress fibre formation was suppressed in c-Myc-expressing cells, but not in vector-expressing cells. Moreover, filopodia and lamellipodia are identified as dynamic cellular features on cell membrane surfaces, requiring actin polymerisation, and are involved in normal cell motility and cancer cell invasion&metastasis. Under a scanning electron microscope, a highly significant reduction or loss of filopodia and lamellipodia (cell protrusion) was observed on the cell surfaces of c-Myc-expressing cells, as strongly supported by a significant reduction in cell motility of c-Myc-expressing PEFs and PDFs compared with control cells.Since RhoA activation induces the formation of stress fibres, Racl stimulates lamellipodium formation and cdc42induces filopodia, pull-down assays were employed to quantify the amount of the GTP-bound active form of RhoA, RhoB, RhoC, Rac1and cdc42. A higher level of the active form of RhoA and RhoC was found in vector-expressing cells compared with that in c-Myc-expressing cells. Similarly, a higher level of active Rac1was observed in vector-expressing cells compared with that in c-Myc-expressing cells. In addition, cdc42was undetectable in c-Myc-expressing cells, while cdc42levels in vector-expressing cells were detectable. These results provide additional evidence that c-Myc plays an important role in regulating cellular events related to cytoskeleton reorganization, MET and cell motility.Collectively, all combined findings from epithelial-like morphological changes, MET-like cellular marker alterations, functional experiments (i.e., in vitro migration assay and cell adhesion assay), phalloidin staining, scanning electron microscope and Rho-GTPase activation assay, for the first time, strongly suggests c-Myc-induced MET in c-Myc-expressing pig fibroblasts, and that cytoskeleton depolymerization and RhoA/Rock pathway inactivation in c-Myc-expressing pig fibroblasts contribute to MET induced by c-Myc.Conclusions:(1) There findings demonstrate, for the first time, the direct conversion of pig, fibroblasts to chondrocyte-like cells by c-Myc only; additionally, c-Myc-expressing fibroblasts which were inoculated subcutaneously in nude mice can form cartilage tissue.(2) Mouse and human fibroblasts can be efficiently and directly reprogrammed in vitro to chondrocyte-like cells by the enforced expression of c-Myc.(3) The enforced expression of c-Myc in fibroblasts can trigger MET to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute, while c-Myc-induced MET may be an early event in c-Myc reprogramming fibroblasts into cartilage-like cells, as remains to be fully characterized.Discoveries and innovations of this investigation:(1) There findings demonstrate, for the first time, the direct conversion of pig, mouse and human fibroblasts to chondrocyte-like cells by c-Myc only; additionally, c-Myc-expressing fibroblasts which were inoculated subcutaneously in nude mice can form cartilage tissue.(2) There findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET.